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Development of methodology for detection of circulating nucleic acids in cancer patients
Kobrle, Lukáš ; Souček, Pavel (advisor) ; Valihrach, Lukáš (referee)
Cancer diagnosis represents one of the most common causes of death in the world, also linked to an ever-increasing incidence of even some more difficult-to-treat variants. One of the most common types of cancer in the world is colorectal cancer, which ranks second in terms of mortality. For these reasons, various methods have been developed for an early diagnosis of the disease. One of the modern and non-invasive methods is the so-called liquid biopsy, based on the detection of circulating DNA and RNA from the blood of patients. This method is also suitable for choosing a therapy, monitoring its effectiveness, and observing the relapse of the disease. In this work, a methodical workflow was created for the monitoring of circulating nucleic acids for its further use in a cohort of patients with colorectal cancer. Various commercial kits were used for the isolation of circulating nucleic acids from plasma of healthy controls, among which the best ones, in terms of quality and quantity, was chosen. The threshold for hemolysis detection by qPCR was also determined using samples from healthy donors. Everything was then applied to RNA samples extracted from plasma of cancer patients, from whichna library of short RNA molecules was prepared for subsequent sequencing. After processing the results, the...
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ADP-ribosylation in ARH3-deficient cells and its impact on cellular functions
Kuttichová, Barbora ; Hanzlíková, Hana (advisor) ; Valihrach, Lukáš (referee)
ADP-ribosylation is a crucial post-translational modification that regulates various cellular processes, including DNA repair. It is catalysed by poly-ADP-ribose polymerases (PARPs) and involves the transfer of ADP-ribose moieties from the redox cofactor NAD+ to proteins, including histones. To maintain cellular homeostasis, ADP-ribose chains need to be rapidly degraded by ADP-ribosyl glycohydrolases. While poly-ADP-ribose glycohydrolase (PARG) is highly efficient, it cannot cleave the terminal ADP-ribose moiety. For the removal of the terminal mono-ADP-ribose, two glycohydrolases, TARG1 and ARH3, are involved. This removal process is necessary because it enables DNA repair factors to access the site of DNA damage. The primary goal of this thesis is to characterise cells derived from patients with homozygous ARH3 mutations and to develop appropriate tools to improve our understanding of the molecular mechanism by which ARH3 mutations affect ADP-ribosylation and how it contributes to the onset of the associated neurological disease. To achieve this, I measured the levels of ARH3 protein and detected increased mono-ADP-ribosylation in ARH3-mutated patient-derived fibroblasts. Furthermore, I assessed the sensitivity of these cells to different PARP inhibitors, which hold potential for the therapeutic...
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Practical aspects of single-cell RT-qPCR analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Pavlínková, Gabriela (referee)
Recent breakthroughs in the RNA quantification of single cells are rapidly transforming the view on biology and medicine. Flexibility and sensitivity of reverse transcription quantitative PCR (RT-qPCR) make it an ideal method for quantification of single-cell material, but its limits had not been yet fully explored. In this thesis, various factors influencing RT-qPCR performance in single-cell application have been assessed, including conditions of sample collection and processing, importance of quality control, performance of reverse transcription, preamplification and role of qPCR assays. We showed that prolonged time for single cell collection as well as repeated freeze-thaw cycles had negligible effect on RT-qPCR data quality. Direct lysis routinely applied for RNA extraction from single cells may be scaled up to 256 cells. The comprehensive comparison of 11 reverse transcriptases in low RNA input conditions identified 2 best-performing enzymes. Decrease in preamplification volume as well as poor primer design resulted in the loss of sensitivity. Finally, the established workflow has been applied to profile gene expression of astrocytes in mouse model of amyotrophic lateral sclerosis (ALS) identifying important components of ALS-induced changes to astrocyte transcriptome. Altogether, the thesis...
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Molecular mechanisms in transcriptional regulation of neurosensory development
Filová, Iva ; Pavlínková, Gabriela (advisor) ; Tureček, Rostislav (referee) ; Valihrach, Lukáš (referee)
The development, maturation, and viability of inner ear neurosensory cells depend on the spatiotemporal expression of multiple transcriptional factors. Based on three mouse models [Tg(Pax2-Isl1)], Sox2CKO, and Neurod1CKO, this thesis investigates the function of three transcriptional factors ISL1, SOX2 and NEUROD1 in the neurosensory development of the inner ear. The mouse mutant [Tg(Pax2-Isl1)] carries transgenic sequence containing Isl1 gene under Pax2 regulatory sequence in its genome. ISL1 ectopic expression driven by Pax2 regulatory sequence resulted in the enlarged cochleovestibular ganglion and accelerated neurite extension in [Tg(Pax2- Isl1)] embryos. In adult mutants, we detected an early onset of age-related hearing loss correlating with the worsening function of outer hair cells. These changes were associated with the loss of medial olivocochlear efferent neuron fibers innervating outer hair cells. For the first time, we showed that the age-related hearing loss (presbyacusis) might be caused by efferent innervation defects besides hair cell loss and spiral ganglion degeneration. In addition to presbyacusis, [Tg(Pax2-Isl1)] mice suffered from hyperactivity that was diminished by the administration of picrotoxin - channel blocker for GABA receptor chloride channels. This indicates that...
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The importance and role of reverse transcriptases in gene expression analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Španielová, Hana (referee)
The continuously advancing field of gene expression analysis enables the evaluation of even the slightest changes that occur in the cell transcriptome. In order to ensure accuracy of the observed biological variances, it is fundamentally important to be aware of the possible biases introduced during sample processing. In gene expression research, the methods of reverse transcription−quantitative PCR (RT−qPCR) and RNA- Sequencing (RNA-Seq) are often the primary choice, mostly because of their high precision and reproducibility. Since these both methods require DNA template, they are coupled with the same initial step - reverse transcription (RT), a reaction producing DNA complementary to its RNA template. It is well known that RT introduces bias. As a result, it is therefore of importance to thoroughly evaluate the effects of these biases. One such annotated source of artifacts is the reverse transcriptase (RTase) itself. However, it has been shown that the enzyme does not account for most of the variance alone. Surprisingly, choice of primers or RNA template may influence the reaction outcome even more than the bias introduced from the enzyme. This is especially the case with recent advances in protein engineering. Production of highly efficient RTases may pronounce the variation originating from...
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