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Characterization of pore opening relevant residues in TM3 and TM4 domains of Orai1
ANDOVA, Ana-Marija
Calcium (Ca2+) ions play a crucial role in almost every aspect of cellular life. The most prominent calcium entry pathway into the cell is the calcium release-activated calcium (CRAC) channel, composed of the Orai1 protein, and the stromal interaction molecule STIM1. The channel is activated through conformational changes upon STIM1 coupling to the C-terminus of Orai1 protein following store depletion, which in turn allows Ca2+ influx into the cell. The abnormal function of the CRAC channel caused by mutations gives rise to distinct pathologies. Since it has not yet been elucidated how the signal propagation moves to the pore upon coupling, this thesis dives into its investigation by focusing on characterizing the TM3 and TM4 domains and their importance in leading to an open permissive conformation of the channel. The pivotal foundation for the creation of novel strategies in the modulation of the Orai1 function lies with the understanding of the dynamics of the Orai1 pore opening.
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Regulation of NMDA receptor trafficking in mammalian cells
Hemelíková, Katarína ; Horák, Martin (advisor) ; Novotný, Jiří (referee) ; Valeš, Karel (referee)
N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...
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Evolution of sensitivity to trail-following pheromones in termites
Száková, Barbora ; Hanus, Robert (advisor) ; Sillam-Dussès, David (referee)
Eusocial insects evolved a sophisticated intraspecific communication, dominated by chemical signals, the pheromones. Termites (Isoptera) represent an excellent example in this respect, having a wide range of pheromones, such as trail-following, sex-pairing, alarm, and other pheromones. It is especially the former category of pheromones which is ubiquitous in termites and which was chemically characterized in many taxa across termite phylogeny. This allowed phylogenetic reconstruction of the chemical diversity of trail- following pheromones and calls for searching of evolutionary patterns of the sensitivity to these pheromones in various lineages across the tree of life, including the search for evolutionary scenario of the emergence of specific olfactory receptor proteins. In most species, the trail-following pheromones are represented by mono-, di- and tri-unsaturated fatty alcohols (3Z)-dodec-3-en-1-ol (DE), (3Z,6Z)-dodeca-3,6-dien-1-ol (DDE), and (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (DTE). My overall aim in this thesis was to contribute to the understanding of the evolution of olfactory detection of C12 fatty alcohol trail-following pheromones in termites. More specifically, my question was whether evolutionarily more basal clades (Kalotermes flavicollis and Neotermes cubanus from the family...
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Hippocampal neuronal representation of a moving object in a novel spatial avoidance task
Ahuja, Nikhil ; Stuchlík, Aleš (advisor) ; Jiruška, Přemysl (referee) ; Telenský, Petr (referee)
In real world environments, animals need to organize their behavior relative to other moving animals or objects; when hunting a predator, when migrating in groups or during various social interactions. In all of these situations, the animal needs to orient relative to another moving animal/object. To understand the role of the hippocampus in this ability we adopted a two-step approach. We developed a task that would mimic important elements of this behavior in the laboratory. The task required the rats to assess not only their distance from the moving object but also their position relative to the object. We further studied how neurons in the hippocampal CA1 subfield encode the subject, the moving object and the environment in the behavioral paradigm and how do these representations interact among themselves. In rats, we aimed to characterize spatial behaviors relative to moving objects and to explore the cognitive mechanisms controlling these behaviors. Three groups of animals were trained to avoid a mild foot-shock delivered in one of three positions: either in front, on the left side, or the right side of a moving robot. Using different variations of the task, we also probed whether avoidance was simply due to increased noise level or size of the retinal image or appearance of the robot. As the...
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Regulation of NMDA receptor trafficking in mammalian cells
Hemelíková, Katarína
N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...
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The effect of sleep on consolidation of episodic-like memory in rats
Petránová, Erika ; Kelemen, Eduard (advisor) ; Svoboda, Jan (referee)
We can notice the positive effects of sleep on many functions of our organism. For a long time we have observed the interconnection between sleep and memory and today we already know, that different sleep phases correlate with an improvement of different memory types. One of the hypotheses, that explain the positive effect of sleep on strengthening of the memory representations, is its irreplaceable active role in the process of memory consolidation. The memory consolidation of episodic type in animals, which processes memories into events with time and space context, could according to this theory occur due to two phase sleep process, in which each phase has a specific role. The theoretical part of this thesis will familiarize the reader with the problematic of organization of time and space in our brain, and introduce him to the foundations of electroencephalography (EEG) and offer a detailed introduction into the discussed hypothesis of active sleep consolidation. The practical part is then focused on the confirmation of the already mentioned hypothesis through the combination of comparison of results from the behavioral task of 2 groups of animals with different sleep manipulation and of the analysis of EEG signal recorded during the experiment before and after the training. The behavioral task...
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Identification of changes in membrane properties of astrocytes in a mouse model of amyotrophic lateral sclerosis
Vaňátko, Ondřej ; Turečková, Jana (advisor) ; Vlachová, Viktorie (referee)
Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder of the central nervous system characterized by loss of motor neurons and voluntary muscle degeneration. Astrocytes play a major role in regulation of the disease onset and progression due to their intimate association with neurons. Regulation of ionic homeostasis is one of their key functions and its failure has been linked to several neurological diseases. The aim of this thesis was to explore differences in membrane properties of astrocytes in ALS. To fulfill this aim, a double transgenic mouse strain with ALS-like phenotype and a specific expression of enhanced green fluorescent protein in astrocytes was generated. To phenotype this strain, two sensorimotor tests, wire grid hang test and rotarod test, were conducted. Immunohistochemistry was used to characterize the strain on a cellular level and to explore changes of specific ion channels. Functional properties of astrocytes were explored using the patch clamp technique. The double transgenic strain has the characteristic ALS-like phenotype and is comparable to the original strain with differences in symptom onset and progression between models and sexes. On the cellular level, there are characteristic ALS features, specifically loss of motor neurons and astrogliosis....
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Microelectrode arrays for mioelectronic
Bráblíková, Aneta ; Vala, Martin (referee) ; Salyk, Ota (advisor)
Organic electronic biosensors are developed as suitable devices that can transform electrochemical processes within the cell membrane into an electronic signal and enable to measure electrical activity of excitable cells and tissues both in vitro and in vivo and thus represent valuable alternative to current cell monitoring methods. In this work we focus on the fabrication of electrophysiological sensors based on organic semiconductors printed by the material printing method. Microelectrode arrays (MEAs) are active components of the device, which can monitore cellular activity and above that stimulating cells with electrical pulses. The proposed platform should be used for cytotoxicity of potential drugs especially on cardiac cells (cardiomyocytes). The experimental part focus on specific production processes of platforms, which were prepared in the laboraty with emphasis on biocompatibility and conductivity of device.
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