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Biophysical interpretation of quantitative phase imaging of live cells generated by coherence-controlled holographic microscopy
Šuráňová, Markéta ; Rösel,, Daniel (referee) ; Vomastek, Tomáš (referee) ; Veselý, Pavel (advisor)
The dissertation thesis deals with the biophysical interpretation of quantitative phase imaging (QPI – Quantitative Phase Imaging) obtained using coherence-controlled holographic microscopy (CCHM – Coherence-Controlled Holographic Microscopy) in the Q-PHASE microscope, Telight, Brno). The theoretical part of this thesis deals with the characteristics of quantitative phase imaging, which provides non-invasive information on the activity of living cells in vitro. The main part of the work consists in elaborating a concept and verifying it of a new methodology (PAMP – Primary Assessment of Migrastatic Potential) for the first critical evaluation of drugs for expected anti-migratory/metastatic potential. The result of this method is considered the first sorting evaluation when considering specific migrastatic agents for future complex oncological treatment. PAMP evaluates the speed of cell migration, the growth of tumor cells and controls the risk of appearance of invasive phenotypes. Furthermore, the correlation microscopy method between the Q-PHASE microscope and the laser scanning confocal microscope (LSCM) is proposed to evaluate cell behavior and the occurrence of focal adhesions after drug application. The quantitative phase image obtained using the Q-PHASE microscope is compared with the quantitative phase image from the HoloMonitor (PHI AB, Sweden), on which the PAMP method has been positively verified.
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Study of the effect of migrastatics on the dynamics of tumour cell migration using a coherence-controlled holographic microscope
Muchová, Nikola ; Netíková,, Irena Štenglová (referee) ; Veselý, Pavel (advisor)
The thesis is focused on the exploitation of the coherence controlled holographic microscopy for the investigation of the influence of the supposed migrastatic drugs on the dynamics of cancer cell migration. The theoretical part briefly describes the history of holographic microscopy and the development of the holographic microscope at the Brno University of Technology in collaboration with Telight, Brno, including an insight into the design and principle of operation of the coherence-controlled holographic microscope. Next a brief description of current cancer treatments introduces new class of anti-cancer drug candidates designated migrastatics that should impair cancer cell migration and thus prevent late metastases formation. The main part of the thesis deals with the design of the experimental observation procedure and data processing using the holographic incoherent Quantitative Phase Imaging technique. The last part of the thesis is the subsequent analysis of the migrastatic effect of the selected drugs on cancer cells in vitro and the comparison of the obtained results with existing studies. Finally, the objectives with magnification 4x and 10x were evaluated and compared for starting “the Rapid Assessment of Cell Growth and Migration”.
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Study of the effect of migrastatics on the dynamics of tumour cell migration using a coherence-controlled holographic microscope
Muchová, Nikola ; Netíková,, Irena Štenglová (referee) ; Veselý, Pavel (advisor)
The thesis is focused on the exploitation of the coherence controlled holographic microscopy for the investigation of the influence of the supposed migrastatic drugs on the dynamics of cancer cell migration. The theoretical part briefly describes the history of holographic microscopy and the development of the holographic microscope at the Brno University of Technology in collaboration with Telight, Brno, including an insight into the design and principle of operation of the coherence-controlled holographic microscope. Next a brief description of current cancer treatments introduces new class of anti-cancer drug candidates designated migrastatics that should impair cancer cell migration and thus prevent late metastases formation. The main part of the thesis deals with the design of the experimental observation procedure and data processing using the holographic incoherent Quantitative Phase Imaging technique. The last part of the thesis is the subsequent analysis of the migrastatic effect of the selected drugs on cancer cells in vitro and the comparison of the obtained results with existing studies. Finally, the objectives with magnification 4x and 10x were evaluated and compared for starting “the Rapid Assessment of Cell Growth and Migration”.
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Regulation of transcription by proteins of the Early growth response and Myb families
Čermák, Vladimír ; Dvořák, Michal (advisor) ; Vomastek, Tomáš (referee) ; Elleder, Daniel (referee)
The regulation of transcription of tens of thousands of genes in a vertebrate organism is an enormously complex phenomenon which entails the participation of thousands of various regulatory proteins. The largest functional category of these regulators is accounted for by sequence-specific DNA-binding proteins known as transcription factors. Proteins of the EGR and Myb families of transcription factors are long-studied regulators of a variety of physiological processes including cellular proliferation and differentiation. The structural and physical aspects of their function have been well characterized. Their cell-type specific participation in complex gene-regulatory networks, on the other hand, is still incompletely understood and represents a major challenge in the respective research areas. Preliminary analysis of gene expression data from metastasizing PR9692 and non- metastasizing PR9692-E9 chicken sarcoma cell lines revealed that the transcription factor EGR1 is expressed at a higher level in metastasizing cells and can thus take part in the regulatory processes that underlie the differences between the two cell lines. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from PR9692...
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The analysis of cancer invasion plasticity in a 3D environment
Škarková, Aneta ; Brábek, Jan (advisor) ; Anděra, Ladislav (referee) ; Bryja, Vítězslav (referee)
iii Abstract Cells have evolved multiple mechanisms of cellular motility ranging from the migration of large cell cohorts to specialized migration of individual cells. The wide range of invasion modes has been exploited by cancer cells to their advantage, which has rendered the metastatic process so difficult to defeat. To allow for a better understanding of cancer invasion plasticity, we have employed studies on cancer cells that adopt the proteolytically active, adhesion-dependent, elongated mesenchymal invasion mode, the protease-independent, low adhesion, rounded amoeboid invasion mode, or combination of both. To study invasion plasticity directly, we have established two model systems of the mesenchymal- amoeboid transition (MAT) that allow for regulated induction of MAT in 3D in vitro environments. Using these systems, MAT was induced in HT1080 fibrosarcoma cells and the acquisition of a motile, invasive amoeboid phenotype was confirmed. We then observed the mesenchymal and amoeboid invasion strategies within 3D collagen in more detail using a digital holographic microscope. Further, HT1080 cells before and after MAT were subject to high throughput proteomic and transcriptomic studies. Comparison of gene expression and protein levels of mesenchymal and amoeboid cells disclosed an inflammatory-like...
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Cancerogenic and metastatic potential of cancer cells with non-functional CRL4 ubiquitination complex
Slámová, Monika ; Procházka, Jan (advisor) ; Grantz Šašková, Klára (referee)
Ubiquitination complex CRL4 (Cullin Ring Ligase) attracts a lot of attention due to its involvement in physiological and pathological processes, especially in the development of cancer. Cullin4 a/b proteins are reported to serve as oncoproteins in various malignancies. Due to their role in the regulation of cancer drugs targeting CRL4 have been identified, including thalidomide and its derivatives inhibiting one of the substrate receptors of the complex, the Cereblon protein. The adapter protein within the CRL4 complex - DDB1, which is involved i.a. in DNA repair, also has a role in cancer. However, the mechanism of this function has not yet been fully elucidated. The subject of this master thesis was to study the effects of elimination and suppression of CRL4 complex functions in the prostate cancer cell line LNCaP. Significantly variable changes in cell proliferation and migration have been observed if the complex functions were affected by thalidomide. The creation of the LNCaP cell line with conditionally suppressed DDB1 function was used to study tumor dynamics in a mouse model. Results show that suppression of DDB1 function has an inhibitory effect on tumor cell proliferation but increases their ability to invade adjacent tissues. Complete deletion of the DDB1 gene in the LNCaP cell line...
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The role of inflammatory signaling in cancer cell invasiveness
Šůchová, Anna-Marie ; Brábek, Jan (advisor) ; Brdička, Tomáš (referee)
Metastasizing is responsible for 90% of death in cancer patients. Metastatic tumour cells have several strategies that they use to invade surrounding tissues - they can migrate together or individually. When individual cells migrate, tumour cells adopt two different morphologies. They are either elongated and migrate using the proteolytically active mesenchymal mode, or they are rounded and migrate in the amoeboid mode. Metastatic tumour cells can switch between these modes, which complicates the development of effective migrastatics. In this work, we focused on the effect of inflammatory signalling on metastatic cell migration. We worked with cell lines of malignant human melanoma, which adopt a mixed morphology and show both amoeboid and mesenchymal phenotype during migration. Upon stimulation of melanoma human cells with interferon beta, a mesenchymal to amoeboid transition occurs. Interferon beta appears to induce amoeboid morphology by maintaining high levels of the ISGF3 complex, which is composed of the heterodimer of STAT 1 and STAT 2 proteins and the IRF9 protein. Upon blocking of Jak / Stat signalling pathway by negative regulators, human melanoma cells return to mesenchymal morphology. Key words - invasiveness, mesenchymal-ameboid transition, interferons, inflammation, migration, metastases
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The localization and transport of extracellular matrix proteases
Lyková, Dominika ; Tolde, Ondřej (advisor) ; Doubravská, Lenka (referee)
Metastasis is the main cause of death from solid cancer. The dissemination of cancer cells from a primary tumour is a very complex process that involves many steps and cells must overcome many obstacles to colonize distant organs. The tumour microenvironment influences the mode and the dynamics of invasion of cancer cells. Cancer cells have the ability to adapt to distinct environmental conditions in order to stay motile. Invasive cancer cells form membrane protrusions called invadopodia that are able to degrade extracellular matrix. The formation of invadopodia by cancer cells is interconnected to the production of matrix metalloproteases (MMPs). Metastasizing tumour cells use MMPs to break through extracellular matrix barriers and migrate in dense matrix. Both invadopodia formation and MMPs secretion is crucial for the degradation of the extracellular matrix. The most important is the membrane bound MMP-14 (MT1-MMP) and soluble MMP-2 and MMP-9. The invasive structures of tumour cells and the proteolytic enzymes in 2D environment is well described. However, a suitable model of localization and transport of MMPs and connection with invadopodia of tumour cells in 3D environment is still lacking. This diploma thesis focused on the extension of current knowledge of these key MMPs and on the...
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Invasive structures of cancer cells in 3D environment
Lyková, Dominika ; Tolde, Ondřej (advisor) ; Libusová, Lenka (referee)
The ability of cells to migrate through tissue barriers plays an important role in physiological and pathological processes including immune response or invasiveness of cancer cells. The cells generate cytoplasmic protrusions called podosomes and invadopodia, collectively known as invadosomes or podosome-type adhesions (PTA), which are thought to be the key structures of cell invasion, especially of cancer cells during metastasis. Invadosomes are F-actin rich cell-matrix contacts with capability to degrade extracellular matrix components and are observed both in normal cells (such as monocytic cells, endothelial cells and smooth muscle cells) and in cancer cells. This bachelor thesis is focused on those in cancer cells, their initiation, regulation, function and morphology in 3D and in vivo and their requirement for tumor metastasis.
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Regulation of transcription by proteins of the Early growth response and Myb families
Čermák, Vladimír ; Dvořák, Michal (advisor) ; Vomastek, Tomáš (referee) ; Elleder, Daniel (referee)
The regulation of transcription of tens of thousands of genes in a vertebrate organism is an enormously complex phenomenon which entails the participation of thousands of various regulatory proteins. The largest functional category of these regulators is accounted for by sequence-specific DNA-binding proteins known as transcription factors. Proteins of the EGR and Myb families of transcription factors are long-studied regulators of a variety of physiological processes including cellular proliferation and differentiation. The structural and physical aspects of their function have been well characterized. Their cell-type specific participation in complex gene-regulatory networks, on the other hand, is still incompletely understood and represents a major challenge in the respective research areas. Preliminary analysis of gene expression data from metastasizing PR9692 and non- metastasizing PR9692-E9 chicken sarcoma cell lines revealed that the transcription factor EGR1 is expressed at a higher level in metastasizing cells and can thus take part in the regulatory processes that underlie the differences between the two cell lines. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from PR9692...
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