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Genomic instability in patient tumors due to excesive AID activity
Vaníčková, Karolína ; Drbal, Karel (advisor) ; Macůrek, Libor (referee)
AID is a member of APOBEC family of mutational enzymes. AID generates U:G mismatches in ssDNA by deaminating cytosine to uracil. In B cells error-prone repair of these mismatches induces a mutational burden in the process of somatic hypermutation of Ig locus during affinity maturation of immunoglobulins (Ig). AID also induces double-strand breaks during Ig class switch recombination or primary Ig diversification through templated gene conversion in some vertebrate species. AID might gain tumorigenic potential in case of insufficient regulation of induction and repair processes, causing genomic instability and possibly leading to tumorigenesis. AID is induced in epithelial tissues by proinflammatory cytokines via canonical NF-B pathway. Both exogenous factors (pathogens Helicobacter pylori or HCV), endogenous factors (bile acid) or even physiological state such as ovulation are the initiating factors. Thus, AID might be the link between inflammation and carcinogenesis. AID is expressed in different stages of carcinomas, mostly during the initial oncogenic transformation. Mice with ectopic AID expression develop lung, gastric, oral and hepatic carcinomas as well as melanomas. AID also regulates epithelial-mesenchymal transition in other tumors. AID is responsible for treatment resistance in both CML...
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Optimization of Processes in Logistics with Visualization Support
Kršák, Martin ; Bidlo, Michal (referee) ; Křivka, Zbyněk (advisor)
The master thesis aims to design, implement, and compare algorithms that optimize processes in logistics, mainly in the planning phase. Heuristics and approximation genetic algorithms will find an near-optimal solution to NP-hard problem, such as the traveling salesman problem, with a delay less than several hours. The role of this algorithm is to plan an efficient route for garbage trucks that collect and distribute large-scale waste to waste yards in a specific city. The goal of the optimization is to minimize the shipping costs.
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Cardiac IKs channel: rate-dependence of the current magnitude
Kachan, Ksenia ; Čmiel, Vratislav (referee) ; Bébarová,, Markéta (advisor)
This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.
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Characterisation of \kur{Drosophila melanogaster} mutated for all genes of the Sirtuin family
PEKÁČOVÁ, Aneta
The aim of my study was to create a Drosophila line lacking the expression of all Sirtuin genes, check its developmental phenotype and characterise its response in stress conditions. The flies had bigger weight than controls, they had decreased fertility and fecundity and they developed more slowly. They showed a trend towards increased resistance to chill coma, but they did not show a significant difference in starvation or oxidative stress assay. Its effect on lifespan is being investigated.
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Genetics of psychic diseases
Krčmářová, Eliška ; Šolc, Roman (advisor) ; Dudová, Iva (referee)
Psychological diseases are very heterogeneus group of diseases, including a complicated and complex multifactorial etiology with a possibility of an important role of a genetic component. Some diaseses got discovered their clear genetic origins, for instance Rett syndrom, where the origins are caused by a mutation in gen MECP2. The origins of a majority of other psychological diseases are more complex - it is chiefly a combination of specific polymorphism of many gens with an impact of an external environment. Among this group we can range diaseases such as anxiety disorder, eating disorder, schizophrenia and bipolar disorder. Autism presents the polymorphism of few different gens, however other mentioned diseases presents mainly polymorphism of genes, connected into neurontransmission. This thesis deals with a selection of potential genetic causes of psychological diseases, illustrated by the specific examples. Keywords: psychological diseases, Rett syndrom, autism, neurotransmitters, anxiety disorder, eating disorders, schizophrenia, bipolar disorder, mutation, polymorphism
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Introduction testing of mutation V600E in the BRAF gene in clinical laboratory
VÍTKOVÁ, Markéta
Nowadays, skin cancer belongs among very frequent tumor diseases. It has been proven that sun and skin cancer are maximally related, and thus it may affect everyone, however, ´light-type´ people having blonde or ginger-coloured hair, light skin and high numbers of freckles are much more likely to be affected. Skin tumors are most frequently localized in body areas that are exposed to sunshine in the long term, such as eyelids, nose, neck, shoulders and hands. The most frequent types of skin cancer are the following: Basaliom, Melanoma and Spinaliom. The aim of my bachelor thesis was to acquire theoretical knowledge about skin cancer, especially about the Malignant Melanoma. The Malignant Melanoma is, unlike the other epitelial tumors, such as Basaliom or Spinaliom, not characterized by local destructive growth, but by the danger of an early haematogenic or lymphogenic metastasis. The Malignant Melanoma often metastasizes into lymph nodes, however, distant metastases may also appear in skin, lungs, the brain and gastro-intestinal tract, mainly in the small intestine. The presence of mutations may be understood to be the biological cause of the Malignant Melanoma. One of the most significant and most described mutations (in connection to the Malignant Melanoma) is the BRAF gene V600E mutation, appearing in position No. 1799 in the area of codon No. 600. Codon GTG>GAG mutation leads to the exchange of valin for glutamic acid. Activating the mutated BRAF protein is a result of conformation changes within the protein structure. The mutated V600E BRAF cells are not subject to apoptosis and aging, the result of which is an incontrollable replicational potential of the cells. The mutated V600E BRAF Melanoma then escapes the organism´s immunity response. The aim of the practical part of my bachelor thesis was to acquire the basic molecularly biological methods in a genetic laboratory. Mainly isolating the genom DNA obtained from a buccal swab as well as peripheral blood, preparing and carriyng out a PCR reaction, preparing agarose gel, and detecting PCR products using gel electrophoresis. Mutation V600E within the BRAF gene examination was carried out via the PCR-ARMS method. The principle of this method relies in using four different primers able to detect both mutated and unmutated DNA sequence during one experiment. The methodology was taken over from Huang et al. (2013) and subsequently adapted to the needs of the laboratory.
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Molecular mechanisms of mutagenesis and resistence in CML cell lineages
Karasová, Dominika ; Čuřík, Nikola (advisor) ; Savvulidi Vargová, Karina (referee)
Chronic myeloid leukemia is a clonal haematopoietic disease, with characteristic BCR-ABL1 fusion gene. Despite the significant improvement in patient treated with tyrosine kinase inhibitors (TKI), 20-30 % of patients develop resistance. One of the main causes of treatment failure are mutations in the BCR-ABL1 kinase domain (KD). The aim of this work was to elucidate the molecular mechanisms of resistance and mutagenesis development in CML using an in vitro CML model KCL-22. The main part of this work was focused on the identification of genes involved in DNA damage response and repair, that could play a role in the process of mutagenesis of BCR-ABL1. We used the RT2 Profiler PCR Arrays method for the group of selected genes regulating DNA damage response and repair. We identified the genes XRCC6 and PARP1 whose gene expression was significantly and specifically decreased during KD BCR-ABL1 mutagenesis. Products of these genes are involved in repairing DNA double-strand breaks through non-homologous end joining (NHEJ). During study of the KD BCR-ABL1 mutagenesis we also found that clones, which developed mutations, did not show the increased BCR-ABL1 expression in the beginning of the culture compared to the clones in which mutations have not evolved. Key words: myeloid leukemia, mutation,...
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Optimization of proces for detection of free tumor DNA in plasma and its clinical utility for colorectal cancer, lung cancer and pancreatic cancer patients
Belšánová, Barbora ; Benešová, Lucie (advisor) ; Tachezy, Ruth (referee)
In current days, examination of circulating tumor DNA (ctDNA) finds new use across different cancers. It is directed at tumor-derived short fragments of DNA present in peripheral blood of patiens (mainly in advanced stages). Due to its minimal invasivity, almost 100 % specificity and relatively high sensitivity in stage IV patients, this approch found its main potential clinical utility especially in early detection of disease relapse or progression after tumor resection (i.e. post-operative follow-up), prediction and monitoring of therapy response and estimation of prognosis. As a result of minute levels of ctDNA on a high background of other non-tumor DNA fragments present in plasma, a suitable method exhibiting highest sensitivity is the key for proper detection of this marker. The approach is predominantly based on initial identification of a mutation in tumor tissue and its subsequent detection in plasma. The present work is aimed at optimization of ctDNA isolation and method of its detection based on PCR amplification followed by heteroduplex analysis by denaturing capillary electrophoresis (DCE) to achieve highest sensitivity for detection of mutated fraction in plasma sample. I have applied the optimized protocol to examine ctDNA in three types of cancers, namely colorectal cancer (122...
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Molecular subgroups of medulloblastoma: the current consensus
Jarošová, Šárka ; Zíková, Martina (advisor) ; Nedvědová, Tereza (referee)
Medulloblastoma is the most common malignant brain tumor in children. In recent years, there has been a steady improvement in understanding of genetic and molecular heterogeneity of this tumor, that has led to better prognosis and improved targeted therapies. This thesis summarizes recent advances in molecular sub-grouping, and the associated gene mutation and copy number variations in medulloblastoma.
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