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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.
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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.
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Nursing care management of patients after intrapleural chemical pleurodesis
KIESEWETTEROVÁ, Renáta
The pleurodesis is a treatment method for patients suffered from the symptomatic malignant pleural effusion. The goal of the pleurodesis is to close a pleural area by connection of visceral and parietal pleura in order to reduce or terminate production of pleural effusion. The sterile talc powder is the world's most used agens within a practice. After a parental analgesic is the powder (as a sllury) either applicated into a pleural cavity through the chest drainage or surgically dispersed over the treated (via abrasion, decortication) pleural space during the thoracoscopy/thoracotomy. This diploma thesis focuses on the treatment technique of bedside talc-pleurodesis through the chest drainage. The nurse has an irreplaceable role in the whole process from a preparation of patient to assistance by intrapreural application, to care of patient after the operation or serves as a physical support during potential complications. The aim of the presented work is to detect priorities in nursing care and find out whether patients need increased care after intrapleural therapy. The qualitative research method was chosen for the analysis where semi-structural interview with open questions were used. We created case reports analysed with the adaptive model of Callista Roy as well. The research sample contained patients chosen with respect to specialization of researched problem. We set two basic focal stimuli according to the analysis of respondents´ reactions. These stimuli serve as a basis for determination of priority parts of the nursing care by patients after bedside pleurodesis. We defined the need to be without pain and the need to ensure safety and certainty as a central focus of the nursing care. Among secondary fields of nursing care were identified sleep disturbances and limited mobility, which leads to reduction of self-care in getting dress, hygiene as well as in defection and miction. We found out within the second goal identical intensity of the nursing care before and after bedside talc-pleurodesis. This statement is based on comparison of interventions before and after aplication analysed via adaptive model by Roy. Nevertheless, causes of interventions were modified to a certain extent. The main goal of diploma thesis was to map requirements for high-quality nursing care related to bedside talc-pleurodesis via chest drainage. The work should extend theoretical basis not only for nurses in clinical practice.
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Vliv teploty vody a intenzity světla na raný vývoj sumečka afrického v komerčním chovu
PROKEŠOVÁ, Markéta
The successful production of quality early fish stages is crucial for artificial rearing fish species. Nevertheless, it is quite a problematic process, because fish as poikilothermic organisms are highly sensitive to various environmental conditions. Moreover, both fish embryos and larvae are more affected by biotic and abiotic factors (such as predation, stocking density, temperature, light, pH, salinity, oxygen saturation, feeding availability, or their interactions) than older juveniles or adults. Most fish processes such as reproduction, metabolism, survival, development and growth can be considerably (positively, negatively or not) influenced by these conditions. Therefore, it is important to understand their impacts on fish stages during embryonic, larval, juvenile, adult, and eventually the senescent period. Then, following the application of new knowledge to the practise, especially species- and age-specific optimal conditions, should result in higher survival, better growth rate, well developed fish without deformities and lower cannibalism. The objectives of the present thesis were to assess the effect of water temperature and light intensity on the early development (embryonic and larval period) of African sharptooth catfish as a perspective fish species for freshwater intensive aquaculture. Due to its great characteristics (for instance fast growth, ability to breathe atmospheric air, rearing at high stocking density) this species can meaningfully contribute to the global aquaculture production which is expected to rise in the coming years. However, there are still some difficulties in the early rearing African sharptooth catfish regarding the cannibalism, feeding, survival and necessity of detailed information about the effects of some abiotic factors and their interactions.
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Inovativní metody v chovu a reprodukci candáta obecného (Sander lucioperca)
BLECHA, Miroslav
The whole Ph.D. thesis includes in total 10 chapters on 146 pages. Chapter 2 9 are specific parts of thesis where following scientific and practical aspects of pikeperch reproduction biology and aquaculture are described. Quality and quantity of pikeperch spermatozoa after varying cold water treatments are presented in the chapter 2 and can help to optimize broodstock management of males with the aim to obtain high quality spermatozoa during a seasonal and an out of season spawning as well. Benefits of hormone treatment of both sexes in semi-artificial reproduction in pikeperch are described in the chapter 3 where the importance of hormonal treatment of both sexes in tank spawning is evaluated for effective production pikeperch larvae. The use of an alcalase treatment for the elimination of pikeperch egg stickiness is being shown in the chapter 4 of this thesis. Post-ovulatory oocyte ageing and its effect on eggs viability rates and occurrence of larval malformations and ploidy anomalies are listed in chapter 5. These results describe the effects of the egg over ripening fertilization process in pikeperch. The first report of heat shock triploidisation in pikeperch is described in chapter 6 with production of 100% pikeperch triploid population. The effect of water surface treatment on survival, swim bladder inflation and growth of larvae is given in the chapter 7 with the aim to optimize the intensive culture of pikeperch larvae. Last two chapters (8 and 9) are describing the adaptation of intensively cultured juveniles to pond culture and the adaptation of pond-cultured juveniles to RAS as a new and effective methods for ongrowing production of pikeperch. In total, four published scientific papers, one handbook, one accepted scientific paper for publication, and two prepared scientific manuscripts are included and discussed in this Ph.D. thesis.
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The effect of pyrethroid based pesticides on fish
RICHTEROVÁ, Zuzana
Pyrethroids are ones of the most used pesticides worldwides. The widespread use and high stability of pyrethroids lead to the assumption of that their occurrence in the environment could be quite frequent. They can reach water ecosystem as pollutants. Residues of pyrethroids are not only detected in the water column, but also in sediments and in fish tissues. The first study was devoted to the product Nexide containing 60 g.l-1 of active substance gamma-cyhalothrin. Tested Nexide concentrations were 5, 25, 50, 100, and 250 µg.l-1. Early life stage test was used.Common carp (Cyprinus carpio) was tested. There were significant mortalities in all concentrations except the lowest concentration during the trial. The lowest concentration tested 5 µg.l-1 only caused a slightly increased mortality. This lowest concentration influenced the growth in length and weight negatively, decelerated ontogenetic development, and made the body surface of the individuals darker. Histopathology of individuals from this concentration revealed dystrophy in liver. Examination of kidney, intestine and gills did not show significant histopathological differences compared with control. The evaluation of selected parameters of oxidative stress demonstrated a significantly higher activity of detoxification enzyme glutathione-S.transferase (GST) and a significantly lower activity of defensive enzyme glutathione peroxidase (GPx) compared with the control group. The other examined parameters of oxidative stress such as catalase (CAT), glutation reductase (GR), and lipid peroxidation determined by using the thiobarbituric acid-reactive substances (TBARs) were comparable to the control group. Changes in oxidative stress parameters suggest that exposure of the organism to the product Nexide in the given concentration leads to dysbalance of defensive enzymes. The second study was devoted to the product Cyperkill 25 EC containing 250 g.l-1. Tested Cyperkill 25 EC concentrations were 7.2, 36, 72, 144, and 360 µg.l-1. The procedure of the trial was the same as the preceded one. There were 100% mortalities in all concentrations except the lowest concentration during the trial. The lowest tested concentration 7.2 µg.l-1 allowed 90% of individuals to stay alive till the end of experiment. The lowest concentration influenced the growth in length and weight negatively and decelerated ontogenetic development compared with the control. Any individual exposed to this concentration did not reach juvenile stage until the end of the trial. Dark pigmentation was visible in 68% of these exposed individuals on the last day. Similar darkening was visible in individuals from higher concentrations shortly before death too. Histological examination did not revealed significant changes in intestine, liver, kidney, and gills compared with the control group. Evaluation of selected parameters of oxidative stress demonstrated significantly lower activities of GST, GR, and GPx. Activities of CAT and TBARS were comparable with the control group. Changes in oxidative stress parameters suggest that exposure of the organism to the product Cyperkill 25 EC in the given concentration could induce oxidative stress and interfere with the activities of antioxidant enzymes. The presented thesis summarises actual data about pyrethroids and their influence on fish. The demonstrated effects confirm high susceptibility of early developmental stages of fish to tested pesticides. When interpreting the results, we have to take into account the fact that studies showed this risk even on single pyrethroid substances. But water organisms are exposed to many other more or less toxic products and substances in a real environment. These xenobiotics could react with each other and their mixture could even potentiate negative effects. The performed studies also clearly show the significant differences in the sensitivity of embryonic and embryolarval tests.
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Iniciace pohybu byčíku, signalizace a regulace pohyblivosti spermií ryb: fyzikální a biochemické řízení
PROKOPCHUK, Galina
The current study attempted to shed light on the regulatory processes and response arrangements of fish spermatozoa during the course of maturation and motility initiation. The first intent of this study was to improve the understanding of the mechanism underlying the acquisition of potential for sperm motility in sturgeon. Up to present work, the physiological process underlying sperm maturation in this species has not been described at all. Our results showed that sperm maturation in sturgeon occurs outside the testes because of dilution of sperm by urine and involves the participation of high molecular weight substances as well as calcium ions present in seminal fluid and/or urine. The second aim of the present study was to investigate the coping mechanisms in fish spermatozoa with osmotic and ionic activating mode, as well as in spermatozoa of euryhaline fishes, to various environmental conditions. We showed that alteration of environmental osmolality might affect the fish sperm in different ways, depending on fish species and modes of spermatozoa motility activation either osmotic or ionic mode. In response to osmotic stress caused by hypotonicity, carp spermatozoa regulated the flow of water across their cell membrane and increased their cytoplasmic volume during their short motility period. In contrast, no indications of sperm volume changes were observed neither in sterlet nor in brook trout spermatozoa, both of which having an ionic mode of motility activation. We also examined the mechanism by which sperm motility triggering in euryhaline fishes can adapt to a broad range of environmental salinity. Our results demonstrated that spermatozoa of euryhaline tilapia, Sarotherodon melanotheron heudelotii, reared in fresh-, sea- or hypersaline water can be activated in hypotonic, isotonic or hypertonic conditions of swimming milieu, provided Ca2+ ions are present at various levels. It was established that the higher the fish rearing salinity or the more hypertonic ambient media at spermatozoa activation, the higher extracellular concentration of Ca2+ ions is required. The results obtained in the present study allow suggesting that osmolality is not the main factor inhibiting sperm motility inside the testis in the S. melanotheron heudelotii. A third aim of this study was investigation of the regulation of motility initiation process and description of flagellar beating initiation in chondrostean spermatozoa. We detected that K+ inhibition of sperm motility in sturgeon can be by-passed due to the pre-exposure of sperm cells to a high osmolality shock prior to its transfer to K+-rich swimming media. Thus, we hypothesized that sturgeon spermatozoa may be activated by use of an unexpected signaling pathway, independent from regular ionic stimulation. The successive activation steps in sturgeon spermatozoa were investigated by high-speed video microscopy, using specific experimental situation, where sperm motility initiation was delayed in time up to several seconds. At motility initiation, the first couple of bends formed at the basal region begins to propagate towards the flagellar tip, but gradually fades when reaching the mid-flagellum. This behavior repeats several times until a stage where the amplitudes of bends gradually reach similar value, what eventually leads to sperm progressive displacement. The total period needed for the flagellum to switch from immobility with rigid shape to full activity with regular propagating bends ranges from 0.4 to 1.2 seconds. In conclusion, the results of the current study bring valuable pieces of information into the general understanding of the processes of maturation of fish spermatozoa, their adaptability to different physical and biochemical circumstances, the extra- and intra-cellular signaling as well as the regulatory mechanisms of motility activation in fish spermatozoa.
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Some Robust Distances for Multivariate Data
Kalina, Jan ; Peštová, Barbora
Numerous methods of multivariate statistics and data mining suffer from the presence of outlying measurements in the data. This paper presents new distance measures suitable for continuous data. First, we consider a Mahalanobis distance suitable for high-dimensional data with the number of variables (largely) exceeding the number of observations. We propose its doubly regularized version, which combines a regularization of the covariance matrix with replacing the means of multivariate data by their regularized counterparts. We formulate explicit expressions for some versions of the regularization of the means, which can be interpreted as a denoising (i.e. robust version) of standard means. Further, we propose a robust cosine similarity measure, which is based on implicit weighting of individual observations. We derive properties of the newly proposed robust cosine similarity, which includes a proof of the high robustness in terms of the breakdown point.
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Comparison of HPLC a GC-MS method for pesticide determination in soil
Chalbia Václavíková, Alena ; Kočárek, Martin (advisor) ; Havlík, Jaroslav (referee)
Physical and chemical properties of pesticides may vary significantly. Pesticides can be acidic, neutral or alkaline. Some compounds containing phosphorus, other sulfur, nitrogen or halogens. These atoms can be important for the detection of pesticides. Some of the compounds are volatile, others on the contrary not evaporate, which is also one of the important properties for chromatography. The diversity of pesticides actually makes it impossible to create a universal method, which is very impractical in terms of quality and time determination. Because that pesticides are one of the most chemicals in most countries. In the food and drinking water are limits on pesticides in order to avoid negative impact on public health. For residues in soil or surface water limits are set. (Lutz et al., 2006)
The objective of this thesis is to evaluate the possibility of using two types of chromatographs with different detectors for the analysis of pesticide. Pendimethalin which was chosen for this work, will be analyzed usinga liquid chromatograph with UV detektor and gas chromatography with mass detector. Both this method will be compared as well two extraction methods (Soxhlet extraction and shaking), and two extraction agents (methanol and acetonitrile).
Another point of the work is to provethe hypothesis that, for the determination of pendimethalin in soil is by gas chromatography using a mass spectrometer in comparison with the high performance liquid chromatography time and more expensive, but will achieve a higher yield and a lower detection limit of the pesticide.
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