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Gene sequencing in patients with cancer anamnesis
MARKOVÁ, Iveta
Cancer is the second most common cause of death in the Czech Republic, of which 5-10% are occupied by hereditary cancer syndromes. They are caused by a congenital - hereditary mutation in one of the alleles of the genes, when after the second random intervention in the other allele hereditary cancer develops. It is important to distinguish between hereditary and sporadic carcinomas due to the high risk of inheritance of mutated alleles in the family. The indication may be, for example, the onset of the disease at a young age or the recurrence of the cancer in the family In my work I focused on the analysis and evaluation of data obtained by Sanger sequencing. The aim was to find mutations in the genes mentioned below and to evaluate their pathogenicity by comparison with databases. In the theoretical part of the bachelor thesis I deal with cancer in general and hereditary cancer. I specify the hereditary breast and ovarian cancer syndrome, including genes, that may cause this syndrome - BRCA1, BRCA2, TP53, PTEN, ATM, PALB2, I also deal with Lynch syndrome and the MMR gene system. Last but not least, I describe a familial adenomatous polyposis associated with the APC gene. In the research part I focused on the examination of selected areas of 18 anonymized samples in the gene PALB2 - exon 13 and in the gene BRCA2 - exon 10/4 and exon 11/12. Using the PCR method, I prepared the samples for Sanger sequencing, which then took place in GenSeq s.r.o. In the last part of my work I deal with the analysis and evaluation of the results using the BioEdit program and the NCBI database. I found a mutation in 5 samples - in 4 it was a deletion of one nucleotide with a conflicting interpretation of pathogenicity, the last mutation was pathogenic - causes hereditary breast and ovarian cancer syndrome, it was a nucleotide duplication.
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The role of WT1 gene and other molecular biological abnormalities in testicular germ cell tumors
Bakardjieva - Mihaylova, Violeta ; Boublíková, Ludmila (advisor) ; Chovanec, Michal (referee) ; Kiss, Igor (referee)
Testicular germinal tumors (TGCT) are relatively rare solid tumors in adults. Even so, they affect more than 700 men a year in the Czech Republic, mostly young patients aged 18- 45 years. A large number of patients are curable by a combination of surgery and chemotherapy, yet about 50 men a year in the Czech Republic succumb to this tumor, in the vast majority of cases due to the development of resistance to chemotherapy containing cisplatin. The rare occurrence and high curability are probably the cause of infrequent molecular and clinical studies carried out in these tumors, and our understanding of the biological processes leading to primary tumor development and the development of cisplatin resistance (CDDP) is still limited. At present, no specific molecular markers that could be used as prognostic or predictive factors and improve patient stratification or treatment tailoring are available in TGCT management. In this work, we studied the molecular-genetic background of TGCT development and CDDP resistance at several levels. To comprehensively study the development of cisplatin resistance, we prepared and analyzed CDDP-exposed TGCT cell lines. Long-term exposure to CDDP increased resistance 10-fold in the NCCIT cell line, while no significant resistance was achieved with Tera-2. The...
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Detection of genetic modifications associated with pancreatic adenocarcinoma
Urbančoková, Alexandra ; Smolková, Katarína (advisor) ; Alán, Lukáš (referee)
Pancreatic ductal adenocarcinoma (PDAC) is a serious oncological disease, which ranks among cancers with the worst prognosis and a three-year life expectancy of 10%. Ex-vivo organoid cultures derived from cancer tissue are popular and reliable research models, which reflect the morphology and histology of the original tissue. Genetic background leading to development PDAC confer typical alterations in genes KRAS, TP53, SMAD4 a CDKN2A. The aim of this thesis was to determine mutations present in organoid cultures derived from human PDAC. We used online genomic databases to estimate specific mutations typical for PDAC. Based on that research we designed protocols for the detection of PDAC genetic alterations and optimized those methods using cultured cells. We applied the approach on primary ex- vivo organoids derived from surgical cancer specimens and detected mutations in KRAS, TP53, SMAD4, or deletion of exons in CDKN2A. Alternatively, we proposed improvements for the analysis of genetic background in PDAC. The data obtained within this thesis will be used for the stratification of metabolomics and biochemical analyses further in the project.
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Vývoj inovativního biosenzoru pro elektrochemickou detekci tumor supresorového genu TP53
Navrátil, Jiří
The theoretical part of the diploma thesis deals with tumour diseases and a general description of tumour suppressor genes. Special attention is paid to the TP53 gene and its influence on the functioning of the organism, including its production of the p53 protein. Based on an analysis and synthesis of findings from specialised sources, the current methods of detecting the aforesaid gene, in particular immunological and elec-trochemical, were described. The last chapter of the theoretical part is devoted to bio-sensors and their classification, with a special focus on electrochemical biosensors. The practical part involves the construction and optimisation of the biosensor, from the cleaning of electrodes, the deposition of gold nanoparticles, the ssDNA bond, the blocking of free binding points and a synthesis of the complementary chain to the resulting measurement and quality testing. This part also presents the concept of an innovative biosensor that offers an easier and more reliable alternative for the detec-tion of the TP53 gene. The last chapter of this part analyses and comments on the aforesaid results.
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The role of WT1 gene and other molecular biological abnormalities in testicular germ cell tumors
Bakardjieva - Mihaylova, Violeta ; Boublíková, Ludmila (advisor) ; Chovanec, Michal (referee) ; Kiss, Igor (referee)
Testicular germinal tumors (TGCT) are relatively rare solid tumors in adults. Even so, they affect more than 700 men a year in the Czech Republic, mostly young patients aged 18- 45 years. A large number of patients are curable by a combination of surgery and chemotherapy, yet about 50 men a year in the Czech Republic succumb to this tumor, in the vast majority of cases due to the development of resistance to chemotherapy containing cisplatin. The rare occurrence and high curability are probably the cause of infrequent molecular and clinical studies carried out in these tumors, and our understanding of the biological processes leading to primary tumor development and the development of cisplatin resistance (CDDP) is still limited. At present, no specific molecular markers that could be used as prognostic or predictive factors and improve patient stratification or treatment tailoring are available in TGCT management. In this work, we studied the molecular-genetic background of TGCT development and CDDP resistance at several levels. To comprehensively study the development of cisplatin resistance, we prepared and analyzed CDDP-exposed TGCT cell lines. Long-term exposure to CDDP increased resistance 10-fold in the NCCIT cell line, while no significant resistance was achieved with Tera-2. The...
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Vztah mezi genetickými polymorfismy DNA reparačních genů a jejich expresí u zdravé populace (s výhledem na stanovení u onkologických pacientů).
Hánová, Monika ; Vodička, Pavel (advisor) ; Bencko, Vladimír (referee) ; Černá, Marie (referee)
DNA damage response is a complex system responsible for protection of a cell against internal and external DNA damaging agents and in maintaining genome integrity. Many of genes participating in DNA damage response pathways are polymorphic. Genetic polymorphisms in coding and regulatory regions may have impact on the function of proteins encoded by the genes. Phenotypic effect of single nucleotide polymorphisms (SNPs) is subject of investigation in connection with the ability of a cell to manage genotoxic stress and subsequently, in relation to cancer susceptibility. The aim of this thesis was to evaluate the association between SNPs in DNA repair genes (hOGG1, XRCC1, XPC) and cell cycle genes (TP53, p21CDKN1A , BCL2 and BAX) and their mRNA expression in peripheral blood lymphocytes from individuals occupationally exposed to styrene and control individuals. The aim was extended to analyses of relationships between mRNA expression levels of the above-mentioned genes and markers of exposure to styrene (concentration of styrene in blood and in air), markers of DNA damage (single strand breaks - SSBs, and endonuclease III specific sites - Endo III sites) and the base excision repair (BER) capacity, by means of γ-irradiation specific DNA repair rates and oxidative repair. Study on the group of healthy...
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Autoimmune and lymphoproliferative diseases: associations and common mechanisms
Dobiášová, Alena ; Daňková, Pavlína (advisor) ; Hušáková, Markéta (referee)
Autoimmune and lymphoproliferative diseases share some etiologic mechanisms. The origin of the diseases is complicated process that involves an accumulation of hereditary and somatic mutations in a hematopoetic cell, which thanks to changed activity overcomes different growth and survival control checkpoints. Such mutations are for example those located in genes coding for transcription factors, apoptotic signaling molecules, costimulatory molecules and secreted exctracellular molecules. All these molecules influence the balance between survival and programmed cell death. Their dysregulated expression enables the cell to overcome defensive mechanisms of the immune system. Therefore, autoimmune and malignant cells are able to survive though, under usual circumstances, they would be selected. The main aim of this work is to shed the light on the influence of the dysregulated expression of the particular molecules on the origin of autoimmune and lymphoproliferative diseases. Key words: autoimmune ilnesess, lymphoproliferative diseases, etiology, AIRE, c-MYC, TP53, FOXP3, Fas, PTEN, Bim, CTLA-4, CD5, CD30, CD40/CD40L, BAFF, α-taxilin, IL- 10.
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