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Study of mesenchymal stem cell migration in the extracellular matrix based on principles of chemotaxis
Scholasterová, Viktorie ; Fohlerová, Zdenka (referee) ; Chmelíková, Larisa (advisor)
This thesis engages in a study of mesenchymal stem cell migration in extracellular matrix based on principles of chemotaxis. First, attention is focused on a theoretical part associated with a clarification of basic terms such as extracellular matrix, migration, confocal microscopy, mesenchymal stem cells or chemotaxis. There is also included a list and a description of some basic methods for monitoring cell migration and a more detailed description of a method called transwell assay, which has been chosen for an experiment in a practical part of this thesis. This part includes protocols of individual steps for the preparation of the experiment, the procedure of data processing obtained by scanning cells with a confocal microscope and a description of the resulting confluence values.
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Study of mesenchymal stem cell migration based on principles of chemotaxis
Pošustová, Veronika ; Skopalík, Josef (referee) ; Chmelíková, Larisa (advisor)
The purpose of this Master thesis is to verify migration of mesenchymal stem cells on the principle known as chemotaxis. First part of this study is focused on cell migration in order to explain the whole migration process. Next part describes various chemotaxis methods and selected studies dealing with clinical applications of mesenchymal stem cells in different medical and biomedical fields. The following step describes confocal microscopy, which is used for acquiring images of the cells. The experimental part is focused on cultivation of mesenchymal stem cells in a laboratory, which is necessary for cell vitality. Furthermore, there are designed two main experiments. Firstly there is a 2D experiment with adherent cells for chemotaxis using -Slide Chemotaxis. Secondly Transwell migration test is designed and executed. Finally, the acquired images from confocal microscope are used for image processing, which was done in Matlab R2020a programming environment. The result of this processing is evaluation of cell confluence and migration. In the end, experimental part of this study was optimized according to recommended studies. The results are summarized in the conclusion with proposal for improvements of those methods.
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Interakce spermií a jiker u sladkovodních ryb: vliv prostředí na fertilizační proces
KHOLODNYY, Vitaliy
Fertilization of fresh water fish occurs in an environment which may affect negatively the gametes, therefore the fish male gametes should reach their counterpart, the female gamete, as soon as possible because spermatozoa become damaged within minutes or less due to osmotic shock. Existence of specific mechanism triggering, supporting and guiding the encounter of gametes would be highly expedient in these conditions. The eggs of many externally fertilizing freshwater fish species are released into the external milieu surrounded by a coat of ovarian fluid (OF) with a composition ideal for supporting and protecting eggs and sperm against the deleterious effect of freshwater. The existing data support the idea that the properties of OF and/or the specific compounds contained in it or released by the eggs could significantly affect the behavior of male gametes and consequently influence the outcome of fertilization in terms of the number of fertilized oocytes. The mechanisms which facilitate and trigger gametes' encounter are also highly demanded in terms of natural selection. It was found that presence of OF affected significantly the behavior of rainbow trout spermatozoa, in particular, their motility traits: higher velocity was supported for longer time and trajectories were straightened, comparing to those observed in water. In the microcapillary spermatozoon accumulation test (test of chemotactic response) the rainbow trout OF showed a trapping effect on activated male gametes which depended on osmotic properties of the activating media. Different molecular weight fractions from OF affected the tactic behavior of the cells in a various way. The most significant trapping effect was rendered by low molecular fraction and the possible chemotactic agent was found to be thermostable. The trapped cells showed specific turn-and-run behavior accompanied by asymmetric bending of flagella and burst-like increase of calcium concentration in the bended area. The in vitro fertilization test revealed the enhancement of spermatozoa performance, especially in the samples from the related individuals, which led to the higher embryo development rate. Presence of particular concentration of ovarian fluid (30% solution in water) had inhibiting effect on sterlet spermatozoa motility initiation. Lower concentrations of the ovarian fluid improved the longevity of spermatozoa and did not affect their trajectories. Test of chemotactic response showed no effect of ovarian fluid on spermatozoa behavior, while at the same time the attracting effect of egg conditioned medium was evident (i.e. due to some substances released from the eggs during their contact with fresh water). The results of in vitro fertilization test showed that presence of ovarian fluid prevented the eggs from losing the fertilizing ability due to the contact with water, as well as promoted the spermatozoa to fertilize the eggs during longer period of time. Presence of common carp ovarian fluid in the activation medium caused the decrease of the velocity of spermatozoa comparing to the OF-free medium and significantly altered the motility pattern from straightforward motility observed in the water to the tumbling in the medium with high OF content (50%). Introduction of OF (in the sperm accumulation test) entailed immediate and prominent chemotactic-like reaction of spermatozoa. The environmental conditions which accompany the encounter of gametes, in particular presence of OF, calcium ion content and osmolarity, significantly affect the performance of male gametes in spermatozoa in rainbow trout, sterlet and common carp in terms of changes in velocity, path linearity and ability to response to the external signals, e.g. attractants. The conducted study allowed to conclude that way how the ovarian fluid affects the behavior of spermatozoa in these species may be associated with their reproduction (spawning) strategy.
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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petr, Jaroslav (referee)
Fertilization is a multiple step process leading to fusion of female and male gametes resulting in a formation of a zygote. Besides direct gamete interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves communication based on chemical molecules triggering various signalling pathways. This work is aimed to characterize chemical communication of gametes of a model organism Mus musculus. For this purpose, modern proteomic and visualisation methods like nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS), selected reaction monitoring (SRM) and immunofluorescent microscopy were used. Lipocalins were identified as candidate proteins involved in communication including those from major urinary proteins (MUPs), LCN lipocalins and fatty acid binding proteins (FABPs). For the first time, we report their presence in the sperm acrosome. Based on lipocalins capacity to bind and transport other molecules, we propose that these proteins have a protective and/or signalling role for gametes. Furthermore, chemical communication between sperm and oocyte is based on chemotaxis which enables their interaction before their fusion. In this work, we detected that spermatozoa show chemotactic responses in the presence of L-glutamate. This amino acid naturally...
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Study of mesenchymal stem cell migration in the extracellular matrix based on principles of chemotaxis
Scholasterová, Viktorie ; Fohlerová, Zdenka (referee) ; Chmelíková, Larisa (advisor)
This thesis engages in a study of mesenchymal stem cell migration in extracellular matrix based on principles of chemotaxis. First, attention is focused on a theoretical part associated with a clarification of basic terms such as extracellular matrix, migration, confocal microscopy, mesenchymal stem cells or chemotaxis. There is also included a list and a description of some basic methods for monitoring cell migration and a more detailed description of a method called transwell assay, which has been chosen for an experiment in a practical part of this thesis. This part includes protocols of individual steps for the preparation of the experiment, the procedure of data processing obtained by scanning cells with a confocal microscope and a description of the resulting confluence values.
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Study of mesenchymal stem cell migration based on principles of chemotaxis
Pošustová, Veronika ; Skopalík, Josef (referee) ; Chmelíková, Larisa (advisor)
The purpose of this Master thesis is to verify migration of mesenchymal stem cells on the principle known as chemotaxis. First part of this study is focused on cell migration in order to explain the whole migration process. Next part describes various chemotaxis methods and selected studies dealing with clinical applications of mesenchymal stem cells in different medical and biomedical fields. The following step describes confocal microscopy, which is used for acquiring images of the cells. The experimental part is focused on cultivation of mesenchymal stem cells in a laboratory, which is necessary for cell vitality. Furthermore, there are designed two main experiments. Firstly there is a 2D experiment with adherent cells for chemotaxis using -Slide Chemotaxis. Secondly Transwell migration test is designed and executed. Finally, the acquired images from confocal microscope are used for image processing, which was done in Matlab R2020a programming environment. The result of this processing is evaluation of cell confluence and migration. In the end, experimental part of this study was optimized according to recommended studies. The results are summarized in the conclusion with proposal for improvements of those methods.
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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petr, Jaroslav (referee)
Fertilization is a multiple step process leading to fusion of female and male gametes resulting in a formation of a zygote. Besides direct gamete interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves communication based on chemical molecules triggering various signalling pathways. This work is aimed to characterize chemical communication of gametes of a model organism Mus musculus. For this purpose, modern proteomic and visualisation methods like nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS), selected reaction monitoring (SRM) and immunofluorescent microscopy were used. Lipocalins were identified as candidate proteins involved in communication including those from major urinary proteins (MUPs), LCN lipocalins and fatty acid binding proteins (FABPs). For the first time, we report their presence in the sperm acrosome. Based on lipocalins capacity to bind and transport other molecules, we propose that these proteins have a protective and/or signalling role for gametes. Furthermore, chemical communication between sperm and oocyte is based on chemotaxis which enables their interaction before their fusion. In this work, we detected that spermatozoa show chemotactic responses in the presence of L-glutamate. This amino acid naturally...
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Sperm cell penetration and membrane fusion with oocyte during fertilization in mammals
Klimková, Veronika ; Krylov, Vladimír (advisor) ; Frolíková, Michaela (referee)
Fertilization is a process involving multiple steps, which are in continuity and complement each other. Spermatozoa become competent to fertilize after capacitation, hyperactivation and acrosome reaction. Oocytes are surrounded by a layer of cumulus cells and give a signals for spermatozoa to activation and orientation. This study focuses on the latest knowledges about a roles of cumulus cells, production of chemoattractants and the possibility that cumulus cells induce the acrosome reaction after binding a novel sperm protein NYD-SP8 to the cumulus. It seems that progesterone induce the acrosome reaction and also is the best chemoattractant secreted by cumulus cells. Next I am focus on gamete fusion including the role of surface proteins such as Juno and IZUMO1. Key words: membrane fusion, cumulus cells, acrosome reaction, chemotaxis sperm, oocyte, fertilization
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Orientation of helminths during host seeking
Vojtová, Terezie ; Mikeš, Libor (advisor) ; Horák, Petr (referee)
The parasitic helminths are often forced in the environment to search for their host quickly in order to continue the ontogenetic development, or else to finish it. To search for and indentify the host, they use various types of receptors which allow them to provoke a taxes corresponding to the oncoming stimulus and to react regarding the current situation. In respect to the concept relevant to the helminths, this work concerns trematodes, whose freely movable stadia are miracidium and cercacia, and then monogeneans which are viparous or oviparous with oncomiracidium larva, and nematodes during which the third larva stage moves. The representatives of the groups of Myxozoans and Ciliophorea are also mentionned, since they use similar mechanisms to look for hosts as helminths. This thesis includes the summary of the existing knowledge related to the orientation of helminths in the environment. On the basis of the data that is already known, it is possible to compare which mechanisms are used for host-seeking by particular species, and according to which signals from the surroundings they most often orientate themselves. Powered by TCPDF (www.tcpdf.org)
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