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Regulation of STING function during murine polyomavirus infection
Šnejdarová, Aneta ; Horníková, Lenka (advisor) ; Pimková Polidarová, Markéta (referee)
Stimulator of Interferon Genes (STING) is the adapter protein of an innate immunity signalling pathway, involved in detection of double-stranded DNA (dsDNA) in the cell cytoplasm, which leads to the expression of pro-inflammatory genes, including the production of type I interferon. Eventhough during the infection with a dsDNA virus, murine polyomavirus (MPyV), the STING protein is activated, the resulting interferon production is moderate. Therefore, it can be assumed that the function of the STING protein is regulated in MPyV-infected cells. The aim of this thesis was to investigate three mechanisms by which the regulation can occur, namely through protein interaction partners, post- translational modifications, or changes in the subcellular localization of the STING protein. A cell-line of mouse fibroblasts stably expressing the STING protein fused with the HA-tag was established to facilitate the research. Furthermore, two plasmids were prepared, that encode the STING protein fused with the green fluorescent protein, facilitating the monitoring of the localization of the protein in the cell, or with a composite tag containing an in vivo biotinylated BioEaseTM -tag enabling effective isolation of the STING protein. The results of colocalization observations and coimmunoprecipitation suggest that...
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Studying the evolution of chromosome rearrangements in pipid frogs using in situ hybridization
Bergelová, Barbora ; Knytl, Martin (advisor) ; Johnson Pokorná, Martina (referee)
In African pipid frogs we can find at least eight polyploidization events in which a lot of levels of ploidy arose. This group includes diploid, tetraploid, octoploid, and/or dodecaploid species. Due to this great variability in ploidy levels, representatives of the genus Xenopus are excellent model organisms for studying the evolution of whole-genome duplications and chromosomal rearrangements. Currently, little information is known about chromosomal rearrangements within the Xenopus species, specifically, there are only two known cases of large-scale rearrangements. The first is a fusion of chromosomes 9 and 10 in the subgenus Xenopus and the other rearrangement is a non-reciprocal translocation occurring between chromosomes 2 and 9 in the subgenus Silurana. In this diploma thesis, we attempted to expand the knowledge about chromosomal rearrangements using the fluorescence in situ hybridization method. We confirmed the fusion of chromosomes 9 and 10 in other species of the Xenopus genus, such as X. pygmaeus, and proposed a hypothesis about the possibility that this fusion occurred in the common diploid ancestors of this subgenus. We also gained further insights into the origin of non-reciprocal translocation within the Silurana subgenus. This translocation was not detected in the species X....
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Plant responses to combined arsenic and cadmium stress
Burešová, Andrea ; Mašková, Petra (advisor) ; Podlipná, Radka (referee)
Arsenic and cadmium are both non-essential, highly toxic and carcinogenic elements that often occur together in the environment. Given the frequent co-contamination of the environment, it is necessary to investigate both plant strategies for dealing with one metal(loid) and the mechanisms that lead to tolerance or, conversely, sensitivity in the presence of both elements. Simultaneous exposure to multiple toxic elements may lead to extensive plant damage, however it may also result in the increasing engagement and intensity of defence strategies. Contamination of the food chain through crops growing on contaminated soils is a major concern, one that poses a risk to both human and animal lives. The aim of this research field is to reduce accumulation and translocation to aboveground edible parts as much as possible, for example by understanding the mechanisms behind heavy metal accumulation and translocation, or by stabilising toxic elements in the soil, or by cleaning up contaminated soil. One method of clean-up is phytoremediation, which usually utilizes plants with high tolerance to toxic elements, called hyperaccumulators. The knowledge of how defence strategies and mechanisms are affected by the interaction of multiple elements is important for identifying plant species capable of activating...
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Mechanisms and aplications of macromolecule translocation across membranes of eukaryotic cells by bacterial toxins
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
Toxin translocation across the cytoplasmic membrane of the eukaryotic cell is a potent virulence factor of bacteria causing disease to eukaryotic organisms. Toxins translocate their domains responsible for the toxic activity inside the cell or create pores in cell membrane allowing the transmembrane traffic of ions, DNA, RNA or proteins. Knowledge of the toxin translocation process enables to characterize the mechanism and also the properties of the pore-forming toxin. Some of these toxins have been described in such a detail that were changed using site-directed mutagenesis and can serve as tools for characterization of the translocated molecules. One of such examples is the transfer of nucleotides or the whole nucleic acid molecules across the membrane through the pore of S. aureus α-hemolysine. Nowadays, this application is commercially used for DNA sequencing. Keywords: translocation, bacterial toxins, plasmatic membrane, nanopore sequencing
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The role of trehalose in mycorrhizal associations
Šoch, Jan ; Ponert, Jan (advisor) ; Hála, Michal (referee)
Mycorrhizal symbiosis is a widely spread phenomenon in nature. A translocation of nutrients occurs between symbionts with disaccharide trehalose playing a key role in the process. However, this saccharide fulfils many important roles in metabolism of fungi and plants. Fungi use trehalose mainly as storage and transport saccharide. On the other hand, trehalose occurs in extremely low amounts in plants where it acts as a signal molecule. Thus it is likely that the saccharide could be used by the fungus to manipulate plant metabolism on a physical interface between symbionts. Trehalose has a similar function in many parasitic interactions. In most cases of mycorrhizal associations trehalose synthesis creates a carbon sink in mycelium which leads to saccharide transfer from the host plant to the fungus. Completely different situation occurs in the types of mycorrhizal symbiosis, where saccharides are translocated from a fungus to a plant. Some plants can utilize trehalose effectively as a sole source of energy. Consequently, the question raises - could such plants gain fungal trehalose on purpose as a source of energy and carbon? This review aims to assess and discuss the mentioned possibilities considering available literature. Key words: mycorrhiza, orchids, parazitism, saccharides, sink, symbiosis,...
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Significance of MLL gene aberrations in patients with acute myeloid leukemia
Šárová, Iveta
In acute myeloid leukemia (AML), predominantly in AML M5a, the most frequent recurrent aberration of chromosome 11 involves region 11q23. Molecular breakpoint studies of several translocations involving chromosomal band 11q23 led to the detection of a gene that was named MLL (myeloid/lymphoid leukemia). This gene is important for the proper HOX gene expression during ontogenesis and hematopoiesis. Chromosomal aberrations affecting the MLL gene occur in 5 - 10 % of AML cases and are very variable. Since that time, more than 70 different translocation partners of the MLL gene have been described. Aberrations of the MLL gene are associated with an aggresive type of the disease and its detection is needed for the treatment decision. Therefore, we investigated the occurrence of MLL abnormalities in bone marrow cells of the 66 newly diagnosed AML patients, using conventional cytogenetic and fluorescence in situ hybridization (FISH) analyses with a commercially available MLL Break Apart Rearrangement probe (Abbott VYSIS). Out of the 66 patients, we proved MLL abnormalities in 9 (13,6%): 5 (7,6%) showed translocation of MLL gene, in 3 (4,5%) we detected MLL gene amplification without any evidence of rearrangement and in 1 (1,5%) pacient only an extra copy of the MLL gene. The FISH results were verified by...
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Correlation of Immunohistochemical and Molecular Methods in Diagnostics of Salivary Gland Tumors
Horáková, Markéta ; Skálová, Alena (advisor) ; Laco, Jan (referee) ; Dušková, Jaroslava (referee)
This doctoral thesis is dealing with the correlation of morphological, immunohistochemical and genetical findings in malignant tumors of salivary glands. The first half of the thesis comprises the summary of current knowledge about salivary malignancies. The second half is presenting the research itself. The research results are divided into three parts. The first part is presenting the method of "2-step diagnostic test" of malignant tumors. This screening test aims to find new, so far not described gene aberrations with a focus on malignant tumors of salivary glands. This method takes place in two consecutive steps. In the first step the material is examined by an immunohistochemical mixture of antibodies, which non-specifically detects aberration in the genes NTRK1-3, ALK and ROS1. In the second step all positive cases are subjected to highly sensitive and specific molecular-genetic examination by the method of next generation sequencing (NGS) using the Archer kit. In the second part of the work there has been designed the approach to the cytological diagnosis of salivary secretory carcinoma by the fine-needle aspiration (FNA). This part is describing to the details the cytomorphology of secretory carcinoma in both, Pap smears and cell blocks, from which additional immunocytochemical and genetic...
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Functional characterization of LACE1 APTase and mitochondrial AAA proteases YME1L and AFG3L2 in mitochondrial protein homeostasis.
Tesařová, Jana ; Stibůrek, Lukáš (advisor) ; Kalous, Martin (referee) ; Pecina, Petr (referee)
Mitochondrial protein homeostasis is crucial for cellular function and integrity. It is ensured by many specific mitochondrial proteases with possible chaperone functions located across the various mitochondrial subcompartments. In the first part, we have focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 and YME1L of the mitochondrial inner membrane complexes m- and i-AAA in HEK293 cells. The double AFG3L2/YME1L knockdown cells showed severe alteration in OPA1 protein processing, marked elevation in OMA1 protease and severe reduction in SPG7. Our results reveal cooperative and partly redundant involvement of AFG3L2 and YME1L in the maintenance of mitochondrial protein homeostasis and further emphasize their importance for mitochondrial and cellular function and integrity. The aim of the second part was to characterize the cellular function of LACE1 (lactation elevated 1) in mitochondrial protein homeostasis. LACE1 protein is a human homologue of yeast Afg1 (ATPase family gene 1) ATPase. We show that LACE1 is a mitochondrial integral membrane protein that exists as a part of three complexes of approximately 140, 400 and 500 kDa. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4, COX5A and COX6A. Using affinity...
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Mechanisms and aplications of macromolecule translocation across membranes of eukaryotic cells by bacterial toxins
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
Toxin translocation across the cytoplasmic membrane of the eukaryotic cell is a potent virulence factor of bacteria causing disease to eukaryotic organisms. Toxins translocate their domains responsible for the toxic activity inside the cell or create pores in cell membrane allowing the transmembrane traffic of ions, DNA, RNA or proteins. Knowledge of the toxin translocation process enables to characterize the mechanism and also the properties of the pore-forming toxin. Some of these toxins have been described in such a detail that were changed using site-directed mutagenesis and can serve as tools for characterization of the translocated molecules. One of such examples is the transfer of nucleotides or the whole nucleic acid molecules across the membrane through the pore of S. aureus α-hemolysine. Nowadays, this application is commercially used for DNA sequencing. Keywords: translocation, bacterial toxins, plasmatic membrane, nanopore sequencing
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Mechanisms and aplications of macromolecule translocation across membranes of eukaryotic cells by bacterial toxins
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
The bacterial protein toxins endowed with the ability to translocate across the plasmatic membrane are often crucial virulence factors of pathogenic bacteria invading eukaryotic organisms. These toxins translocate either their own protein domains carrying toxic activity or can form pores transferring other substances like small ions, DNA, RNA or proteins. By observing the translocation of these molecules together with other artificially prepared agents on synthetic membranes it allows detailed understanding of mode of action of individual pore-forming toxins. Some of the toxins were actually described in such a detail that can serve as tools for drug delivery or characterization of new translocated molecules. One of such examples is the transfer of nucleotides or the whole nucleic acid molecules across the membrane pore of S. aureus α-hemolysine. Nowadays, this application is commercially used for DNA sequencing. Keywords: translocation, bacterial toxins, plasmatic membrane, nanopore sequencing
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