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Targeting insulin storage forms in pancreatic β-cell secretory granules
Asai, Seiya ; Jiráček, Jiří (advisor) ; Skořepa, Ondřej (referee) ; Koblas, Tomáš (referee)
In this dissertation, we focused on a comprehensive investigation of insulin production, storage and secretion by pancreatic -cells. We successfully developed a new assay for the rapid and sensitive determination of insulin concentration in biological samples. This assay, based on the competition of the measured sample with a radioligand for the insulin receptor, helped us to determine the influence of several low molecular weight compounds, as well as peptides, on insulin secretion. We found that arginine and ornithine have a dose-dependent stimulatory effect on glucose- stimulated insulin secretion from -cells, but that dopamine inhibits insulin secretion. The effect of serotonin on insulin secretion was ambiguous. We also studied the effects of the bone protein osteocalcin and its fragments on insulin secretion. We found that these peptides do not stimulate insulin secretion from -cells, but that osteocalcin may have proliferative properties. We also tested the effect of tryptophan and its metabolites and found that these compounds do not stimulate insulin secretion but that some of them may inhibit secretion at higher concentrations. An important result of the study is the experimental confirmation of the presence of crystalline insulin in the secretory granules of -cells. This is the first...
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The development, constitution and functions of the gland apparatus in schistosome cercariae and endopeptidases in its contents
Titlová, Lucie ; Mikeš, Libor (advisor) ; Konečný, Lukáš (referee)
Flukes of the family Schistosomatidae are blood parasites with two-host life cycles involving aquatic snails as intermediate hosts and avian or mammalian definitive hosts. Cercariae, as invasive aquatic stages of schistosomes, enter the lumen of vessels by penetrating the skin of the definitive host. During their short life, cercariae possess a glandular apparatus consisting of three types of glands with external secretion: penetration glands, escape glands, and a head gland. These glands secrete granules containing, among other things, proteolytic enzymes, which play an important role in the process of penetrating the host's skin, but at the same time they occupy many other functions during the life of the cercaria and other life stages of schistosomes. This thesis summarizes basic knowledge about the anatomy, function and development of the gland apparatus of schistosome cercariae and further focuses on the representation of proteolytic enzymes in these glands, specifically endopeptidases. At the end, it briefly compares the different representation of endopeptidases in some members of the family.
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The Role of selected exocyst subunits in response of plants to pathogen
Sabol, Peter ; Kulich, Ivan (advisor) ; Burketová, Lenka (referee) ; Dagdas, Yasin (referee)
In the recent years, there has been a growing number of publications indicating at the involvement of plant secretory pathway in defense against phytopathogens. Specifically, roles of plant exocyst complex have been explored in deeper detail in current research. Yet, exactly how exocyst- mediated exocytosis contributes to secretion of antimicrobials and cell wall-based defense remains unclear. In the presented Dissertation, I provide both experimental evidence and devise further hypotheses on selected exocyst's subunits in plant immune reactions. Particularly, I show that EXO70B1 exocyst subunit interacts with immunity-related RIN4 protein. Cleavage of RIN4 by AvrRpt2 Pseudomonas syringae effector protease releases both RIN4 fragments and EXO70B1 from the plasma membrane when transiently expressed in Nicotiana benthamiana leaves. I speculate on how this might have an implication in regulation of polarized callose deposition. In a co-authored opinion paper, we also hypothesize that EXO70B1-mediated autophagic degradation of TN2 resistance protein prevents its hyperactivation and lesion mimic phenotype development. In addition, in collaboration with my colleagues, I present data on EXO70H4's engagement in PMR4 callose synthase secretion, required for silica deposition. Representing a possible...
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Effectors of RAB GTPases and their role in plant secretion
Růžičková, Martina ; Hála, Michal (advisor) ; Motyka, Václav (referee) ; Klíma, Petr (referee)
Rab GTPases are small signaling molecules that play an important role in vesicle trafficking in eukaryotic cells. Correct signaling through small GTPases allows orchestration of vesicle transport among cellular organelles and also to the cell wall providing cell wall material for cell growth and elongation. Engagement of Rab GTPases in the regulation of endomembrane trafficking is one of the evolutionary conserved aspects of secretion regulation. The network of Rab GTPases interaction includes also various downstream effectors. One of them is the exocyst complex involved in vesicle docking at the plasma membrane. It is a complex composed of eight different subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Exocyst was discovered as Sec4p Rab GTPase effector in yeast and also data from animal models describe the Sec15 exocyst subunit as the Rab-interacting partner, but data from plants are missing. On the other hand, numerous studies identified exocyst role in tip growth of pollen tube and root hairs, seed coat formation, cell plate and cell wall formation, hypocotyl elongation, and importantly also PIN auxin efflux carriers recycling and polar auxin transport. There are two paralogues of SEC15 in the Arabidopsis genome, SEC15a and SEC15b, the previous one already shown to be...
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Conventional and Novel Functions of the Exocyst Complex in Plants
Kulich, Ivan ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Exocyst is an octameric protein complex, conserved across all Eukaryotes. Its role, originally described in yeast, resides in a tethering of the secretory vesicles to the plasma membrane prior to the membrane fusion of the two membranes. Subunits SEC3 and EXO70 are believed to be spatial landmarks for the vesicles delivery. While yeast genome encodes single EXO70, we find dozens of them in land plants (23 in Arabidopsis). This work is focused at a role of the exocyst complex in plant cells. Its first part documents, that exocyst is essential for delivery of the cell wall components, namely pectins, but also for pathogen induced secondary cell wall thickening. Second part reveals an unconventional role of EXO70B1 subunit harboring exocyst subcomplex at an autophagic pathway to the vacuole and raises many questions about plant secretory pathway.
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Morphological and functional variability of secretory glands in cercariae of chosen trematode groups
Krčmářová, Veronika ; Bulantová, Jana (advisor) ; Mikeš, Libor (referee)
Trematodes are characterized by their complex life cycles that include definitive hosts and variable number of intermediate hosts. Transfer of the parasite from the first intermediate host to the other is usually realized by larval stage called cercaria. After finishing of their development within the first intermediate host, morphology and fate of these larval stages vary according to the way of infection of the next host. Some cercariae actively penetrate directly to their definitive hosts trough their body surface, other encystate in the outer environment where they wait in a form of metacercariae to be ingested by definitive host. Both of these ways can be combinated and cercariae encystate inside second intermediate host after they actively penetrate them. Exceptionally, cercariae do not leave the sporocyst in which they were developing inside the first intermediate host. They encyst there waiting for ingestion by the definitive host. Various types of secretory glands have been developed in cercariae for successful direct infection of next hosts, survival of parasite in adverse conditions of outer environment or for transformation of one larval stage to subsequent one. Variability in morphology and function of these secretory glands in cercariae is closely connected with differences in life...
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The role of RTX domain in the activity of adenylate cyclase toxin from Bordetella pertussis
Klímová, Nela ; Bumba, Ladislav (advisor) ; Konopásek, Ivo (referee)
The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein comprising an amino-terminal adenylate cyclase (AC) domain and a carboxy-terminal Repeat-in-Toxin (RTX) domain. The RTX domain is a hallmark of the family of RTX proteins, which are secreted from the cytosol of Gram-negative bacteria to the cell environment through the Type I Secretion System (T1SS). The RTX domain of CyaA consists of five blocks of RTX nonapetide repeats with a consensus sequence X-(L/I/V)-X-G-G-X-G- X-D. The aim of this work was to determine the role of the RTX domain in biological activities of CyaA and its role in the secretion of the toxin molecule from Bordetella pertussis. Systematic deletion analysis revealed that none of the prepared CyaA constructs was able to translocate its AC domain across the cytoplasmic membrane of host cells and make pores in target membranes. Moreover, deletion of individual RTX repeat blocks resulted in a very low efficacy of secretion of CyaA mutants into cell exterior. These data suggested that structural integrity of the RTX domain of CyaA is essential not only for cytotoxic activities of the toxin molecule but also for its secretion through the T1SS.
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Mechanisms of epidermal cells polarization in plants.
Vojtíková, Zdeňka ; Žárský, Viktor (advisor) ; Soukup, Aleš (referee)
Plant epidermal cells form contact area of the plant, they protect it from impacts of surrounding environment and they mediate the communication with its neighbourhood. In the epidermis there are evenly distributed several cell types with quite specialized morphology (pavement cells, trichomes and guard cells) due to the polarization mechanisms. The cytoskeleton and signal molecules of ROP GTPase family promote the polarized growth. Thanks to polarized growth the cells reach their shapes. The cytoskeleton responds to the signal by expanding the cell, helps with targeting of the secretion to the sites of active growth and mediates polarized formation of the cell wall. On the upper side of the epidermis the cuticle and layer of epicuticular waxex is secreted. The secretion of cuticular components is baso-apicaly polarized. This work summarizes the mechanisms of the polarization in plant epidermal cells discoverd untill now.
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Secretion and autophagy in plant defence against microbes
Dobešová, Karolína ; Žárský, Viktor (advisor) ; Burketová, Lenka (referee)
Plants are sessile organisms and when attacked by microbes, they cannot easily run away. For this reason, they have developed sophisticated defensive mechanisms, that allow them to defend themselves. Since plants, unlike mammals, do not have any special immune cells, their defense takes place in each cell separately. The key moment during a microbial infection is the recognition of the microbe by the plant through its released molecular patterns (mostly proteins) associated with microbes (MAMPs). MAMPs trigger signaling cascades that lead to the secretion of antimicrobial compounds to the site of an attack. The process of autophagy is also important in the defense against microbes, which not only maintains a cellular homeostasis and controls the level of phytohormones and defense proteins in the plant cytoplasm, but also participates in the secretory activity of the cell. Recent analyzes of plant secretome have shown that plants secrete many proteins (including defensive ones) independently of the signal peptide and compartments of a conventional secretion. During exocytosis a vesicle fuses with the cytoplasmic membrane. The octameric protein complex exocyst and SNARE proteins take part in this process. The exocyst complex is highly diversified in plants - especially it's EXO70 subunit, which is...
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The role of exocyst subunit AtEXO70E2 in autophagy and secretion
Moulík, Michal ; Sabol, Peter (advisor) ; Janda, Martin (referee)
Exocyst is a protein complex composed of eight subunits, evolutionarily conserved in yeasts, animals, and plants. The main function of exocyst is to mediate the tethering of secretory vesicles to the plasma membrane. However, the involvement of exocyst in some other processes, especially in autophagy, has been recently discovered. Plant exocyst is specific because most of its subunits have multiple paralogs. The most diversified subunit is EXO70, which is encoded by 23 paralogous genes in Arabidopsis thaliana. In this thesis, I dealt with subunit AtEXO70E2 (AT5G61010), which has been localized to double-membrane compartments considerably reminiscent of autophagosomes. These compartments were named EXPOs (for exocyst-positive organelles) and described as a component of unconventional protein secretion pathways. There are also hints that EXO70E2 could play a role in autophagic processes. However, details of this relationship remained unexplored. For my experiments, I used stably transformed lines of A. thaliana and transiently transformed leaves of Nicotiana benthamiana. I performed numerous colocalization experiments, applied various pharmacological treatments to the studied lines, and analyzed a mutant line in the EXO70E2 gene. According to my observations, protein EXO70E2 is expressed especially...
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