|
|
Identification and characterization of main genetic components involved in phototransduction and vision of the cubozoan jellyfish Tripedalia cystophora
Liegertová, Michaela ; Kozmík, Zbyněk (advisor) ; Černý, Robert (referee) ; Kostrouch, Zdeněk (referee)
(English) Many of the metazoan phyla sense light by an opsin-based photopigment present in a photosensitive receptor cell (photoreceptor), with Cnidaria being arguably the earliest branching phylum containing a well-developed and complex visual system (advanced eyes morphologically similar to those of vertebrate). The evolutionary history of phototransduction and visual components (ranging from light-sensing opsins to structural genes of the lenses) is a long standing question. In this work, we decided to address this issue by applying a comprehensive multidisciplinary approach combining modern molecular biology methods with bioinformatics. Comprehensive genome-wide inspection of a cubozoan jellyfish Tripedalia cystophora, was complemented with gene expression analyses, together with functional (cell culture based assays) and behavioural (pharmacogenetics) testing. First, genome analysis uncovered the presence of a surprisingly large number of opsin genes with distinct tissue- and stage-specific expression. Our extensive phylogenetic analysis classified cubozoan opsins as a sister group to c-type opsins and documented a lineage-specific expansion of opsin gene repertoire. Functional tests in cell cultures provided evidence for the use of Gs-cAMP signalling pathway only in a small subset of opsins,...
|
|
|
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...
|
|
|
Orphan Nuclear Receptor TLX (NR2E1) in Regulation of Cell Reproduction and Differentiation
Raška, Otakar ; Kostrouch, Zdeněk (advisor) ; Otová, Berta (referee) ; Nedvídková, Jara (referee)
Nuclear receptors constitute a large family of transcription factors that are powerful regulators of animal tissue metabolism, homeostasis, tissue maintenance and development. They are particularly attractive for their ability to respond to the binding of hormones, metabolites, xenobiotics and artificially prepared molecules and transmit the interaction with these small lipophylic molecules to specific regulatory potential. In search for nuclear receptors that are likely to be critical for neural tissues in invertebrates and conserved during the evolution of animals, we have identified a close homologue of vertebrate TLX in a planarian Schmidtea mediterranea. Planaria represent very promising biological model systems for studies on tissue maintenance and regeneration. Planaria are able to resorb their tissues and use them as sources of energy during fasting and they re-build their bodies from neoblasts when food is plentiful. Our search in Schmidtea mediterranea's publicly accessible genome sequencing data indicated that planarian genome contains at least one gene with a high degree of similarity to vertebrate TLX. We cloned full length CDS (coding DNA sequence of cDNA) and characterized the gene functionally. This showed that the planarian and vertebrate NR2E1 are highly similar...
|
|
|
Retinoid regulation of target gene transcription and antitumor activity of retinoid related molecules
Raška, Ivan ; Broulík, Petr (advisor) ; Kostrouch, Zdeněk (referee) ; Pokorný, Jaroslav (referee)
Nuclear Retinoic Acid (RA) Receptors (RARs) activate gene expression through dynamic interactions with coregulators in coordination with the ligand and phosphorylation processes. In the first part of this work, we showed that during RA-dependent activation of the RARq isotype, the pi 60 coactivator pCIP/ACTR/AIВ-1 /RAC-3/TRAM-1 /SRC-3 was phosphorylated by p38MAPK. SRC-3 phosphorylation was correlated to an initial facilitation of RARa-target genes activation, via the control of the dynamics of the interactions of the coactivator with RARa. Then phosphorylation inhibited transcription via promoting the degradation of SRC-3. In line with this, inhibition of p38MAPK markedly enhanced RARa-mediated transcription and RA-dependent induction of cell differentiation. SRC-3 phosphorylation and degradation occurred only within the context of RARa complexes, suggesting that the RAR isotype defined a phosphorylation code through dictating the accessibility of the coactivator to p38MAPK. We proposed a model in which RARa transcriptional activity was regulated by SRC-3 through coordinated events that are fine- tuned by RA and p38MAPK. The retinoid related molecules (RRMs), ST1926 and CD437, are promising anti-cancer agents. In the second part of this work, we compared the RAR trans-activating properties of the two RRMs...
|
|
| |
|
|
Functional genomics of nuclear hormone receptors and their cofactors: Connection between metabolism and development by diversified nematode nuclear hormone receptors
Pohludka, Michal ; Kostrouch, Zdeněk (advisor) ; Mandys, Václav (referee) ; Raška, Ivan (referee)
The aim of the study was to contribute to the understanding of regulatory functions of nuclear hormone receptors by studies focused on a group of multiplied nuclear receptors present in the genome of Caenorhabditis elegans, specifically nhr-40 and nhr-60. The work presented in this thesis was a part of an effort to characterize the selected nuclear receptors systematically by means of Caenorhabditis elegans functional genetics and genomics. Studies constituting this thesis were meant as an application of proteomics in the functional analysis of the selected nuclear receptors in the frame of the complex biology of Caenorhabditis elegans. The proteomic scope of this thesis was aimed at understanding of the selected receptors at the level of the protein, the protein nature, localization and dynamics as well as the proteomic characterization of the consequences of the protein loss of function.
|
|
|
Image analysis of fluorescently labeled nuclear structures: DNA replication in focus
Mašata, Martin ; Raška, Ivan (advisor) ; Kostrouch, Zdeněk (referee) ; Kozubek, Michal (referee)
In this thesis, we expanded the knowledge about some aspects of DNA replication in human cell nucleus with the help of microscopic techniques and advanced mathematical approaches. DNA replication was studied from several perspectives: Fluorescence detection of newborn DNA on the stretched DNA fibers, ultrastructural mappings of individual replication domains, fluorescence analysis of replication foci re-distribution in early S-phase and cold-dependent immunodetection of replication-coupled chromatin modulation. We started with measurements of average speed of a replication fork movement during S- phase. In agreement with previous studies on DNA spreads with radioactive labeling, the replication speed was two to three times slower in early S-phase than in late S-phase. Moreover, the exogenous dNTP supply accelerated the replication in the early S-phase while additional dNTPs had no effect on the speed of replication forks in late S-phase. Therefore the availability of dNTPs seems to be a rate-limiting factor for replication speed at the beginning of DNA synthesis. We then focused on in situ replication pattern at the EM level. Using an improved method for detection, replication domains, identified by clusters of many silver particles, were observed throughout the S-phase. While replication foci in early...
|
|
|
The role of evolutionarily conserved proteins BIR-1/Survivin and SKP-1 in the regulation of gene expression
Kostrouch, David ; Kostrouch, Zdeněk (advisor) ; Dráber, Pavel (referee) ; Pacák, Karel (referee)
SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...
|
|
| |
|
| |
guest ::
