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Mechanisms of transmembrane auxin transport in a broader evolutionary context.
Rubešová, Magdaléna ; Petrášek, Jan (advisor) ; Tylová, Edita (referee)
Auxin, represented by the molecule indole-3-acetic acid (IAA), is one of the main phytohormones involved in the regulation of plant development. Its intercellular transport establishes concentration gradients in individual cells that control gene expression and a number of downstream processes. In plants, a complex mechanism for efficient IAA transport has evolved, involving both long-distance transport and intercellular transport within individual tissues. Because our understanding of the auxin transport mechanisms is still incomplete, this thesis attempts to summarize the literature data on all modes of auxin transport across cell membranes that have been recognized to date and places them in a broader evolutionary context. The presence of IAA in many prokaryotic and eukaryotic organisms, together with the similarly wide occurrence of carriers from "auxin efflux carrier" transporter family, evolutionarily related PIN-FORMED-like carriers, points to the possibility that IAA transport may also be evolutionarily very ancient and may functionally derive from more general mechanisms of ions or amino acids.
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The role of actin cytoskeleton in the targeting of auxin carriers to the plasma membrane.
Kebrlová, Štěpánka ; Petrášek, Jan (advisor) ; Pernisová, Markéta (referee)
Auxin plays an important morphogenic role in plant development, mainly through its effect on gene expression, but also through a number of faster processes that are directly dependent on its concentration. Therefore, in many plant tissues, directional auxin transport using specific transporters in the plasma membrane, is important for the coordination of morphogenic stimuli. The amount of auxin carriers in the plasma membrane directly affects the resulting auxin concentration inside the cell. Although the localization of auxin transporters and their abundance in the plasma membrane could be determined primarily by the actin cytoskeleton and its involvement in vesicle transport processes, this relationship is currently still unclear. Therefore, in this study, we were interested in how the localization and function of auxin transporters is affected when the function of the actin cytoskeleton is affected in a given cell type. To this end, the localization of the auxin transporters PIN3, PIN4, PIN7, and AUX1 was studied in epidermal cells of cotyledons in young seedlings of Arabidopsis thaliana whose morphogenesis was affected by mutations in subunits of the actin nucleation complex ARP2/3. Crosses of mutants in the ARP2/3 complex subunits with marker lines carrying fluorescently labeled auxin carriers...
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The effect of ethinylestradiol on Na+, K+ - ATPase
Kettnerová, Karolína ; Svoboda, Petr (advisor) ; Driák, Daniel (referee)
This diploma thesis is oriented to analysis of physiological effect of synthetic estrogen ethinylestradiol (EE), which represents the main component of steroid-based substance used in hormonal contraception. From wide range of physiologically important protein molecules, which might be effected by this steroid, thesis focuses to the study of the sodium plus potassium activated, magnesium dependent adenosinetriphosphatase (Na+, K+ - ATPase), which is selectively inhibited by cardiac glycosides such as ouabain (g strophantine). Na+, K+ - ATPase represents an important plasma membrane bound enzyme, which catalyzes the active transport of sodium and potassium across plasma membrane. In the first part of this work, Na+, K+ - ATPase was determined by binding of radioactively labeled selective inhibitor of this enzyme [3H]ouabain, used for this purpose. In the second part of this work, plasma membrane fluidity was analyzed by steady-state fluorescence anisotropy of DPH. The effect of EE on [3H]ouabain binding was studied first under in vitro conditions by using human embryonic kidney cells (HEK293) which were cultivated for 24 hours in the presence of EE in tissue culture medium. Second, the effect of EE was also studied under in vivo conditions, by subcutaneous application of EE to the female rats of Wistar...
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Caveolae and caveosoms
Galica, Tomáš ; Černý, Jan (advisor) ; Forstová, Jitka (referee)
Caevolae are remarkably stable structures at the plasma membrane. They form specific domains distinct in lipid composition from the rest of plasma membrane. Many diverse functions are assigned to Caevolae. They play role in modulation of cellular surface, signalization and well regulated endocytosis. Caveosomes suppose to be large intracellular vesicular structures potentialy new membrane organels. They are derived from internalized caveolae. Tohether with caveolae they are proposed to form a separeted system of intracellular vesicles. However recent evidence suggests that caveolae can fuse with endosomes immediately after internalization. If this is true, then the system of vesicles derived from caveolae, including caveosomes, can be considered a regular component of endosomal system. Isolation of caveosomes from endosomes has been seen mainly in experiments where polyomavirus SV40 was used. Thus the question, if this isolation is not just a result of SV40 infection, arises. It has been shown recently that SV40 virus is capable of inducing caveosome-like structures even in the absence of caveolae. Consequently existence and properties of caveosomes are being questioned. The problem of high importance is the genesis of caveosomes and their existence in SV40 non-infected cells. In this thesis...
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Analysis of endomembrane markers in the cortical cytoplasm and their co-localization with Arp2/3 complex
Jelínková, Barbora ; Schwarzerová, Kateřina (advisor) ; Fendrych, Matyáš (referee)
ARP2/3 is an evolutionarily conserved heteroheptameric protein complex. Its main activity lies in the nucleation of dendritic actin filaments that are involved in membrane remodeling. ARP2/3 takes part in plasma membrane remodeling and the formation of cytoplasmic protrusions that serve in the amoeboid motion of mammalian cells and some protists and plays role in exocytosis and endocytosis of animal and yeast cells. The main objective of this work was to find a connection between the ARP2/3 complex and the regulation of the plant endomembrane system. Using TIRF microscopy we visualized the localization of the ARP2/3 complex in the cortical layer of plant cells and compared it to the localization of several endomembrane markers from the Rab family and an exocytotic marker Exo84b. In the vicinity of the plasma membrane, the ARP2/3 complex subunits localized to dynamic dots very similar to the localization of Exo84b protein. Colocalization analysis showed that a small portion of Exo84b marker and ARP2/3 complex signals colocalize and this result was seconded by the biochemical approach of coimmunoprecipitation. Key words: ARP2/3, endomembrane system, cortical layer, RabA1g, RabC1, RaD2a, Exo84b
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Effect of amyloid β on the function of endosomes and lysosomes
Tmějová, Monika ; Rudajev, Vladimír (advisor) ; Búran, Peter (referee)
Amyloid β peptide is produced by proteolytic processing of amyloid precursor protein. Accumulation of toxic Aβ in lysosomes and endosomes is considered to be one of the earliest signs of Alzheimer disease. Alzherimer disease was first described in 1907 by doctor Alois Alzheimer. This disease is most common in elderly people over the age of 65 and it is currently the most common cause of dementia. Although significant progress has been made in recent years, the key mechanism of formation of senile plaques and neurofibrillary tangles still remains unclear. Vesicular trafficking plays fundamental role in regulation of APP and generation of Aβ. This thesis summarizes molecular mechanisms of pathological effect of Aβ on the endolysosomal complex.
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Interaction of amyloid β with neuronal membrane proteins
Manová, Blanka ; Rudajev, Vladimír (advisor) ; Černá, Barbora (referee)
Amyloid b peptide is cleaved from the amyloid precursor protein by b and γ secretases. According to the amyloid hypothesis i tis the main cause of the early pathogenetic events of Alzheimer's disease (AD) which is the most common neurodegenerative disease in the world without an effective treatment. The main pathogenesis of AD is considered to be the loss of synapses, disruption of neuronal plasticity and neurodegeneration. Amyloid b can bind directly to the membrane or mediate neuronal damage indirectly via toxic inflammatory mediators (e.g., reactive oxygen intermediates, nitric oxide and cytokines) by activating microglia and astrocytes. In addition to interacting with various membrane receptors, Ab can also bind to the cell surface directly, disrupting membrane integrity or forming selective cation channels. This thesis summarizes key interactions with membranes of synapses and mechanisms of amyloid- induced toxicity through receptors.
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Cell signalling and molecular complexes of the TRH receptor
Drastichová, Zdeňka
The first part of this thesis is preoccupied with the identification of protein alterations in the membrane fraction of HEK293-E2M11 cells after prolonged TRH treatment. The isolated membrane fraction enriched in plasma membranes contained high amounts of Na+ ,K+ -ATPase, TRH receptor and G-proteins compared to the postnuclear supernatant. By using 2D electrophoresis and mass spectrometry, the levels of 42 proteins were identified to be altered in samples of PM-enriched fractions from TRH-treated (16 h; 10 μM) cells. Out of these proteins only ezrin and stomatin-like 2 are known to be localized in the plasma membrane. Five proteins (mitofilin, MTHSP75, prohibitin, stomatin like-2, peroxiredoxin III) whose levels were increased after the prolonged TRH treatment represent proteins localized in mitochondria. All of them are important for proper structure and function of mitochondria. The ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was markedly higher in cells treated with TRH compared to control cells. Hence, it can be concluded that prolonged TRH treatment may significantly affect mitochondrial membrane and function of mitochondria. The second part of this thesis deals with the identification of molecular protein complexes of TRH-R and/or Gq/11 protein. The presumed effects of TRH on the...
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Modulatory effect of monovalent ions on δ-opioid receptors
Vošahlíková, Miroslava ; Svoboda, Petr (advisor) ; Jakubík, Jan (referee) ; Kršiak, Miloslav (referee)
The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.
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Evolutionary-developmental study of membrane proteins
Vosolsobě, Stanislav ; Schwarzerová, Kateřina (advisor) ; Baluška, František (referee) ; Štorchová, Helena (referee)
Evolutionary-developmental study of membrane proteins Mgr. Stanislav Vosolsobě Abstract Using a plethora of experimental approaches for phylogenetical and functional study on several membrane signalling proteins, I brought new evidences supporting a hypothesis that the molecular evolution of protein families is a highly dynamic, not conservative, process. In DREPP family of calcium-binding peripherally-associated plasma-membrane proteins I found a broad flexibility in protein-membrane binding manners coupled with a many independent duplication of this Euphyllophyta-clade specific plant gene. In three families of auxin transporting proteins, PIN-FORMED, LAX and PILS, I showed that emergences of these proteins are uncorrelated and placed on different levels of the plant kingdom phylogenetic tree. However these proteins ensure very fundamental plant morphogenetic processes, like cell differentiation, organ formation or tropisms, with strong effects of their deleterious mutations, I found many gene radiations and losses on a all taxonomic levels in these families, evidencing that key and shared physiological processes may be realised by genes touched by a recently undergoing evolution. Evolutionary-developmental synthesis of a functional and phylogenetic data must be done with caution due to high risk of...
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