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Emergent properties of the G1/S network
Dražková, Jana ; Tomášek, Petr (referee) ; Palumbo,, Pasquale (advisor)
Tato práce se zabývá buněčným cyklem kvasinky Saccgaromyces cerevisiae. Oblastí našeho zájmu je přechod mezi G1 a S fází, kde je naším cílem identifikovat velikosti buňky v době počátku DNA replikace. Nejprve se věnujeme nedávno publikovanému matematickému modelu, který popisuje mechanismy vedoucí k S fázi. Práce poskytuje detailní popis tohoto modelu, stejně jako časový průběh některých důležitých proteinů či jejich sloučenin. Dále se zabýváme pravděpodobnostním modelem aktivace replikačních počátků DNA. Nově uvažujeme vliv šíření DNA replikace mezi sousedícími počátky a analyzujeme jeho důsledky. Poskytujeme také senzitivní analýzu kritické velikosti buňky vzhledem ke konstantám popisujícím dynamiku reakcí v modelu G1/S přechodu.
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Coherence-controlled holographic microscope in cell's life cycle research
Bartoníček, Jan ; Chmelík, Radim (referee) ; Uhlířová, Hana (advisor)
The subject of the bachelor thesis is live-cell imaging in a transmitted-light holographic microscope which was designed at the Institute of Physical Engineering BUT and comparing this imaging method with the phase-contrast microscopy. The first part is dedicated to a basic description of used imaging techniques and a cell biology. A description of an experiment preparation follows. In the part dedicated to a data analysis the method of dynamic phase differences is described and the method of growth monitoring is proposed. Both methods were used for the analysis of experiments which are described in the last part of this work. Experiments were focused on acquiring time-lapse data of a cell’s cycle and particularly the mitosis.
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Modelling of Cell Colony Dynamics
Bělehrádek, Stanislav ; Škutková, Helena (referee) ; Sedlář, Karel (advisor)
The content of the thesis is a description of intracellular processes responsible for cell cycle regulation and reactions of cells to external and internal stimuli. Thoroughly described are important signaling pathways with appropriate methods, which can be used to simulate them in silico. From these cellular processes, a cell cycle model is created and implemented in a tool programmed in C ++ with OpenGL used for visualization. The model is then tested for various cell processes including HeLa cells growth. Finally, the results are compared with the behavior of living cells.
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The effect of synthetic modified mRNAs induced proliferation on pancreatic beta cells
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a chronic disease caused by the loss of pancreatic beta cells due to autoimmune destruction or increased apoptosis. Beta-cell deficiency results in reduced insulin production, which plays an important role in glucose metabolism. The number of beta-cells in the body is one of the main factors that influence the development of this chronic disease. Therefore, it is necessary to find a way by which the number of beta-cells of the organism can be increased and thus the insulin production can be restored in a natural way without any need for the use of insulin infusions. However, the ability of beta-cells to divide decreases with age and is virtually nil in adulthood. The study of the cell cycle, especially the early and late cyclins and cyclin-dependent kinases, which act as cell cycle regulators, thus appears to be a promising way to restore natural insulin-producing tissues. In order to increase the number of beta cells entering the cell cycle, we focused on studying the effect of in vitro transcribed (IVT) mRNAs, encoding cyclins type D and cyclin dependent kinases 4 and 6 on stimulating cell division of isolated beta-cells. We found that transfection IVT mRNAs for type D cyclins in combination with cyclin-dependent kinases 4 and 6 significantly increased the proliferation of beta-cells...
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The foundation of maternal factors in sturgeon: from oocyte to embryo
POCHERNIAIEVA, Kseniia Kostyantynivna
The effective application of embryo engineering to endangered sturgeon species requires fundamental knowledge of its embryonic development and information about structure and characteristics of sturgeon oocyte itself. To reveal intracellular geometry, mechanisms of maternal determinants organization and its later reorganization and morphogenetic aspects we used several techniques such as qPCR tomography, inhibition of transcription and visualization of nucleuos. The qPCR tomography was discovered as reliable technique to determine the role of the genes detected in the animal and vegetal hemispheres of the sturgeon oocyte, and to identify profiles of these genes during early developmental stages of sturgeon embryos. The 12 selected maternal genes were investigated. Two groups of transcriptomes categorized as animal or vegetal with evident gradient profile were identified. The primarily germplasm markers such as dnd, vasa, ddx25 were localized toward the extreme vegetal pole. This finding reveals localization of primordial germ cells in the body plan of the sturgeon oocyte. Another aspect of applying such technique was comparative analysis of RNA profiles in the oocyte of distantly-related species Xenopus laevis and Acipenser ruthenus. We found clear similarity in the localization of mRNA molecules in Acipenser ruthenus and Xenopus laevis, which revealed significant aspects of early development that have been conserved during evolution. Such similarities in expression profiles of distantly related species indicate that their ancestors could have arisen from more closely related lineages. The maternal to zygotic transition (MZT) is a separate developmental period that begins with the elimination of maternal transcripts, continues through the production of zygotic transcripts, and concludes with the first major morphological requirement for zygotic transcripts in embryo development.The alpha-Amanitin as transcript inhibition factor was used to determine the zygotic genome switch in sterlet embryos. The transition in sterlet was observed after the tenth cleavage during late blastula, when blastomeres in the animal pole are surpassed 1000 cells. Mid-blastula transition (MBT) in early embryogenesis can be defined as a time point characterized by cell cycle lengthening, loss of synchrony and acquisition of cell motility. We opted to use oocytes of crosses sterlet A. ruthenus and Russian sturgeon Acipenser gueldenstaedtii, since the hybridization results in increased DNA content in their hybrid offspring compared to parental species A. ruthenus making the embryo a useful model for investigation of changes in the timing of early development. Nucleous vizualization by 4'-6-diaminido-2-phenylindole (DAPI) staining showed that cells divided synchronously at a constant rate until MBT at the ninth cell cycle in control sterlet embryos that corresponds to 1000 cell stage (13 hpf). The sterlet x Russian sturgeon hybrid embryos showed transition from synchronous to asynchronous division at the eighth cell cycle which is the 512 cells stage (12 hpf). In both sterlet and hybrid embryos, the transition occurred within 1 h. Thus, our study confirmed hypothesis the MBT in sturgeon is governed by the ratio of nucleus to cytoplasm, which can be controlled using hybridization, induction of polyspermy or injecting plasmid DNA Embryos of sturgeon injected with alpha-Amanitin also showed cell cycle kinetics similar to controls, with no delay or malformation during cleavage, which most likely indicates that MBT in the sturgeon proceeds independently of onset of zygotic transcripts production. The results and observations presented in this study demonstrate the path from an egg to a developed embryo, which are the basis for improving the production methods and preservation of sturgeons listed in the IUCN Red List, and which is equally important, provide the fundamental knowledge about the nature of sturgeons.
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The role of truncated PPM1D/Wip1 phosphatase in cancer
Martiníková, Andra-Stefania ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Mistrík, Martin (referee)
When encountering damage, the cells activate the DNA Damage Response (DDR) pathway and stop the cell cycle until the DNA is repaired. PPM1D/WIP1 phosphatase resumes the cell cycle after the damage has been repaired, by directly dephosphorylating DNA damage markers. The DDR pathway prevents genome instability or cancer development. Mutations in the Ppm1d gene encoding PPM1D result in an overstable and truncated protein observed both in cancer patients and in cancer cell lines. In this thesis, we used an "in-house" transgenic mouse model in which mutations in the exon 6 of the Ppm1d gene resulted in a truncated PPM1D protein. First, we observed high PPM1D levels and impaired DDR to gamma ionizing radiation (IR) in the mouse thymi having truncated PPM1D (Ppm1dT/+ ). We then bred the Ppm1dT/+ mice with the Trp53+/- heterozygote knock-out mice which are prone to thymic lymphoma. The Ppm1dT/+ Trp53+/- double-mutants had a higher frequency of developing IR-induced T-cell lymphomas, compared to the single Trp53+/- mutants. Moreover, truncated PPM1D leads to a defective cell cycle checkpoint activation in human non-transformed RPE cells (RPE1), which then proliferate despite the presence of DNA damage. RPE1 cells also display increased proliferation after replication stress. RPE1 or U2OS cells with...
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Characterization of pancreatic beta cells after their in vitro proliferation induced by synthetic modified mRNA
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Černá, Věra (referee)
The origin and development of type I. and II. diabetes mellitus is directly related to homeostasis of proliferation and apoptosis of pancreatic β-cells. Any imbalance that leads to a decrease in the number of β-cells consequently increases the pro- bability of developing this disease. Patients suffering from diabetes mellitus are de- pendent on partial or complete exogenous insulin replacement, as their pancreas is unable to meet the body's insulin needs. Therefore a need for restoration of normal β-cell mass in diabetic patients leads to the attempts to develop new therapeutic approaches that could expand remaining β-cells of the organism and restore phys- iological insulin production. A major obstacle in this regard is a low sensitivity of terminally differentiated β-cells to mitogenic stimuli that could induce the entry of β-cells into the cell cycle. Activation of β-cell proliferation is associated with the G0/G1/S cell cycle transi- tion, which is under the control of retinoblastoma protein (RB). In order to activate cell cycle entry RB must be phosphorylated. RB phosphorylation is provided by specific cell cycle regulators, particularly cyclin-dependent kinases 4 and 6, which associate with family D cyclins. In accordance with the aim of this Diploma thesis, the effect of these cell cycle...
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Role of RecQ helicases in maintenance of genomic stability during mitosis
Černoch, Marek ; Janščák, Pavel (advisor) ; Půta, František (referee)
Helicases are proteins capable of unwinding nucleic acids, their malfunction can be dangerous for genome stability of the cell. Five RecQ-family helicases identified in human cells participate in many cellular events during the whole cell cycle, including mitosis, and therefore are very important for correct functioning. The mutations in RecQ helicases can cause them to malfunction and seriously damage various cell processes, for example DNA replication, DNA damage control or sister chromatids separation. The mutations can also lead to dangerous syndromes, with the hallmark symptom of increased risk of cancer.
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Regulation of cell cycle in Bacillus subtilis.
Zelenka, Tomáš ; Lichá, Irena (advisor) ; Harant, Karel (referee)
2 Abstract Relations between several events running in bacterial cell during cell cycle were the subject of many studies during last years. More advanced techniques showed, that bacterial cell life has much more variable factors, than we supposed before. Relatively recent researches managed to reveal function and in few events molecular principle of several mechanisms coordinating those events such as progression of replication and its initiation, segregation of newly replicated chromosomes and after all synchronization of complex cell division machinery. Furthermore it showed variability of those events during changing living conditions of the cell. Keywords: Cell cycle, regulation, initiation, replication, segregation of chromosome, cytokinesis, Bacillus subtilis
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Analysis of embryotoxic effect of hydrocortisone using chick embryotoxicity screening test (CHEST).
Janíková, Michaela ; Peterka, Miroslav (advisor) ; Hovořáková, Mária (referee)
Cleft lip is one of the most common human birth deffects. Its etiopathogenesis is multifactorial and many aspects of its occurrence remain unknown in the fields of both genetics and teratology. One of the set of known negative external factors causing cleft lip is chemical hydrocortisone. Its effect on cell proliferation is highly heterogeneous and depends on attributes of a specific cell population. In this work we studied the cleft beak origin after the hydrocortisone treatment on the basis of Chick Embryotoxicity Screening Test (CHEST). Our main aim was to detect cell cycle changes in the chick frontonasal process after hydrocortisone injection via flow cytometry analysis. Hydrocortisone caused S phase arrest within a minor subpopulation of highly granular cells with specific cell cycle. This sensitive subpopulation was localized in the areas of previously defined proliferative centers within the frontonasal process using immunohistochemistry of frozen sections. Quantitative analysis of cells in these areas revealed significant decrease of M phase portion in the hydrocortisone treated samples in comparison with the control samples. The TUNEL staining of histological sections was used to determine the apoptotic rate in the frontonasal process. The comparison between the control and the...
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