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Cell layer cultivation in the microfluidic system
Kachan, Ksenia ; Svoboda, Ondřej (referee) ; Chmelíková, Larisa (advisor)
The theoretical part of this thesis describes the principles of cell culture in vitro and modern in vitro models for endothelial cell layer cultivation. It also describes the principles of confocal and fluorescence microscopy. The practical part is dedicated to working with cell cultures and realization of experiment with cell cultivation in microfluidic system. Cell layer was photographed using laser confocal scanning microscope Leica TCS SP8 X during the entire experiment. For processing and analysis of the obtained images, an algorithm in the software MATLAB was proposed.
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Mutual communication and relationship between tumor cells and the tumor microenvironment
Novák, Štěpán ; Szabo, Pavol (advisor) ; Krška, Zdeněk (referee) ; Grobárová, Valéria (referee)
The present dissertation addresses two main objectives. First, the interaction between four pancreatic ductal adenocarcinoma (PDAC) cell lines and conditioned media from normal/healthy fibroblasts was investigated, HF) and cancer- associated fibroblasts (CAFs) derived from PDAC (PDAC-derived CAFs, PANFs), cells derived from ascitic fluid of patients with end-stage PDAC and primary PDAC tumor. Subsequently, the genome-wide profile of PANFs was determined. The second aim was to differentiate squamous cell carcinoma (SCC) cells, surgical resection margin (MSR) cells and normal mucosa (NM) cells in patients operated for head and neck squamous cell carcinoma (HNSCC) by immunohistochemistry, using detection of tenascin (Ten), fibronectin (Fn) and galectin-1 (Gal-1), and to correlate these results with their clinical characteristics. Subsequently, whole-genome profiling of SCC, MSR and NM samples was performed based on Ten expression stratification. Results Cultivation of the four PDAC lines under the influence of conditioned media from HF and PANF was heterogeneous. After stimulation, the most aggressive behavior was obtained in the Panc-1 cell line while the PaTu-8902 line was rather inhibited by the media. The PANF transcriptome showed increased expression of some genes (e.g. IL-6, IL-8, MFGE8,...
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Spontaneous Regression of Melanoma in Pigs of the MeLiM Strain
Plánská, Daniela
Melanoma is a skin tumour arising from melanocytes - skin cells bearing pigment melanin. Melanoma belongs among immunogenic tumours, which is probably associated with a relatively high incidence of partial spontaneous regression (SR). Melanoma-bearing Libechov Minipigs (MeLiM) represent a specially bred animal model that is mostly affected by nodular melanomas, which fully regressed in about 2/3 of the affected animals. Our interest was to examine immune response (associated with melanoma cell destruction) and the role of proteins related to the extracellular matrix (reflecting tissue remodeling) during SR of MeLiM melanoma. We performed an extensive time-lapse study of skin melanomas taken from individuals of the MeLiM strain at 3, 4, 6, 8, 10, 12, 20 and 32 weeks (5-10 samples in each age category) in which we immunohistochemically detected the expression of collagen IV, laminin, fibronectin, tenascin C, as well as MMP-2 and we monitored the proportion of basic immune subpopulations in blood and tumour by flow cytometry. The higher expression of collagen IV, laminin and MMP-2 positively correlated with the appearance of melanoma cells. The expression of collagen IV and laminin indicates a possible survival of tumour cells due to the interaction with these proteins, the presence of MMP-2 in these...
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Spontaneous Regression of Melanoma in Pigs of the MeLiM Strain
Plánská, Daniela ; Horák, Vratislav (advisor) ; Smetana, Karel (referee) ; Bartůňková, Jiřina (referee)
Melanoma is a skin tumour arising from melanocytes - skin cells bearing pigment melanin. Melanoma belongs among immunogenic tumours, which is probably associated with a relatively high incidence of partial spontaneous regression (SR). Melanoma-bearing Libechov Minipigs (MeLiM) represent a specially bred animal model that is mostly affected by nodular melanomas, which fully regressed in about 2/3 of the affected animals. Our interest was to examine immune response (associated with melanoma cell destruction) and the role of proteins related to the extracellular matrix (reflecting tissue remodeling) during SR of MeLiM melanoma. We performed an extensive time-lapse study of skin melanomas taken from individuals of the MeLiM strain at 3, 4, 6, 8, 10, 12, 20 and 32 weeks (5-10 samples in each age category) in which we immunohistochemically detected the expression of collagen IV, laminin, fibronectin, tenascin C, as well as MMP-2 and we monitored the proportion of basic immune subpopulations in blood and tumour by flow cytometry. The higher expression of collagen IV, laminin and MMP-2 positively correlated with the appearance of melanoma cells. The expression of collagen IV and laminin indicates a possible survival of tumour cells due to the interaction with these proteins, the presence of MMP-2 in these...
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Influence of transcription regulatory elemets on pre-mRNA splicing
Volek, Martin ; Staněk, David (advisor) ; Malík, Radek (referee)
In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...
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Influence of transcription regulatory elemets on pre-mRNA splicing
Volek, Martin ; Staněk, David (advisor) ; Malík, Radek (referee)
In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...
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Cell layer cultivation in the microfluidic system
Kachan, Ksenia ; Svoboda, Ondřej (referee) ; Chmelíková, Larisa (advisor)
The theoretical part of this thesis describes the principles of cell culture in vitro and modern in vitro models for endothelial cell layer cultivation. It also describes the principles of confocal and fluorescence microscopy. The practical part is dedicated to working with cell cultures and realization of experiment with cell cultivation in microfluidic system. Cell layer was photographed using laser confocal scanning microscope Leica TCS SP8 X during the entire experiment. For processing and analysis of the obtained images, an algorithm in the software MATLAB was proposed.
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