|
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.
|
|
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.
|
|
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.
|
|
Expression of selected proteins in sperm in men with normal and pathological spermiograms using monoclonal antibodies
Pěknicová, Jana ; Čapková, Jana ; Dorosh, Andriy ; Margaryan, Hasmik ; Kubátová, Alena ; Děd, Lukáš
Recent studies show that infertility in human populations it affects an estimated 15% of couples of reproductive age. Male infertility is the primary cause for 60% of these cases. For these reasons, we analyzed the acrosomal and sperm surface proteins in men with normal and pathological spermiograms. We found that intra-acrosome proteins: TERA (Transitional endoplasmic reticulum ATPase), GAPDHS (Sperm Glyceraldehyde-3-phosphate dehydrogenase), and PRKAR2A (C-AMP-dependent protein kinase II, PRKAR2A), which can be identified using our monoclonal antibodies, are different express in healthy men and men with astenozoospermia (with reduced sperm motility), and with a significantly reduced expression in the astenozoospermia. These proteins are involved in energy metabolism and apoptosis of the cells, and some of them in the sperm-egg interaction; therefore, they have an important role in reproduction. On the other hand, there were no statistically significant differences in the expression of surface proteins (Appolipoprotein J (Clusterin) and Semenogelin). Our findings show that astenozoospermia as a complex disorder of the semen is often combined with other pathological conditions that are not diagnosed by the semen analysis. Therefore monoclonal antibodies are so suitable instrument for the detection of proteins associated with the pathology of the sperm in the semen with low sperm motility. In general, monoclonal antibodies against the sperm proteins are an appropriate tool to detect sperm quality in reproductive medicine.
|
|
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.
|
|
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.
|
|
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.
|
|
Fluorescent analysis of the differential protein expression in normozoospermic and asthenozoospermic sperm samples
Děd, Lukáš ; Čapková, Jana ; Kubátová, Alena ; Teplá, O. ; Pěknicová, Jana
Asthenozoospermia is one of the main seminal pathologies underlying male infertility. Previous proteomic studies have demonstrated the significant differences in the protein profiles between normozoospermic and asthenozoospermic sperm samples. Since these studies were primarily focused on the identification of differentially expressed proteins by mass spectrometry, we aimed to evaluate the ability of our diagnostic antibodies to detect the differential expression of selected protein markers by fluorescent microscopy and flow cytometry techniques. Therefore, we analyzed sperm samples from 30 men with normal and 30 men with astheno spermiograms, average by the panel of our diagnostic anti-human sperm (Hs) antibodies. Fluorescent microscopy and flow cytometry analysis revealed quantitative differences in the protein abundances between normo and astheno sperm samples, namely, in GAPDHs, evaluated with Hs-8 MoAb, VCP, evaluated with Hs-14 MoAb, and ATP synthase, evaluated with MoAb Hs-36. From the methodological point of view, we observed very high correlation between the data obtained by fluorescent microscopy and flow cytometry techniques and therefore both methods are useful for evaluation of protein differences associated with asthenozoospermia. From the clinical point of view, we observed the strong association of the low sperm motility in the sample with the expression of proteins, playing an important role in sperm energy metabolism (expected), but also with the expression of all tested intra-acrosomal proteins. These findings further demonstrate asthenozoospermia as a complex semen disorder frequently associated with other semen pathologies, which are not diagnosed by basic semen analysis, and the possibility to use monoclonal antibodies as a tool for diagnosis of protein associated sperm pathologies in the semen with the low sperm motility.
|
|
High normal pressure and hypertension of children and adolescents
KRÁLOVÁ, Kateřina
In children and adolescents, high normal blood pressure is often present. People with high normal blood pressure show a short time prerequisite to the formation of arterial hypertension. Hypertension occurs most often in children as asymptomatic. Increased blood pressure in children is most often discovered during preventive examinations by a general practitioner for children and adolescents. This thesis contains information about the anatomy and physiology of the cardiovascular system, a blood pressure measurement sister, evaluation of blood pressure, high normal pressure, high blood pressure in children, the role of nurses in primary care pediatricians in the area of communication and education, and treatment of high normal pressure and hypertension. Objectives The first goal was to explore how children adhere regime measures for the prevention of high normal pressure and hypertension. It used this research question: How children adhere regime measures for the prevention of high normal pressure and hypertension? The second objective was to determine the role of nurses in the care of children with high normal pressure and hypertension. The second research question is: What is the role of the nurses in taking care of children with a normal high pressure and hypertension? Methods used In the research part of the thesis, we used the method of qualitative research, questioning method and technique-depth interviews. The interviews were recorded on a Dictaphone and then transcribed literally. Four sisters and three mothers did not wish to be recorded, so they were interviewed at polling literally written on paper. For research, two research files were created. The first sample consisted of six nurses, who were working in GP surgery for children and adolescents in the South Region. Second survey consisted of six people selected from among parents of children with high normal pressure and hypertension, also from South Bohemia. Results The transcribed interviews were investigated by open coding method, pencil and paper. Resulted in 9 categories: Cardiovascular disease, role of the nurse, pressure measurement, blood pressure, hypertension, pressure measurement, recommendation, Lifestyle, Leisure. Individual categories were assigned to subcategories, which are encoded core data. Conclusion The first research confirms nurses working in a pediatrician's office for children and adolescents, the most common cardiovascular disease in the general hypertension. They argue that the role of nurses in primary care pediatricians is properly measure blood pressure and make the education of children and parents in the area. The respondents concurred in the principles of how to properly measure blood pressure measurement are available for at least three cuff sizes. Informants have basic information about high normal pressure and hypertension, and said that, in the offices of primary care pediatricians to conduct checks blood pressure of preventive examinations or troubled child. Informants reported that blood pressure control and education on blood pressure occurs, but in the second research study showed that children and adolescents do not comply with the recommended recommendations in this area, perhaps because they are less motivated. Neither sister nor the parents have not mentioned, they would receive educational aids such as brochures and sample menus.The second research showed that parents most often found elevated blood pressure in primary care pediatricians, either for reasons of preventive examinations or difficulty a child. On the control measurements came at least three times. Respondents agreed on the recommendations they received in the office GPCY such as reducing the weight of the child, regular physical activity, restriction of salt and proper diet the child, but not all children and adolescents comply with these recommendations. The research results will be provided to the journal Pediatrics practice.
|
|
The Role of Nurse in the Prevention of Disease of Civilisation
PAVLOVÁ, Martina
The current state Prevention is a complex care of a patient when we are trying to prevent from the occurrence or development of a disease, or when we want to prevent from its consequences and mitigate them. Recommendations for prevention set by a doctor must be on a scientific basis, and they must be a part of all branches of medicine. Their effectiveness depends on the cooperation between patients and medical staff. The basic and most efficient method of prevention is the health promotion and health education, which is closely associated with it. The term health promotion involves a set of activities and precautions for the purpose of making health better and sustaining it. Diseases of civilisation (hereinafter "DoC") are groups of diseases which are distinguished by their association to our lifestyles. Human body is more and more threatened from unfavourable influences of external environment, such as the quality of atmosphere, work environment and environment itself, the lack of physical movement, unhealthy diet, smoking, and alcohol consumption. The list of diseases of civilisation varies and changes together with our society and the development of our lifestyles. Among the most common or frequent diseases of current time are cardiovascular diseases, which involve atherosclerosis, hypertension, myocardial infarction, stroke, coronary artery disease and obesity. Furthermore, between civilization diseases include diabetes mellitus type 2 or cancer, etc. The goal of the thesis There are two goals set for this paper. The first goal was to find out what the role of a nurse in the prevention of diseases of civilisation was. The second goal was to find out what the above mentioned role of a nurse was from the point of view of patients. Methodology For the purpose of the research part of this thesis, qualitative research using semistructured interviews was carried out. Respondents were divided into two groups; the first one consisted of eight nurses working at internal medicine and neurological department of the hospital in České Budějovice and a nurse working in a private office of a general practitioner for adult patients. The second group consisted of eight patients of the above mentioned health facilities. Results According to our findings, nurses and patients have different oppinions on the role of a nurse. Nurses in disease prevention see themselves as an equivalent to a doctor when undertaking this practice and giving guidance. The patient speaks only to the nurse who emphasized what they were told by the doctor and reiterated the main points. They make available to them leaflets and brochures or Internet resources. In contrast, most patients see a nurse as a more important source of information than the doctor. Patients have the feeling that the doctor does not have enough time or does not want to be botherd with questions. Rather the patient turns to his nurse, which they sees as more appropriate and someone to whom they can confide in without bothering. Summary The goal of the thesis was to focus on prevention, DoC, and the role of a nurse in the prevention of such diseases, as well as to study this issue, look at it from the point of view of patients and nurses via the interviews carried out in this paper, and to analyse such information. Based on this analysis are published recommendations for nurses and patients.
|
guest ::
