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Characterization of proteins interacting with proteolytic system of ticks
Matoušková, Zuzana ; Mareš, Michael (advisor) ; Hudeček, Jiří (referee) ; Perner, Jan (referee)
(EN) Ticks are ectoparasites found worldwide that feed on the blood of their hosts and transmit several important pathogens to humans and domestic animals. Tick saliva contains bioactive molecules that are injected into the host tissue to aid in successful blood feeding. Among these molecules, proteinaceous protease inhibitors are being extensively studied for their potential biomedical applications. This work focuses on novel protease inhibitors from the saliva of the ticks Ixodes ricinus, vector of Lyme disease and tick-borne encephalitis, and Ornithodorus moubata, vector of relapsing fever and African swine fever. Research has focused on the biochemical and structural characterization of three members of the protease inhibitor families of cystatins, serpins, and tyropins, and has attempted to elucidate their biological function in tick-host interactions. Cystatin OmC2 from O. moubata was identified as a broad-spectrum inhibitor of host cysteine cathepsins with both endopeptidase and exopeptidase activity. Crystal structure determination allowed description of the relationship between the structure of OmC2 and its inhibitory specificity. The ability of OmC2 to modulate the host immune response was demonstrated in bioassays and a suppressive effect on O. moubata in vaccination experiments. The...
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Female Footbinding: A Comparison of Attitudes of the Chinese and of the Western Missionaries in China
Chobotská, Eva ; Hudeček, Jiří (advisor) ; Lomová, Olga (referee)
The bachelor thesis evaluates the influences that missionaries and activists, mainly from Great Britain, had on the Chinese anti-footbinding movements. Its particular focus is on the works of writer and activist Alicia Little, and missionaries Timothy Richard and John MacGowan, comparing their positions and arguments with the writings of Chinese reformers and activists Liang Qichao and Kang Youwei. The purpose of the thesis is to highlight, based on contemporary sources, that Western criticism was primarily and mostly voiced by women who sought to improve conditions for their Chinese counterparts, while Chinese criticism was formulated by men as part of reforms and efforts to modernize China.
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Therapeutic strategies in cystic fibrosis
Křesťan, Jan ; Bořek Dohalská, Lucie (advisor) ; Hudeček, Jiří (referee)
Cystic fibrosis is an autozomal recessive disease caused by mutations in the CFTR gene. The aim of this work is conclusion of therecent knowledgefrom theusage of CFTR modulators in the treatment of cystic fibrosis. The first part of this bachelor thesis is dedicated to the disease itself, its history and symptoms. In the form of description of the CFTR gene and its mutations which lead to the cystic fibrosis disease, the causes of the disease are described. Function of CFTR protein is also mentioned. The second part of this thesis is focused on conclusion of the current knowledge of the CFTR modulators treatment. In the last chapter of the work, possible future changes and ways of treating patients with CFTR modulators therapy are considered.
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Structure and stability of lysozyme - effects of the environment
Toupalová, Kateřina ; Hudeček, Jiří (advisor) ; Martínek, Václav (referee)
In this Thesis, we studied the stablity of lysozyme solutions with temperature and the influence of alcohols (ethanol, propan-1-ol, 2-methoxyethanol) on lysozyme structure. Within the limits of pH 3 to 10, the protein is rather stable and is not much influenced by concentrations up to 40 % of alcohol (for ethanol and propanol), or 9% of 2-methoxyethanol. Lowering or increase of pH (to 1.9 or 11, respectively) destabilizes the protein to some extent; this effect is more pronounced for the longer alkyl chain of propanol. The temperature stability of lysozyme up to 55 řC observed for solutions with pH 3 and 7 is in accordance with the data in literature, as well as the observation of some thermal denaturation starting between 45 an 50 řC in acidic solution (pH 1.9). Also at alkaline pH, the stability is lowered, and the protein is partially precipitated. Key words: derivative spectroscopy, differece spectroscopy, influence of alcohols, stability of lysozyme
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Macrocyclic complexes as models for copper(II) bonding in cupredoxins: Proof of concept
Ulrichová, Tereza ; Hermann, Petr (advisor) ; Hudeček, Jiří (referee)
Currently, modified electron transport metaloproteins with presence ion Cu(II) in their active site are most commonly used to study kinetics of electron transfer. One of the most studied model protein is cupredoxin azurin from P. aeruginosa with standard redox potential approximately 310-360 nm (depends on experimental conditions and techniques). Ion Cu(II) in azurin is coordinated by the side chain atoms of His46 , His117 , Cys112 , Met121 and carbonyl of Gly45 . The aim of this work was to prepare macrocyclic ligand, 1-[1H-imidazol-2- yl)methyl]-1,4,7-triazacycklononanu (LIM ), which would be able to form stable complexes with both of copper ions Cu(II) and Cu(I). Such as macrocyclic complexes could be utilized as low molecular models of active sites in proteins. The ligand was prepared using five-step synthesis employing protection of macrocycle, reduction and subsequent bromation of 1H- imidazol-2-karbaldehyd, and following alkylation reaction. Deprotection of macrocycle revealed final product of ligand. All reaction intermediates and prepared ligand were characterized by NMR and MS. Potentiometric titrations determined ligand protonation constant (log K1 = 10,62, log K2 = 6,65, log K3 = 4,91), which describe acid-base properties of a ligand in an aqueous solution. Similarly, the coordination...
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Interactions of prosthetic material with proteins
Osifová, Zuzana ; Hudeček, Jiří (advisor) ; Martínek, Václav (referee)
This bachelor thesis deals with the problem of ocular prostheses. This problem is in thesis widely researched. Emphasis is placed on protein adsorption onto synthetic surface. Experimental part of the thesis focuses on lysozyme adsorption onto polymethylmethacrylate surface. Protein adsorption was lower than the specified limit of detection. According to experimental data was this amount lower than 5,37 μg/cm2 . In the thesis was conducted survey on secondary education students' awareness of ocular prosthesis. The results show slightly greater awareness of science-oriented students.
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Occurence and importance of the "molten globule" structure in proteins
Plutinský, Martin ; Hudeček, Jiří (advisor) ; Martínek, Václav (referee)
One part of my work focus on literature review on molten globule theme. This conformational state is generally formed in solution of some proteins under mild denaturation conditions as a thermodynamically stable state. Molten globule-like intermediate is also transiently formed during refolding of proteins. It is assumed and even it was proved for some proteins that kinetic (refolding) intermediate and equilibrium unfolding molten globule is identical. The second part of my work presents conformational study of horse heart ferricytochrome c under acidic conditions in low and high ionic strenght (addition of 0,5 M sodium chloride). Cytochrome c is a mitochondrial protein which mediates electron transfer in respiratory chain. These conformational changes were monitored by UV/VIS and derivative spectrophotometry in four wavelength ranges of absorption spectra - aromatic amino acids absorption range, Soret band, Q-band and CT band. Under high ionic strength probably a molten globule state is stabilized in the pH range 1,90-2,60. However, under low ionic strength, changes in polarity of tryptophane and tyrosine residues vicinity (between pH 2,60 and 2,30) and in spin state of iron atom (around pH 2,10) were observed due to putative denaturation of the protein. (in Czech) (in Czech)
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Functional and structural study of thermally activated TRP ion channels: The role evolutionarily conserved motifs in the TRPA1 modulation
Kádková, Anna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee) ; Obšilová, Veronika (referee)
Ankyrin receptor TRPA1 is an ion channel widely expressed on primary afferent sensory neurons, where it acts as a polymodal sensor of nociceptive stimuli. Apart from pungent chemicals (e. g. isothiocyanates, cinnamaldehyde and its derivatives, acrolein, menthol), it could be activated by cold temperatures, depolarizing voltages or intracellular calcium ions. TRPA1 channel is a homotetramer in which each subunit consists of cytoplasmic N and C termini and a transmembrane region. The transmembrane part is organized into six alpha- helices connected by intra- and extracellular loops. The N terminus comprises a tandem set of 16 to 17 ankyrin repeats (AR), while the C terminus has a substantially shorter, dominantly helical structure. In 2015, a partial cryo-EM structure of TRPA1 was resolved; however, the functional roles of the individual regions of the receptor have not yet been fully understood. This doctoral thesis is concerned to elucidate the role of highly conserved sequence and structural motifs within the cytoplasmic termini and the S4-S5 region of TRPA1 in voltage- and chemical sensitivity of the receptor. The probable binding site for calcium ions that are the most important physiological modulators of TRPA1 was described by using homology modeling, molecular-dynamics simulations,...
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Preparation of recombinant human cytochrome b5
Hálková, Tereza ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Cytochrome b5 is a small heme protein. There are three isoforms present in human organism, one is located in the membrane of endoplasmic reticulum (microsomal cyt b5), second in the outer mitochondrial membrane and the third (soluble form) was found in cytoplasm of matured erythrocytes. The main role of cytochrome b5 is to transport single electron in various reactions including cytochrome P450-dependent reactions. First aim of the thesis was to prepare and to isolate the soluble form of rabbit microsomal cytochrome b5, using heterologous expression in Escherichia coli strain BL-21 Gold. The plasmid pET22b containing synthetic gene for rabbit microsomal cytochrome b5, lacking the sequence encoding the membrane associated C-terminal domain, was used as an expression vector. The second aim was to synthesize the expression vectors carrying genes for human microsomal and erythrocytic cytochromes b5. These genes were prepared by gene synthesis, ligated to cloning vector pUC19, amplified in E. coli DH5α competent cells and their sequences were verified by DNA sequencing. Consequently the pET22b expression vectors containing genes for human microsomal and erythrocytic cytochrome b5 were constructed and finally their suitability for heterologous expression was evaluated. Keywords: Heterologous expression,...
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Pulse proteolysis in evaluation of conformational stability of cytochromes b5
Maroušková, Růžena ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Mixed-function oxidases play a major role in the metabolism of xenobiotics. The main component of this system is the cytochrome P450, it oxidizes substrates coming into our body to more polar products. Another component of mixed-function system - the cytochrome b5 (cyt b5) is able to modulate the function of cytochrome P450, the mechanism of this modulation is yet unknown. However, it is believed that it could be mediated via transfer of electron or allosteric modulation of cytochrome P450 caused by interaction with cyt b5. The aim of this thesis was to find and prepare analogs of cyt b5, which are unable to transfer electrons to cytochrome P450 and simultaneously are structurally very similar to native cyt b5. The conformational stability of cyt b5 and its analogs was monitored using pulse proteolysis. This method employs proteases to cleave the evaluated protein at varying concentration of a denaturant. For soluble proteins, urea is typically used as denaturant in combination with thermolysin as protease. While for membrane proteins, sodium dodecyl sulfate (SDS) is usually used as denaturant together with subtilisin as protease. The aim of this thesis was to use these methods to compare a conformational stability of the native human cyt b5 with apo-cyt b5 and analogs of the cyt b5 reconstituted...
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