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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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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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Role apoptózy a nekrózy makrofágů mléčné žlázy skotu
Klimešová, Monika
This bachelor thesis is aimed to the role of apoptosis and necrosis of bovine mamma-ry gland macrophages provides an overview of macrophages that are part of nonspecific immunity. It describes their development from pluripotent bone marrow cells via monocytes to macrophages and their importance in the bovine mammary gland, which inc-ludes the phagocytosis of microorganisms and dead cells, the production of cytokines, and the stimulation of cells that regulate inflammatory responses. In the next part the bachelor thesis is focused on individual types of cell death (apoptosis and necrosis). They describe their structural, biochemical and genetic features and subsequent events that take place after cell death, ie. the removal of dead cells from the organism. The effects of cell death on the surrounding tissue are also of no signifi-cance. At the end of the work are summarized the basic differences between apoptosis and necrosis and their roles during the inflammation of the bovine mammary gland.
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Interakce makrofágů a buněk rakovinné prostatické linie PC-3
Mazalová, Lenka
This dissertation on the theme Interaction of macrophages and the prostate cancer cell line PC-3 was focused on the initial mapping of interactions between human mac-rophages and the prostate cancer cell line PC-3. Microscopic and molecular-genetic methods have been used for analysis. The structure and ultrastructure of macrophages and PC-3 cells have been de-scribed. The description of cell morphology in interaction with each other has been de-voted to processes associated with adherence of macrophages on PC-3, eferocytosis and phagocytosis. Cultivation of PC-3 with plumbagine showed an increase of apoptotic cells. Cultivation of macrophages with PC-3 supernatant has shown that solubiling fac-tors in supernatant may have an effect on inducing cell death in macrophages. In our study, the supernatant did not affect the production of TNF alfa, IL-12, IL-10 or IL-6 by macrophages. A time lapse video was created to show physical interactions of macro-phages and invasive cell. All obtained results show that there are interactions between the cancer cells and immune system cells that have not yet been discovered.
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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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