|
|
Genomics and cell biology of oxymonads
Treitli, Sebastian Cristian ; Hampl, Vladimír (advisor) ; Brune, Andreas (referee) ; Beneš, Vladimír (referee)
Oxymonads are a group of poorly studied protists living as intestinal endosymbionts in the gut of insects and vertebrates. In this thesis I focused on the study of phylogeny, genomics and cell biology of oxymonads. Using culture-based approaches, we uncovered the hidden diversity of small oxymonads and described one new genus and six new species. In Monocercomonoides exilis, the only oxymonad with a published genome, we investigated the genome organization using fluorescence in situ hybdridization (FISH) against the telomeric regions and single-copy genes. Our results show that the genome is most probably haploid being organized in 6-7 chromosomes. Annotation of the genome revealed that the DNA replication and repair mechanisms in M. exilis are canonical and they seem more complete than those of other metamonads whose genomes are available. Although M. exilis lacks in any traces of mitochondria, its genome annotation revealed that other cellular systems do not markedly differ from other eukaryotes. Our taxon-rich phylogenetic analyses suggested that the genus Monocercomonoides is closely related to the oxymonad Streblomastix strix, which is found exclusively in the gut of the termites. Streblomastix strix, as opposed to M. exilis, is highly adapted to harbour bacterial ectosymbionts. Since S. strix...
|
|
|
Metabolic control of the cell cycle in bacteria
Valtová, Aneta ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Metabolic control of cell cycle has been studied for a long time in bacteria, but it is not still fully elucitated. The mechanisms described for several decades have been described in more detail and find new connections between basic metabolism and the cell division process itself. Cell cycle regulation, depending on metabolism and nutritional conditions, takes place over all steps of the cycle. The most mechanisms are studied at the level of bacterial division formation. Nutritional deprivation induces stress responses that use low-molecular substances which are involved in signaling pathways and which regulate the cell cycle. One of the most studying is the molecule of guanosine (penta)tetraphosphate (p)ppGpp, which affects cell cycle at the level of genes expression, at the level of proteins involved in the process of creating divisome, even at the level of replication. Recent research revealed that some enzymes with their already known enzymatic function in major metabolic pathways (glycolysis and TCA), also have a function as sensors that transmit a signal about the nutritional change directly to the division apparatus of the cell. These enzymes regulate the formation of the Z ring through the protein FtsZ or its auxiliary proteins most often and have been found in Gram-positive (Bacillus...
|
|
|
The cell cycle and differentiation of haematopoietic stem and progenitor cells.
Páral, Petr ; Šefc, Luděk (advisor) ; Horváthová, Monika (referee) ; Kokavec, Juraj (referee)
Haematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analysed the cell cycle and cell production rate in HSPCs in murine haematopoiesis. The labelling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled the determination of the cell cycle flow rate into the G2-phase, the duration of the S-phase and the average cell cycle time in Sca-1+ and Sca-1- HSPCs. The determination of cells with 2n DNA content and labelled during the preceding S-phase was used to establish the cell flow rates in the G1-phase. Our measurements revealed a significant difference in how Sca-1+ and Sca-1- HSPCs self-renew and differentiate. The division of Sca-1+ progenitors led to the loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. In contrast both Sca-1- progenitors, arising from mitotic cell division, entered a new round of the cell cycle. This corresponds to symmetric self-renewing cell division. The novel data also enabled us to estimate the cell production rates in the Sca-1+ and in three subtypes of Sca-1- HSPCs. We focused on adult murine erythroid differentiation in the next part of our study. We introduced an original flow cytometry approach for identifying and studying erythroid...
|
|
|
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...
|
|
|
Activation and regulation of cell death in senescent cancer cells.
Holíček, Peter ; Anděra, Ladislav (advisor) ; Drbal, Karel (referee)
Cellular senescence is a distinct cell state, characteristic by cessation of cell proliferation and it is accompanied by specific morphological and biochemical alterations. Increasing and persisting incidence of senescence cells has been shown to have detrimental effect on an organism largely contributing to its ageing. Senescent cells also positively support tumour growth and can even stimulate carcinogenic transformation of surrounding cells. Moreover, senescence can be induced even in tumour cells spontaneously or by chemotherapy. Regardless of an initial stimuli and type of cells, there are two main senescence inducing pathways p16/pRb and p53/p21. Both senescent cells as well as senescent cancer cells seems to have modified apoptotic signalling at the level of mitochondria and Bcl-2 family proteins. In this study, we aimed to analyse effect of senescent state as well as pre-senescent (growth arrested state) induced by p16/pRb and p53/p21 signalling pathways on the response of H28 mesothelioma cancer cells-derived clonal cultures to various cell death-inducing stimuli. By inducible expression of p16 and p21 proteins in doxycycline-dependent manner, we forced cells to acquire senescent-like phenotype, which we detailly characterised. Our results showed that senescent-like phenotype, manifests...
|
|
|
Study of dysregulation of DLX1 protein in myeloid leukemia cells in in vitro and in vivo models
Jelínková, Alena ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
The heterogeneous nature of acute myeloid leukemia (AML) worsens the results of patients treated with standard therapy. Understanding the processes of leukemogenesis can contribute to identification of more appropriate treatment. Family of DLX genes (Distal-less homeobox), belonging to the homeobox genes, are associated with haematological malignancies and solid tumors. In the analysis of expression data, the low level of the DLX1 gene was associated with a worse prognosis of patients with AML. In this work we studied phenotypic changes of cell lines with different expression of the DLX1 gene. We silenced the DLX1 gene in AML cell line (sh cells) and compared it to the parental line with higher expression of DLX1 (NSC cells). By cell cycle analysis and apoptosis assays in vitro and in vivo, we have observed the arrest of sh cells in the G0 phase and a lower number of apoptotic cells. Differences were found when measuring the absolute number of cells in time. In in vitro conditions there were less sh cells, in in vivo environment there was significantly higher number of sh cells engrafted in comparison to NSC cells. Further results have shown that sh cells have lower levels of pro-apoptotic proteins and exhibit a higher level of TGF-β targeting PAI-1 gene that activates replicative senescence. We...
|
|
|
Role of Smarca5 (Snf2h) chromation remodeling ATPase in hematopoitic development and erythropoiesis
Kokavec, Juraj ; Stopka, Tomáš (advisor) ; Divoký, Vladimír (referee) ; Kořínek, Vladimír (referee)
The Imitation Switch (ISWI) nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells (HSPCs) accumulated but their maturation towards erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S-18) second with CBP/p300 (K376Ac), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4- OHT-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis.
|
|
|
Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
|
|
|
CIrcadian regulation of miRNA and clock-controlled genes in tumorigenesis
Balounová, Kateřina ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee)
The circadian clock generates circadian rhythms, which participate on regulation of a number of signalling pathways. Disruption of the circadian regulatory mechanism is linked to a development and a progression of certain types of cancer including colorectal tumorigenesis. Progression of tumorigenesis depends on the cell cycle machinery related to cell proliferation and apoptosis. MiRNAs play a role in initiation and progression of tumorigenesis because they interfere in regulatory pathways associated with tumorigenesis. The aim of the thesis was to determinate existence of circadian rhytms in clock controlled genes (Tef, Dbp), miRNAs (miR-1-3p, miR-16-5p, miR-34a-5p, miR-155-5p, miR-192-3p) and genes of the cell cycle machinery (Ccnd1, Ccne1, Ccna1, Ccnb1) and apoptosis (Casp3, Bcl2, Bad). Further, to compare detected circadian rhythms during aging and neoplastic transformation of colon by quantitative RT-PCR. We have observed circadian expression of Tef, Dbp, Ccne1, Ccna1, Ccnb1, Casp3 and Bcl2 in young mice colon, Tef, Dbp, miR-1-3p, Ccne1, Ccna1 in old mice colon and Tef and Dbp in colorectal tumors. In summary, circadian expression of clock controlled genes varied but was maintained in mice colorectal tumors. In aging we demonstrated weakening of circadian rhythms of the genes of the cell...
|
|
|
Cytokinines and their role in plant cell division, with accent on G2/M transition
Prášilová, Jana ; Ševčíková, Hana (advisor) ; Bíšová, Kateřina (referee)
The eukaryotic cell cycle is well understood mainly in yeasts and animals. Basic regulatory mechanisms, with cyclin-dependent kinases (CDKs) playing crucial roles, are similar in all eukaryotes including plants. CDKs operate mainly at the key cell cycle checkpoints, G1/S and G2/M. Phosphorylation and dephosphorylation of CDKs by kinases and phosphatases have both negative and positive effect. Negative regulator at the G2/M transition is WEE1 kinase which phosphorylates conserved amino acid residues T14 and Y15 of CDK. Phosphatase CDC25 removes this inhibitory phosphate in yeasts and animals and forces cells into mitosis. Plant cell cycle exhibits remarkable differences. Importantly, it is controlled by phytohormones, and some key points of regulation remain obscure - a functional plant homologue of yeast CDC25 phosphatase has not been found in plants yet though Y15 inhibitory phosphorylation by WEE1 kinase blocks mitosis entry in plants as well. Thus, the regulatory mechanism of G2/M transition in plant cells is still to be found. Phytohormones play a key role, not only in the plant cell cycle, but in whole plant development. Interplay between the two groups of phytohormones: auxins and cytokinins, is crucial. Especially cytokinins significantly influence the regulation of G2/M checkpoint. It is...
|
guest ::
