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MicroRNAs encoded by polyomaviruses.
Zachovalová, Veronika ; Bruštíková, Kateřina (advisor) ; Malík, Radek (referee)
MicroRNAs are small regulating molecules of RNA that are encoded by orgamism's genome. Biogenesis of microRNA takes place partly in the nucleus and partly in the cytoplasm. Result of this biogenesis is a 22 nt long microRNA molecule. They are able to silence the genes thanks to sequence- specific degradation of a target mRNA or thanks to the repression of translation of target, complementary mRNA. In mammalian cells the mechanism of translational repression is more common. During this mechanism the microRNA molecule is not entirely complementary to 3'UTR of its target mRNA. Polyomaviruses are small, non-enveloped dsDNA viruses with a circular genome and icosahedral capsid composed of VP1 protein pentamers. These viruses belong in a group called onkoviruses, which can transform infected cells and contribute to development of serious illnesses such as Merkell cell carcinoma. Their genome encodes regulating proteins called T antigens, structural capsid proteins and also microRNAs. My main focus in this thesis will be SV40, MPyV, MCPyV, BKPyV and JCPyV encoded microRNA molecules. Key words: polyomaviruses, small interfering RNA, microRNA, siRNA, RNA interference, mouse polyomavirus, BK virus, JC virus, SV40
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The Role of oncogenic microRNA - 155 and proto - oncogen MYB in chronic lymphocytic leukemia
Vargová, Karina
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B-CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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Delineating aggressiveness of acute myeloid leukemia in a mouse model carrying mutations of Spil (PU.1) and Trp53.
Bašová, Petra
PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...
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Messenger RNA stability and microRNA activity in mouse oocytes
Flemr, Matyáš ; Svoboda, Petr (advisor) ; Motlík, Jan (referee) ; Hampl, Aleš (referee)
The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...
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Diagnostic and prognostic markers in the era of targeted treatment of CRC.
Veškrňová, Veronika ; Büchler, Tomáš (advisor) ; Mohelníková Duchoňová, Beatrice (referee) ; Valík, Dalibor (referee)
Introduction: Colorectal cancer (CRC) is the most common malignant tumor in both sexes in the Czech Republic. Prognostic factors in CRC can be classified as clinical (stage at the time of diagnosis, histological type of tumor), genetic (RAS, BRAF), immunological (Immunoscore)and biochemical (CEA, CA 19-9, miRNA). MicroRNAs (miRNAs) regulate the expression of oncogenes and tumor suppressors. The regulatory function of miRNAs is influenced by single nucleotide polymorphisms (SNPs) of target miRNA binding sites (miRSNPs). Aims: To evaluate the currently available prognostic factors for CRC patients treated using targeted therapies and assess the role of novel ones, including miRNA. Methods: The thesis includes clinical works focused on targeted treatment of colorectal cancer, original work focused on the role of miRNA in colorectal cancer pathogenesis and especially as a prognostic and predictive marker, work focused on functional polymorphisms of DNA repair genes and a review article summarizing biochemical factors influencing the effect of fluoropyrimidine cytostatics in the treatment of colorectal cancer. Results: We have identified miR-17/92 as a non-invasive biomarker for predicting post-treatment prognosis in patients with a higher risk of relapse, as well as miRSNPs rs8679 polymorphisms as a...
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Biomarkers of subchondral bone damage caused by inflammation in axial spondyloarthritis.
Bubová, Kristýna ; Pavelka, Karel (advisor) ; Horák, Pavel (referee) ; Hrnčíř, Zbyněk (referee)
Background: Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease affecting primarily the spine and its adjacent structures. The disease is characterized not only by destructive joint changes but also by excessive osteoproduction, which can lead to gradual ankylosis of the spine and thus significantly reduce the mobility and quality of life. The pathogenesis of the disease is not yet fully understood, but a strong genetic background is suggested, along with dysregulation of tissue metabolism resulting from an imbalance of pro- and anti-inflammatory immune mechanisms. We are still lacking biomarker with sufficient sensitivity and specificity which could help to identify early diagnosis, to monitor subchondral damage, and to differentiate rapidly progressing patients. The aim of this work was to determine the levels of potential biomarkers of connective tissue metabolism, fat metabolism and new promising biomarkers for both disease subtypes, their relationship to disease activity and progressive structural changes. Results: We have shown increased serum/plasma levels of connective tissue metabolism biomarkers (especially matrix metalloproteinase mediated metabolites), which were able to differentiate patients with early and late forms of axSpA from healthy individuals (HC), were...
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Pathophysiology of colorectal cancer. Colorectal cancer screening effect and the role of microRNA in pathophysiology of colorectal cancer.
Král, Jan ; Špičák, Julius (advisor) ; Keil, Radan (referee) ; Mohelníková Duchoňová, Beatrice (referee)
Colorectal cancer is a serious malignant disease with an incidence of over 1.8 million new cases per year worldwide. There are about 8 000 patients diagnosed with CRC in the Czech Republic each year, and about half of them present with an advanced disease. Screening program identifies patients in the early stages of CRC resulting in overall better prognosis and survival. There is also a lack of biomarkers of early CRC detection and of response to treatment. The first aim of our project was to conduct a national multicentre prospective observational study to evaluate the impact of CRC screening within the framework of a Czech population screening programme. Between March 2013 and September 2015, a total of 265 patients were enrolled in 12 centres across the Czech Republic. Patients were divided into screening and control groups and compared for pathology status and clinical characteristics. Screening was defined as a primary screening colonoscopy or a colonoscopy after a positive FOBT in an average-risk population. The distribution of CRC stages was significantly favourable in the screening group compared with the control group (stages 0, I and II, 63% versus 43.3%; p <0.001). The presence of distant (M1) and local metastases (N1 and N2) was significantly less prevalent in the screening group (0%,...
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The role of miRNA in injury and regeneration of spinal cord tissue
Šprincl, Vojtěch ; Romanyuk, Natalyia (advisor) ; Mrózková, Petra (referee)
MicroRNAs are small non-coding RNA molecules of a length about 20-24 nucleotides, that regulate gene expression post-transcriptionally. They interfere mRNA molecules via base- pairing with complementary sequences. Recently it was shown that they play an important role in injury and regeneration of nervous tissue. The aim of this bachelor thesis is to describe possible role of miRNAs in central nervous system injury with focus on spinal cord injury.
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Entropically driven cascade hybridization reactions for detection of microRNA
Runová, Alžbeta ; Cígler, Petr (advisor) ; Kožíšek, Milan (referee)
The emerging potential of miRNA molecules as diagnostic biomarkers calls for the development of a new quantification method. Current approaches usually require time-consuming and costly miRNA isolation for proper sample analysis. In this thesis, a new, isolation-free, oligonucleotide- modified gold nanoparticle (AuNP/DNA) system is proposed and designed for miRNA detection and quantification in living cells. This cascade, entropy-driven, and enzyme-free amplification system provides fluorescence signal upon selective interaction with the target miRNA. For this purpose, citrate-stabilized gold nanoparticles were synthesized, and their diameters were determined by dynamic light scattering and transmission electron microscopy. The AuNP/DNA conjugates were prepared following a recently published "freezing method". Their reaction kinetics with the target miRNA and selectivity to various miRNAs were compared with those of an analogous DNA system without AuNPs in a series of fluorescence measurements. Furthermore, stability experiments in glutathione environment were conducted, as well as DNA electrophoresis, demonstrating the mechanistic aspects of the reaction. The reaction yields and selectivity to target miRNA of 42.31 ± 2.91 nm AuNP/DNA constructs, containing approximately 25 DNA complexes per AuNP,...
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Diagnostic and prognostic impact of miR-122 and miR-142 in colorectal cancer
Kalkušová, Kateřina ; Červená, Klára (advisor) ; Král, Jan (referee)
Colorectal carcinoma is one of the most frequent cancers and it is also one of the most common causes of cancer related death. It is often diagnosed at a late stage which is associated with a worse prognosis. Therefore, there is an effort to identify new biomarkers that allow earlier diagnosis. These biomarkers could predict prognosis of disease and patient's response to treatment. Short noncoding RNAs - microRNA - appears to be promising candidates, these molecules are involved in many cellular processes through RNA interference. The aim of this thesis is to summarize knowledge about application of miR-122 and miR-142 as diagnostic and prognostic biomarkers for colorectal cancer. Keywords: microRNA, biomarkers, colorectal cancer, diagnostics, prognosis
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