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Regulation of protein expression non-specific immunity in Caenorhabditis elegans
Kaštánková, Iva ; Kostrouch, Zdeněk (advisor) ; Cmarko, Dušan (referee)
6 Abstract Lipopolysaccharides are composed of covalently bound saccharides. They are a characteristic component of the cell wall of gram-negative bacteria. They are the cause of severe sepsis in humans and complications in human medicine. Lipopolysaccharides are a constant part of the infections of gram-negative bacteria. We expect an evolutionarily conserved non-specific immune response and protection. The question is whether there is an immune response in the model organism Caenorhabditis elegans. If so, what mechanism is controlled and regulated. We submitted lipopolysaccharides from the bacteria Pseudomona aeruginosa with the bacteria Escherichia coli OP50 and observed the influence of lipopolysaccharides on the expression of selected genes. We examined metabolism and development. We have shown the influence of lipopolysaccharides on gene expression of C-type lectine clec-60 a clec-71, nextna lys-5, hsp-60 a F44G.3.2.1 genes. We incubated Caenorhabditis elegans on some components of lipopolysaccharide. We found regulation of these selected genes with hydrophobic components of lipopolysacharide, lipid A. We did not observe regulation with saccharide components of lipopolysaccharide, glucose and galatose. The metabolism of lipids had changed. We demonstrated a reduction of neutral lipids and changes in...
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DNA replication and chromatin: From 3D to function
Ligasová, Anna ; Koberna, Karel (advisor) ; Cmarko, Dušan (referee) ; Nedvídek, Josef (referee)
We characterized the spatio-temporal organization of replication sites. In agreement with the previous studies, five different replication patterns were observed during the S phase in HeLa cells at the level of light microscopy. Using the electron microscopy approach, we observed the close association of numerous silver grains at many sites in the early S phase. The labeled sites had discrete sizes in terms of both area and diameter. Moreover, the much larger heterochromatin domains that replicate in the late S phase, and to a lesser extent in the mid S phase, were actually composed of closely-associated, labeled RS with virtually identical diameters and areas as the early-S-labeled RS. The replication sites labeled in the early S phase were maintained as similarly labeled clusters of colloidal gold particles later in the S phase and in the next cell generation. The number of replication sites was similar in the early- and mid-S-phase cells with a slight decrease in the late S phase. The electron microscopy tomography revealed that most of the observed replication sites corresponded to individual replicons. We found that the replisomes operate as tightly-associated couples during replication. Our results concern only human HeLa cells, but considering that the findings on prokaryotic cells and budding yeast...
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Structure-function organization of the cell nucleus.Microscopical analysis of nuclear subcompartments.
Jůda, Pavel ; Cmarko, Dušan (advisor) ; Mokrý, Jaroslav (referee) ; Kučera, Tomáš (referee) ; Smetana, Karel (referee)
Pavel Jůda - Abstract The cell nucleus is a complex cellular organelle. The nucleus and nuclear processes are organized into functionally and morphologically separated nuclear subcompartments. This thesis is particularly concerned with the three following nuclear subcompartments: sites of DNA replication, Polycomb bodies and nuclear inclusions constituted of inosine monophosphate dehydrogenase 2 (IMPDH2). First, we examined the relationship between MCM proteins and DNA replication. Using immunofluorescent labeling of cells extracted prior fixation and applying cross-correlation function analysis, we showed that MCM proteins are present at the sites of active DNA synthesis. Our results contributed to the solving of the first part of so-called MCM paradox. Second, we studied the structural basis of the Polycomb bodies. Based on fluorescence microscopy studies, Polycomb bodies have been considered to be the nuclear subcompartments formed by the accumulation of Polycomb proteins in the interchromatin compartment. In our work, using correlative light electron microscopy and experimental changes in macromolecular crowding, we clearly showed that a Polycomb body is a chromosomal domain formed by an accumulation of heterochromatin structures, rather than a typical nucleoplasmic body. Third, we were interested in...
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Characterization of the Caenorhabditis elegans pop-1 gene
Jakšová, Soňa ; Vacík, Tomáš (advisor) ; Cmarko, Dušan (referee)
The human proteom diversity is caused by the ability of a single gene locus to encode more protein isoforms. The TCF/LEF genes produce a broad spectrum of protein variants, which consequently leads to a great functional diversity of the TCF/LEF proteins. The TCF/LEF transcriptional factors regulate the canonical Wnt signaling target genes. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1
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Study of the mechanism of gene expression regulation at the level of functional organization of chromatin domains.
Hornáček, Matúš ; Cmarko, Dušan (advisor) ; Kučera, Tomáš (referee) ; Stixová, Lenka (referee)
- 1 - ABSTRACT Nucleoli are formed on the basis of genes of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In our work we study changes of FC/DFC units in the course of cell cycle. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60 to 80 %. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis but become again active...
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Structure-function organization of the cell nucleus.Microscopical analysis of nuclear subcompartments.
Jůda, Pavel ; Cmarko, Dušan (advisor) ; Mokrý, Jaroslav (referee) ; Kučera, Tomáš (referee) ; Smetana, Karel (referee)
Pavel Jůda - Abstract The cell nucleus is a complex cellular organelle. The nucleus and nuclear processes are organized into functionally and morphologically separated nuclear subcompartments. This thesis is particularly concerned with the three following nuclear subcompartments: sites of DNA replication, Polycomb bodies and nuclear inclusions constituted of inosine monophosphate dehydrogenase 2 (IMPDH2). First, we examined the relationship between MCM proteins and DNA replication. Using immunofluorescent labeling of cells extracted prior fixation and applying cross-correlation function analysis, we showed that MCM proteins are present at the sites of active DNA synthesis. Our results contributed to the solving of the first part of so-called MCM paradox. Second, we studied the structural basis of the Polycomb bodies. Based on fluorescence microscopy studies, Polycomb bodies have been considered to be the nuclear subcompartments formed by the accumulation of Polycomb proteins in the interchromatin compartment. In our work, using correlative light electron microscopy and experimental changes in macromolecular crowding, we clearly showed that a Polycomb body is a chromosomal domain formed by an accumulation of heterochromatin structures, rather than a typical nucleoplasmic body. Third, we were interested in...
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Study of the organization and dynamics of the membraneless cell compartments
Blažíková, Michaela ; Heřman, Petr (advisor) ; Cmarko, Dušan (referee) ; Hašek, Jiří (referee)
of Doctoral Thesis Title: Study of the organization and dynamics of the membraneless cell compartments Author: Michaela Blažíková Institute: Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Supervisor: Doc. RNDr. Petr Heřman, CSc., Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Abstract Eukaryotic cells contain many organelles and specific bodies. Beside the membrane delimited organelles such as nucleus, mitochondria or Golgi apparatus there are other structurally and functionally distinct membraneless structures in the cells. In this work we studied the self-organization processes, i.e. the processes that do not require specific interactions, of membraneless structures in nuclei, cytoplasm and plasma membrane of mammalian cells and yeast. The research was focused on the formation of nucleoli and Cajal bodies in mammalian cell nulei and processing bodies (P- bodies) in the cytoplasm of mammalian cells. The organization of MCC domains in the yeast plasma membrane (Membrane compartment of Can1) was studied as well. It was shown that nonspecific interactions as the result of macromolecular crowding could be one of the main driving forces in formation and stabilization of these...
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Regulation of protein expression non-specific immunity in Caenorhabditis elegans
Kaštánková, Iva ; Kostrouch, Zdeněk (advisor) ; Cmarko, Dušan (referee)
6 Abstract Lipopolysaccharides are composed of covalently bound saccharides. They are a characteristic component of the cell wall of gram-negative bacteria. They are the cause of severe sepsis in humans and complications in human medicine. Lipopolysaccharides are a constant part of the infections of gram-negative bacteria. We expect an evolutionarily conserved non-specific immune response and protection. The question is whether there is an immune response in the model organism Caenorhabditis elegans. If so, what mechanism is controlled and regulated. We submitted lipopolysaccharides from the bacteria Pseudomona aeruginosa with the bacteria Escherichia coli OP50 and observed the influence of lipopolysaccharides on the expression of selected genes. We examined metabolism and development. We have shown the influence of lipopolysaccharides on gene expression of C-type lectine clec-60 a clec-71, nextna lys-5, hsp-60 a F44G.3.2.1 genes. We incubated Caenorhabditis elegans on some components of lipopolysaccharide. We found regulation of these selected genes with hydrophobic components of lipopolysacharide, lipid A. We did not observe regulation with saccharide components of lipopolysaccharide, glucose and galatose. The metabolism of lipids had changed. We demonstrated a reduction of neutral lipids and changes in...
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Formování sestřihových snRNP v buněčném jádře
Novotný, Ivan ; Staněk, David (advisor) ; Cmarko, Dušan (referee) ; Forstová, Jitka (referee)
1 ABSTRACT There are many structures, suborganelles and bodies in the eukaryotic cell nucleus. These domains provide the nucleus with many specific functions. Nucleolus is specialized compartment serves to ribosomes assembly, nuclear speckles or Splicing Factors Compartment play an important role in RNA processing and best studied of them, Cajal bodies (CBs), are involved in snRNP maturation. However, non-membrane substructures are not unique for cell nucleus; processing bodies (P bodies) found in the cytoplasm are proposed to be important places in mRNA degradation pathway. This work is a compilation of four projects focused on non-membrane cellular bodies; namely, nuclear CBs and cytoplasmic P bodies. Both CBs and P bodies are dynamic structures that continuously exchange their components with surrounding environment. In addition to a widely accepted role of CBs in snRNP biogenesis, we show that the CB serves as a place where snRNPs are regenerated after each round of splicing. Thus, CBs are important nuclear compartment involved in snRNP recycling. To further characterize tri-snRNP assembly in CBs we applied kinetic experiments combined with mathematical modeling and created a kinetic model of tri- snRNP formation in the CB that determined kinetic parameters of tri-snRNP formation. Moreover, our kinetic...
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