|
|
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...
|
|
|
New molecular mechanisms involved in cell cycle control
Aquino, Cecilia ; Macůrek, Libor (advisor) ; Anger, Martin (referee) ; Braun, Marcus (referee)
Cecilia Aquino Perez, M. Sc. Doctoral thesis abstract In this doctoral, thesis we aimed to find and study novel mechanisms regulating cell cycle phase transitions in non-stressed conditions and in context of the cell response to various types of stress. First, we focused on studying Polo-like kinase 3 that has previously been implicated in activation of the cell cycle checkpoint after DNA damage. For this, we employed CRISPR/Cas9- mediated gene editing to knock-out PLK3 in RPE cells while in parallel performing RNA interference assays and submitting the cells to different types of stress. The main observation was that in both systems PLK3 was disposable for response to DNA damage, hypoxia and osmotic stress. Through mass spectrometry analysis of purified EGFP-PLK3 we identified PP6 and its regulatory subunits PPP6R1 and PPP6R3 as novel PLK3 interactors. We observed that PLK3 is phosphorylated in its conserved residue Thr-219 and that PP6 depletion boosted PLK3 phosphorylation status but did not affect its kinase activity. The possible regulation of PLK3 trough PP6 is interesting and its biological relevance will be addressed by future research. Next, we performed a transcriptomic analysis in human RPE-FUCCI cells aiming to identify new regulators of the cell cycle. We selected Family with sequence...
|
|
|
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.
|
|
|
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
|
|
|
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...
|
|
|
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.
|
|
|
Transcription factors CSL and their role in the yeast Schizosaccharomyces pombe
Oravcová, Martina ; Převorovský, Martin (advisor) ; Heidingsfeld, Olga (referee) ; Krásný, Libor (referee)
Proteins of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) act as effectors of the Notch signalling pathway in metazoan organisms. They function as repressors or activators of gene transcription in the framework of this pathway and influence many developmental processes. Metazoan CSL proteins can regulate gene expression Notch-independently as well. Notch-independent functions of CSL proteins might be evolutionarily ancestral and in cells and organisms may be important equally as Notch-dependent functions. Presence of CSL proteins was identified in several fungal species, organisms lacking the Notch signalling pathway components and most of known metazoan interacting partners of CSL proteins. CSL paralogs of the fission yeast Schizosaccharomyces pombe, cbf11 and cbf12, are non-essential genes encoding proteins localized in the nucleus of the cell. They exert antagonistic effects on regulation of processes like coordination of nuclear and cellular division and cell cycle progression, ploidy maintenance, cell adhesion and other. In this study, we have proved that both CSL paralogs are able to sequence-specifically bind the CSL-response element DNA in vitro and Cbf11 in vivo as well. Both proteins could activate gene expression in vivo and perform the function of transcription factors....
|
|
|
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...
|
|
|
Connections between intermediary metabolism and acetylation of histones
Zach, Róbert ; Převorovský, Martin (advisor) ; Bieberstein, Nicole (referee)
Acetylation of histone proteins affects chromatin structure and functions as a coactivating signal for transcription. Acetylation of histone lysine residues is mediated by histone acetyltransferases, which utilize molecule of Ac-CoA as a donor of acetyl group. Ac-CoA is located in the centre of intermediary carbon metabolism, where it fuels citric acid cycle and fatty acid synthesis. Level of intracellular Ac-CoA fluctuates in response to changes in availability of utilizable carbon sources and metabolic activity of the cell. Since changes in intracellular concentration of Ac-CoA positively correlate with histone acetylation level, Ac-CoA might contribute to transcriptional modulation in response to nutritional stress. Moreover, Ac-CoA takes part in process of differentiation and seems to be important for cell cycle regulation. Key words: Ac-CoA, histone acetylation, nutrition, intermediary metabolism, regulation of transcription, cell cycle, glucose
|
|
| |
guest ::
