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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.
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...
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...
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...
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.
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...
Heřmanová, Ivana ; Doležal, Pavel (advisor) ; Berka, Pavel (referee)
1. ABSTRACT The objective of this study is to optimize a nortryptiline hydrochloride patch previously designed (1) with the intended aim to develop controlled drug release system as a smoking cessation aid. Patches of 5%, 7.5% and 10% NTH concentrations were assayed for physical experiments. Measured thickness of the patches remains constant regardless of the NTH concentration. Obtained values are 31.88±5.0 µm, 32.33±5.1 µm and 32.00±3.8 µm for 5%, 7.5% and 10% patch respectively. The variability is lower than 20 % in every case. Release and penetration studies were performed using Franz type of diffusion cells. The maximum cumulative amount released from assayed patches at 7 hours (last sampling point) varies as a function of the NTH concentration. Two kinetic models, power law and first order kinetics, are useful in order to predict the maximum amount to be released and the rate at which the process will be developed. Taking first order results into account, the maximum amount to be released (Qinf) is 7.185±0.30[mg/cm2 ]; 12.359±0.69[mg/cm2 ]; and 29.333 ±1.97 [mg/cm2 ] for 5%; 7.5% and 10%patch respectively. Considering the power law fitting, the main conclusion is that the release mechanism is basically fickian diffusion as the exponent is about 0.5. Estimated permeation parameters are Kp=0.019·10-3...
Permeation of nanoparticles across sublingual membrane 4
Horejšová, Lenka ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
Charles University in Prague, Faculty of Pharmacy in Hradci Králové Department of: Pharmaceutical technology Consultant: doc. RNDr. Pavel Doležal, CSc. Student: Lenka Horejšová Title of Thesis: Permeation of Nanoparticles Across Sublingual Membrane 4 The theoretical part describes the anatomy and physiology of oral cavity and sublingual drug administration. It also describes theory of nanotechnology and the characterization of nanoparticles with a focus on nanoparticle drug forms and there are also mentioned methods and mesuring device. . The experimental part describes in vitro permeation experiment of nanoparticles (Nano beads based on PD, Chromeon 470 marked, carboxylated). Permeation of this nanoparticles through porcine sublingual membrane (1 cm2) . Thesis compares nanoparticles transfer of phosphate buffer pH 6.6 and citrate buffer pH 6.6 to the acceptor phase consisting of phosphate buffer pH 7,4. First permeation experiment works with frozen sublingual membrane, a second experiment with the native membrane. The dimension of nanoparticles and aggregates of nanoparticles in dispersions were measured before the start of permeation by the dynamic light scattering. This work demonstrates that the choice of donor buffer has not an important influence. Key words: nanoparticles, fluorescent...
DSC of Lipidic Excipients
Gillová, Martina ; Musilová, Marie (advisor) ; Doležal, Pavel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate Mgr. Martina Gillová Consultant RNDr. Marie Musilová, CSc. Title of Thesis DSC of lipidic excipients In this work was evaluated the interaction of the lipidic components with water by using DSC (differential scanning calorimetry). In the first part of this work, we used samples which contained a phospholipid (DMPE). The samples which we used in the second part of the work, contained cutaneous lipids presented by pseudoceramides (14S24 and H6524) and cholesterol. Our attention was especially payed to the vindication of the existence of different structure organizations of hydrated lipidic components. By the samples which contained the phospholipid DMPE, we registered the most of the phase transitions presented in the literature on our calorimeter by using slightly modificated conditions. In similar conditions of the measurement we registered only the crystallization peak from the different phase transitions by the commercially produced pseudoceramide H6524. By the synthetic pseudoceramide 14S24 by used conditions of the measurement we didn't prove any phase transitions.
Characterization of the protein import into Giardia Intestinalis mitosomes
Pyrihová, Eva ; Doležal, Pavel (advisor) ; Tsaousis, Anastasios (referee) ; Stairs, Courtney (referee)
Mitochondrial endosymbiosis was a key event in the evolution of eukaryotes. Its proteome evolved into a unique combination of inherited bacterial components as well as novel eukaryotic inventions. Today, mitochondria show a huge variety across eukaryotic species - from aerobic mitochondria with cristae and complex protein apparatus for maintaining its own genome to hydrogen-producing hydrogenosomes and tiny anaerobic mitosomes without their own genome and with only a single metabolic pathway. Comparing the existing spectra of mitochondria is beneficial for studying their evolution. The only ubiquitous and unifying features are double membrane, ISC pathway for iron-sulfur cluster synthesis and the core of protein import pathway. Therefore, these features could be considered as truly ancestral and essential to mitochondria. Mitosomes of various parasitic protists have evolved independently from complex mitochondria, since they are present in completely unrelated species and yet their evolution led in a surprisingly similar composition of protein import pathway. These retained components are thus believed to be functionally essential and for some reason hard to be replaced by alternate proteins. Mitosomal import pathways were considered very minimalistic for a long time. Nevertheless, the latest...

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7 DOLEŽAL, Pavel
49 DOLEŽAL, Petr
49 Doležal, Petr
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