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Development of Biophysical Interpretation of Quantitative Phase Image Data
Křížová, Aneta ; Jákl, Petr (referee) ; Vomastek, Tomáš (referee) ; Chmelík, Radim (advisor)
This doctoral thesis deals with biophysical interpretation of quantitative phase imaging (QPI) gained with coherence-controlled holographic microscope (CCHM). In the first part methods evaluating information from QPI such as analysis of shape and dynamical characteristics of segmented objects as well as evaluation of the phase information itself are described. In addition, a method of dynamic phase differences (DPD) is designed to allow more detailed monitoring of cell mass translocations. All of these methods are used in biological applications. In an extensive study of various types of cell death, QPI information is compared with flow cytometry data, and preferably a combination of QPI and fluorescence microscopy is used. The DPD method is used to study mass translocations inside the cell during osmotic events. The simplified DPD method is applied to investigate the mechanism of tumor cell movement in collagen gels.
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Analysis of Microscopic Images of Cancer Cells
Vičar, Tomáš ; Matula,, Petr (referee) ; Sladoje, Natasa (referee) ; Kolář, Radim (advisor)
Tato disertační práce je zaměřena na analýzu různých forem mikroskopických obrazových dat nádorových buněk (statické 2D snímky, statické 3D obrazy, 2D časosběrné zobrazování živých buněk). Hlavní pozornost je věnována datům získaným koherencí řízeným holografickým mikroskopem, který je relativně novou modalitou schopnou kotrastních záznamů živých buněk bez barvení (label-free) a poskytuje kvantitativní informaci (kvantitativní fázové zobrazení - QPI). V práci je popsán základní postup analýzy těchto snímků a jsou vytvářeny nové metody a zdokonalovány metody pro jednotlivé kroky této analýzy. Největší část práce je věnována segmentaci buněk, kde jsou shrnuty klasické metody i metody založené na hlubokém učení. Jsou také vyvinuty nové metody vhodné právě pro QPI data. Část práce je také věnována segmentaci 3D fluorescenční jader a detekci DNA zlomů pomocí hlubokého učení. Práce se zabývá i dalším zpracování v podobě sledování buněk, extrakce příznaků a následné analýze, kde je detekována buněčná smrt a jsou vytvořeny vhodné interpretovatelné příznaky pro klasifikaci buněčné smrti na apoptickou a lytickou. Celkově tato práce přispívá k rozvoji jednotlivých kroků analýzy obrazu nádorových buněk a odráží současný pokrok v oblasti analýzy obrazu, zejména přístupy hlubokého učení, což je také demonstrováno na několika výzkumných aplikacích.
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The role of a specific miRNAs in the regulation of apoptosis during physiological and pathophysiological processes in the CNS
Kaslová, Tereza ; Romanyuk, Natalyia (advisor) ; Klassen, Ruslan (referee)
MicroRNAs are small non-coding RNAs of 20 to 24 nucleotides in size that are able to post- transcriptionally regulate gene expression by binding to mRNA. This paper focuses on how these microRNAs are generated and how they are able to regulate at the level of proteins involved in programmed cell death - apoptosis. By what mechanisms apoptosis occurs, what proteins are involved and what changes the cell undergoes are further discussed in this thesis. The precise influence of this post-transcriptional regulation is presented by using selected microRNAs that influence apoptosis during the development of the central nervous system, as well as during and as a consequence of the neurodegenerative diseases and damage that can affect it. Finally, it will also introduce the use of microRNAs as potential biomarkers, due to changes in their levels associated with various diseases, and as direct therapeutic targets. Keywords Apoptosis, microRNA, cell death, central nervous system, neurodegenerative diseases, gene expression regulation
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Molecular consequences of electron transport chain deficiency in proliferating and quiescent cells
Magalhães Novais, Silvia Carina ; Rohlena, Jakub (advisor) ; Panicucci Zíková, Alena (referee) ; Eelen, Guy (referee)
Oxidative phosphorylation (OXPHOS) system is best known for producing ATP in mitochondria. As a major metabolic hub, OXPHOS also supports other biological functions that are unrelated to ATP synthesis. Are these additional roles of OXPHOS unique to different cell states, such as to proliferation and quiescence, two of the most contrasting states of a mammalian cell? In this thesis we used genetic models of OXPHOS deficiency to show that in proliferating cells and in tumors OXPHOS primarily supports biosynthesis of nucleotides, whereas in quiescent cells OXPHOS provides oxidative stress resistance. Mechanistically, in growing tumors OXPHOS drives dihydroorotate dehydrogenase (DHODH), an enzyme crucial for de novo synthesis of pyrimidines. Complex II of OXPHOS also acts as a sensor of mitochondrial dysfunction and regulates pyrimidine synthesis for energy conservation. In contrast, in quiescent cells OXPHOS maintains autophagy by continuous low level ROS generation and together with the antioxidant defense provides constitutive oxidative stress protection. In summary, beyond ATP production OXPHOS has additional vital roles tailored to the particular requirements of quiescence and proliferation, with relevance for pathophysiology in cancer and vascular biology.
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Analysis of Microscopic Images of Cancer Cells
Vičar, Tomáš ; Matula,, Petr (referee) ; Sladoje, Natasa (referee) ; Kolář, Radim (advisor)
Tato disertační práce je zaměřena na analýzu různých forem mikroskopických obrazových dat nádorových buněk (statické 2D snímky, statické 3D obrazy, 2D časosběrné zobrazování živých buněk). Hlavní pozornost je věnována datům získaným koherencí řízeným holografickým mikroskopem, který je relativně novou modalitou schopnou kotrastních záznamů živých buněk bez barvení (label-free) a poskytuje kvantitativní informaci (kvantitativní fázové zobrazení - QPI). V práci je popsán základní postup analýzy těchto snímků a jsou vytvářeny nové metody a zdokonalovány metody pro jednotlivé kroky této analýzy. Největší část práce je věnována segmentaci buněk, kde jsou shrnuty klasické metody i metody založené na hlubokém učení. Jsou také vyvinuty nové metody vhodné právě pro QPI data. Část práce je také věnována segmentaci 3D fluorescenční jader a detekci DNA zlomů pomocí hlubokého učení. Práce se zabývá i dalším zpracování v podobě sledování buněk, extrakce příznaků a následné analýze, kde je detekována buněčná smrt a jsou vytvořeny vhodné interpretovatelné příznaky pro klasifikaci buněčné smrti na apoptickou a lytickou. Celkově tato práce přispívá k rozvoji jednotlivých kroků analýzy obrazu nádorových buněk a odráží současný pokrok v oblasti analýzy obrazu, zejména přístupy hlubokého učení, což je také demonstrováno na několika výzkumných aplikacích.
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Cell death-regulating micro RNAs and their role in the development and pathological processes.
Běhounek, Matěj ; Anděra, Ladislav (advisor) ; Seifertová, Eva (referee)
MicroRNAs are small protein non-coding, ~ 22 nucleotides long dsRNAs. Their main task is suppression of gene expression via removal/destabilization of mRNA or its targeting to degradation. These small molecules play an important role in the regulation of many cellular processes and have been found to affect expression of more than 30% of human genes. Among the processes affected or regulated by miRNAa belongs also programmed cell death. Although this work is mainly focused on the analysis and characterization a role of distinct miRNAs in the regulation of apoptotic cell death, miRNAs can also participate in the regulation of autophagic cell death or programmed necrosis. MiRNA can enhance cellular sensitivity to apoptosis by suppressing the expression of death receptor genes, but can also drive cells to apoptosis by regulating expression of anti-apoptotic protein Bcl-2. In many different organisms were already discovered and described thausends of micro RNAs anddozens of them participate in the regulation of cell death. Poor or impaired function of miRNAs and related disturbance in apoptotic signaling could lead to a number of pathological processes as tumorigenesis or disturbances in tissue development and homeostasis. . Understanding how miRNA functions in cell death and possible practical...
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Charakterizace vlivu senescence na indukci a regulaci smrti nádorových buněk
Nováková, Gita ; Anděra, Ladislav (advisor) ; Truksa, Jaroslav (referee)
4 Abstract Senescence is a specific cell state distinquished by cessation of cell division and proliferation and changes in gene expression. Normal cells enter senescence after distinct number of cell divisions or in case of an unrepairable damage. Senescence in cancer cells can be induced by subliminal stress as sublethal treatment with certain drugs. Senescent cancer cells persist in the tissue and may secrete a number of factors and nutrients affecting surrounding cells. Senescence can thus change the response of cancer cells to various apoptogens during cancer therapy. In this study, we focused on the elucidation of presumed differences between normal proliferating and senescent cancer cells in their response to selected apoptogens. Implementing bromodeoxyuridine (BrdU)-mediated replication stress in cancer cells derived from pancreatic (PANC-1) or mesothelioma (H28) tumors, we efficiently forced these cells to acquire senescent phenotype. We document that these senescent cells gain higher resistance to combined TRAIL and homoharringtonine (HHT) treatment and enhance sensitivity to other apoptogens such as FasL, camptothecin and mVES. These cells also showed increased expression of anti-apoptotic protein c-FLIP in senescent cells and changes in the expression of some Bcl-2 family proteins....
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Development of Biophysical Interpretation of Quantitative Phase Image Data
Křížová, Aneta ; Jákl, Petr (referee) ; Vomastek, Tomáš (referee) ; Chmelík, Radim (advisor)
This doctoral thesis deals with biophysical interpretation of quantitative phase imaging (QPI) gained with coherence-controlled holographic microscope (CCHM). In the first part methods evaluating information from QPI such as analysis of shape and dynamical characteristics of segmented objects as well as evaluation of the phase information itself are described. In addition, a method of dynamic phase differences (DPD) is designed to allow more detailed monitoring of cell mass translocations. All of these methods are used in biological applications. In an extensive study of various types of cell death, QPI information is compared with flow cytometry data, and preferably a combination of QPI and fluorescence microscopy is used. The DPD method is used to study mass translocations inside the cell during osmotic events. The simplified DPD method is applied to investigate the mechanism of tumor cell movement in collagen gels.
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The role of mitochondrial dynamics in cell death
Novotná, Eliška ; Rohlena, Jakub (advisor) ; Alán, Lukáš (referee)
Mitochondria form a dynamic reticulum, which fragments in apoptosis. It is assumed that proteins of mitochondrial dynamics participate in the intrinsic pathway of apoptosis and remodel mitochondrial membranes to release cytochrome c to the cytosol. The most important role in this process is played by Opa1, a protein involved in mitochondrial fusion, and by Drp1, which induces mitochondrial fission. During apoptosis, Opa1 remodels cristae in the inner mitochondrial membrane, which is crucial for effective release of cytochrome c to the cytosol. The role of Drp1 is less clear and is a subject of intense debate. Upon initiation of apoptosis Drp1 is recruited to mitochondria where it facilitates apoptotic pore formation and triggers fission. However, it appears that recruitment of Drp1 is not absolutely required for successful execution of apoptosis. In addition, mitochondrial dynamics is influenced by Bcl-2 family proteins. Recruitment of proapoptotic Bcl-2 proteins to mitochondrial outer membrane leads to inhibition of mitochondrial fusion, which enhances fragmented morphology of mitochondria. Although mitochondrial fragmentation in apoptosis is known for decades, its precise purpose remains to be elucidated.
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Molecular mechanism of MST1 kinase activation in cancer cells
Smetanová, Jitka ; Vališ, Karel (advisor) ; Grobárová, Valéria (referee)
MST1 kinase is an internal part of the Hippo signal pathway. The Hippo pathway is an evolutionary conserved regulator of tissue and organ growth and affects proliferation and apoptosis. Active MST1 kinase phosphorylates YAP and TAZ oncoproteins, which regulate the activity of transcription factors in their unphosphorylated state, including TEAD and SMAD. Furthermore active MST1 kinase phosphorylates FOXO transcription factors and induces their translocation into the cell nucleus. Finally the activation of MST1 kinase leads to cell apoptosis or halt cell cycle in G1 phase. Activation of MST1 protein depends on its auto-phosphorylation and cleavage. Recently, there are several articles which take interest in the issue of activation of MST1. Some of them describe the activation of MST1 by the effector caspase-3 and -7, on the other hand the latest articles argue that MST1 kinase itself is responsible for the activation of caspases. The molecular mechanism of MST1 kinase activation was studied in this bachelor thesis. We used the biologically active compounds GDC-0941 for the activation of MST1 protein. The activity of caspase was inhibited by specific inhibitor Z-DEVD. Using electrophoresis and Western blot it was demonstrated that MST1 is active in the case when caspases are inhibited. This fact...
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