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Crystallographic study of the iron-regulated outer membrane lipoprotein (FrpD) from Neisseria meningitidis
SVIRIDOVA, Ekaterina
Neisseria meningitidis (N. meningitidis) is a Gram-negative commensal bacterium colonizing nasopharynx of about 10 % of healthy individuals, which can cause invasive diseases, such sepsis and meningitis, upon occasional penetration into bloodstream. Pathogenesis of N. meningitidis appears to be directly related to conditions of limited iron availability. Under these conditions two proteins of unknown function: FrpC and FrpD, are synthesized. FrpD is a highly conserved lipoprotein of N. meningitidis anchored to the bacterial outer membrane. It is known that FrpD tightly binds the FrpC protein, which belongs to the Repeat-in-Toxin (RTX) protein family and may act as bacterial exotoxin. However, the mechanism of FrpD-FrpC interaction and the exact function of this complex are unknown due to the absence of structural information on these proteins. Therefore, we set out to determine the structure of FrpD and provide insights into its interaction mechanism with FrpC and structure-functional relationships of these two proteins. We determined the first crystal and solution structures of the FrpD protein. We found that atomic structures of FrpD reveal a novel protein fold. We uncovered the structure-function relationships underlying the mechanism of interaction between the FrpD and FrpC proteins and tested the putative function of the FrpD-FrpC1-414 complex in vitro. Finally, we proposed the putative function of the FrpD-FrpC1-414 complex as a new minor adhesin of N. meningitidis, which mediates the bacterial adhesion to the host epithelial cells and facilitate the colonization. Our work constitutes the first step in clarifying the molecular basis of the FrpD-FrpC interaction and sets the base for further investigation of the role of FrpD and FrpC in the virulence mechanism of N. meningitidis.
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Mechanochemical Preparation of Alumina-Ceria
Jirátová, Květa ; Spojakina, A. ; Tyuliev, G. ; Balabánová, Jana ; Kaluža, Luděk ; Palcheva, R.
Ceria containing catalysts play an essential role in heterogeneous catalytic processes. However, ceria shows poor thermal stability and low specific surface area and therefore, many studies have been done to improve its properties by combination with other oxides. Alumina-ceria is substantial component of the three ways catalysts, due to the ceria ability to function as the buffer of oxygen and to enhance the oxygen storage capacity of the catalysts. Ceria in these catalysts also functions as structural promoting component, increasing alumina stability towards thermal sintering. Promising method of oxides preparation, very interesting and simple but not sufficiently studied yet is a mechanochemical synthesis. Here we report on the synthesis of nano-sized alumina, ceria and ceria-alumina of various compositions by a wet solid phase mechanochemical reaction of hydrous aluminum, and/or cerium nitrate with ammonium bicarbonate after addition of a small amount of water. The aim of this contribution is to study processes being in progress during synthesis of the mixed oxides, interaction between components and their mutual effect on the properties of resulting products. The phase evolution during mechanical milling and the subsequent heat treatment of precursors were studied by X-ray diffraction, DTA/TG, H2-TPR, NH3-TPD, CO2-TPD, N2 adsorption at -195°C, IR, and XPS spectroscopy. Alumina and mixtures of alumina with different quantities of CeO2 (1- 18 wt. %) were synthesized by mechanochemical method from aluminum nitrate, cerium nitrate and ammonia bicarbonate.
Fulltext: content.csg -  PDF
Plný tet: SKMBT_C22015083108560 - PDF
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Human and social capital in primary education
KOKŠTEIN, Jiří
The thesis contains a theoretical insight into the studies that the issue of human and social capital involved from different perspectives, engage their evolution over time, their measurability, examine them, and complemented with other evidence. This part contains relation of human and social capital in relation to the aging of the human population and focuses on the human capital of older workers. Theoretical study ends with an insight into the basic education, the role of teacher and seeks to provide information on surveys already conducted in the area of education, which in the application of serve for me compared to the data obtained from the survey. The application part is focused on the teaching staff of primary schools. Education in currently globalized world, changing not only its content but also methods and forms of work. Irreplaceable role in this process is played by educators. To meet all the demands placed on them, it is essential that they pay close attention to their continuing education and sharing of acquired education, which is essential to the growth of human and social capital, because they are interacting. The work examines selected sample of teachers and approaches to this issue by detecting what is education motivates them whether they are satisfied with the benefits of educational events, what type of education they prefer etc. Working findings further analyzes looking for relationships between age and sex educators according to the hypotheses . Educator is someone who is in constant contact with other people. This of course also formalizes its attitudes and experiences. As a human being perceives the views of society and shapes on her own views, which it subsequently may influence the exercise of his profession. Therefore, in this part of the thesis is dedicated to exploring and bridging social capital, which determines the size of the circle of people with whom the teachers generally meet and interact. The last research that is possible to find thesis, it is perceptions of social status of teachers by themselves.
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Distribution of CD46 and β1 integrin molecules with respect to different membrane structures of the sperm head
Šebková, Nataša ; Frolíková, Michaela ; Děd, Lukáš ; Dvořáková-Hortová, Kateřina
CD46 protein plays an important role during fertilization and its role is associated with acrosome stability. CD46 is probably involved in signalling pathways triggering the acrosome reaction (AR). It also associates through membrane integrins with specific MAP kinases involved in the AR. Our aim was to monitor the dynamics of relocation of CD46 and β1 integrin during sperm maturation and its preparation for the fertilization. The dependence of this localization changes on the dynamic of actin cytoskeleton was studied. Our results show the changes in the localization of these proteins associated with the AR and their co-localization was observed using proximity ligation assay. After the AR CD46 and β1 integrin spreads across the sperm head, entering the post-acrosomal compartment, and permeates the borders of different domains. It was shown previously that actin dynamics is necessary for acrosome reaction-associated translocation of Izumo1 protein that is required for sperm-egg fusion. Therefore Latrunculin A was used during sperm incubation. The co-incubation of capacitated sperm with Latrunculin A leads to a decrease of the percentages of sperm, which express relocation pattern after induced AR. 3D models and visualizations of potential membrane processes responsible for the relocation of proteins from the acrosomal area to the other compartments of the sperm head were prepared. Our results deliver new information that proteins CD46 and β1 integrin undergo dynamic relocation towards the sites of sperm-egg fusion during the AR in vitro. The inhibitor of actin dynamics abrogates significantly the AR-associated changes in proteins localization. We speculate that this relocation is of importance for the successful sperm-egg interaction, adhesion and subsequent gamete fusion.
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Estrogen receptor beta (ERβ) in testicular cells and sperm
Dostálová, Pavla ; Žatecká, Eva ; Děd, Lukáš ; Dorosh, Andriy ; Postlerová, Pavla ; Jonáková, Věra ; Dvořáková-Hortová, Kateřina ; Pěknicová, Jana
Estrogen is a steroid hormone that plays an important role during sperm development in the male and female reproductive tract. Estrogen signalling is a complex process that depends on cell milieu and presence of receptors. Thanks to the steroid nature of estrogens, they can pass through the plasmatic membrane and bind to the intracellular estrogen receptors (ERs). Within the cell, there are several pools of ERs. One of them is localized to the cell nucleus and their activation leads to direct or indirect binding to DNA and ultimately to alternation in gene expression (genomic pathway). Other pools of ERs are associated with plasma membrane or are located in cytosol. Activation of membrane associated ERs leads to rapid non-genomic responses. Nowadays, two classical estrogen receptors are known – ERα and ERβ. Since ERβ is a predominant variant in testes, we focused our study on expression of ERβ variants in murine testes and sperm. We detected two variants of ERβ at mRNA level in both, testes and sperm. These variants differ in 54 nucleotids within the ligand binding domain and this variability results in different affinity to estrogen. We analyzed individual testicular cell types (spermatogonia, spermatocytes, spermatids, Sertoli cells) by RT-qPCR. Our results suggest that both ERβ variants are coexpressed in the same cell type and may therefore interact together. This may have consequences in mediating of estrogen signalling. Moreover, ERβ is expressed more in the later stages of spermatogenesis suggesting the role of ERβ in these stages or alternatively in spermatozoa alone. At the protein level, we detected ERβ in nuclear, membrane and cytosolic fraction prepared from testicular tissue suggesting the involvement of both, genomic and non-genomic, pathways of estrogen signaling in testes. In sperm, anti-ERβ antibodies localized ERβ in acrosome region and tail which is in accordance with the known role of estrogen on capacitation, acrosome reaction and motility.
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Possible role of spermatogenic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) in mammalian sperm
Margaryan, Hasmik ; Dorosh, Andriy ; Čapková, Jana ; Postlerová, Pavla ; Philimonenko, Anatoly ; Hozák, Pavel ; Pěknicová, Jana
Sperm proteins are important for the structure and function of these specific, highly differentiated cells. Certain of these proteins play a role in sperm-egg recognition during primary or secondary binding at zona pellucida glycoprotein matrix. The aim of this study was to characterize the acrosomal sperm protein recognized by a monoclonal antibody (MoAb) Hs-8, prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperm, and to test the possible role of this protein in gamete interaction. MoAb Hs-8 specifically labelled a 45 kDa protein from the sperm extract in the immunoblotting test. Sequence analysis identified this Hs-8 protein as GAPDHS. In order to perform a control tests, a commercial mouse anti-GAPDHS MoAb was applied. Both antibodies showed similar staining patterns using immunofluorescence labelling, transmission electron microscopy and immunoblot analysis. Moreover, both Hs-8 and commercial anti-GAPDHS antibodies blocked the secondary sperm-zona pellucida binding. Generally, GAPDHS was considered mainly as sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility and its role in the sperm head was unknown. In this study, we confirmed the potential additional role of GAPDHS as a binding protein that is involved in the sperm-zona pellucida interaction.
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CD46 and β1integrin interaction in mouse sperm head
Šebková, Nataša ; Frolíková, Michaela ; Dvořáková-Hortová, Kateřina
CD46 protein plays an important role during fertilization and its role is associated with acrosome stability. CD46 is probably involved in signalling pathways triggering the acrosome reaction. Integrins interact with many cytoskeletal proteins such as actin, therefore changes in the actin cytoskeleton before and after AR may lead to changes in the association and localization of CD46 and β1integrin. Our aim was to monitor mutual CD46 and β1integrin interaction detected by the proximity ligation assay. It generates a localized signal in a form of spots revealing the exact position of the recognition event. Proteins interaction was study in freshly released sperm and sperm during the acrosome reaction, during which there is a gradual relocation of these proteins towards the equatorial segment and the whole sperm head. Proteins α and β tubulin were used as a positive control, α tubulin and β1 integrin as a negative control. In situ PLA showed a distinct spotted signal indicating the mutual interaction of CD46 and β1integrin. A positive response was demonstrated not only in freshly released sperm but also in sperm during the acrosome reaction. Freshly released sperm were distinctively labelled in the acrosome region and the neck, similarly to the positive control. Sperm during the acrosome reaction showed the signal across the whole sperm head region. No signal or sporadic nonspecific staining was detected in the case of the negative control. In summary, our results deliver new information that proteins CD46 and β1 integrin interact with each other. These results suppose the theory that β1 integrin can mediate a connection between CD46 and sperm cytoskeleton thereby molecules of signalling pathways leading to activation of the acrosome reaction.
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Dynamics of mouse sperm capacitation and acrosome reaction
Dvořáková-Hortová, Kateřina ; Frolíková, Michaela ; Děd, Lukáš ; Šebková, Nataša
Capacitation followed by the acrosome reaction (AR), is a very complex event of molecular changes, including acrosome matrix rearrangement and actin polymerization, which mammalian sperm must undergo in the female reproductive tract in order to obtain the ability to penetrate and fertilize the egg. CD46 and β1-integrin belong to specific proteins, which are predicted to interact during molecular reorganization of capacitating sperm. The IZUMO1 as the primary fusion protein of the mammalian sperm is also involved in this dynamic network. We investigated the relationship between the Izumo, CD46 and β1 integrin relocation in the sperm head during the capacitation and AR in vitro. We have already successfully monitored by immunofluorescent labelling the dynamics of proteins CD46 and β1-integrin. The changes in the localization of these proteins associated with the AR and their mutual co-localization was observed. The original β1-integrin location in the freshly released epididymal sperm is in the acrosome and it relocates during the AR further through the sperm head compartments into the equatorial segment and over the whole sperm head. Its density over the equatorial segment is decreasing with the extended time of the AR. Also its presence in the perforatorium of the mouse sperm head is very prominent. The pattern for protein CD46 is extremely similar if not identical in both aspects such as compartment localization and time progress during capacitation and AR in vitro. The molecular interaction of CD46 and β1-integrin was investigated using the Proximity Ligation Assay and Super resolution microscopy STED. The data were statistically analysed. The newly obtained results from CD46 and β1-integrin relocation are in correlation with IZUMO1 dynamics and giving a substantial knowledge on the studied protein network rearrangement during capacitation and AR in mouse spermatozoa.
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Interaction of humans and the environment in residential hauses with walls from inorganic masonry
Brada, Lukáš ; Kocourková, Jana (advisor) ; Zemanová, Lucie (referee)
Interaction of humans and the environment, is subject to the occurrence of mold in
residential buildings. Mold development has been the ultimate result of a variety of
phenomena, processes and causes that have penetrated in various stages of preparation,
implementation and operation of buildings. The negative effects which the mold in a residential area buildings exhibit is primarily a reduction in the use of comfort living
space in the form of health risks in the form of allergic reactions and possibly causing
chronic problems. Chronic problems over time can lead to serious illness. A big role in
the occurrence of mold also plays a reduction of aesthetic values of residential spaces
requiring repair and remedy these deficiencies. When removing adverse events leads to
an unnecessary additional environmental burden.
Problems of fungi in ground buildings are very expansive and interdisciplinary
problems, which incorporates structural, termomechanic, chemical and biologic aspects.
In this undergraduate work I concentrated on the origin of fungi in ground buildings
with inorganic materials and I am discribing at least some important sources and aspects
of fungi origin in each phases origin and exploitation building, with usage of specialised
literature. Model compultations and examples of origin fungi are performed.
Autocephalous origin of fungi in residencial buildings is yet fallback situation, which
can be possibly prevented. But due to conditions for origin fugi can begin long ago this
fallback situation. Store of bias each phases living buildings is accentuated on possibly
fugi production in buildings from project up to operation.
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Evaluation of Marketing Communication
Sýkorová, Lucie ; Pokorná, Jitka (advisor) ; Tereza, Tereza (referee)
The paper focuses on the issues of the marketing communication. The main goal of the paper is to suggest recommandation for the improvement of the marketing communication of the company Cajova kvetina s.r.o. (Ltd) with a focus on Matcha Tea product. The theory part delimits the terms marketing, marketing mix and the marketing communication. Later there is the preparation of the effective marketing communication defined. The practical part analyses the marketing mix with a focus on marketing communication of Matcha Tea product followed by a survay research in the form of personal questioning. The result shows that 37 per cent of the respondents are aware of the existence of Matcha Tea product, 81 per cent of them has already tried it, and 87 per cent of those are satisfied. In the conclusion part there is an evaluation of the returnability of the discount coupons that received all the respondents after filling in the questionaire. The 15% discount coupon for one packaging of Matcha Tea + shaker is used by 29% ofrespondents. Based on the questionaire research it is suggested and recommanded to improve marketing communication for Matcha Tea product. The primary goal of the suggestion is the increase of the Matcha Tea sales by 10% during the following six months and the secondary goal is the increased visibility on the market. In order to reach these goals, the following models of marketing communication were chosen: personal selling, sales promotion, word-of-mouth marketing, interactive marketing and advertising.
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