Die Lebertransplantation bietet gegenwärtig die beste Therapiemöglichkeit des Hepatozellulären Karzinoms und der Leberzirrhose im Endstadium. Für die positiven Resultate ist die rechtzeitige Diagnose- und Indikationsstellung entscheidend. Bei einem Japanese Integrated System (JIS) Score von 0 liegt die 5-Jahres-Überlebensrate nach einer Transplantation bei 73 %, bei einem Anstieg des Scores auf den Wert 3, sinkt sie auf 13 %[74]. Die Sensitivität der gegenwärtigen Diagnostik aus bildgebenden Verfahren und der Bestimmung des AFP zur Detektion eines HCC schwankt zwischen 20 % und 94 %[47,48,69]. Der für die Listung zur Transplantation entscheidende MELD-Score vernachlässigt ebenso wie die Milan-Kriterien die genetische und ätiologische Komponente dieser Tumorerkrankung, welche aber maßgeblich das rezidivfreie Überleben bestimmen[16–18,70]. Im Rahmen dieser Dissertation wurden Gewebeproben aus explantierten Lebern auf mikroRNA-Expression untersucht. Des Weiteren wurde zum ersten Mal analysiert, ob mit Hilfe binär-logistischen Regression und der Entscheidungsbaumklassifikation Algorithmen aus mikroRNA-Profilen und laborchemisch-klinischen Parametern zur Detektion und ätiologischen Differenzierung des HCC und der Leberzirrhosen entwickelt werden können. In der vorliegenden Arbeit konnte dargestellt werden, dass zwischen dem HCC-Gewebe und dem tumorumgebenden Gewebe, zwischen tumorumgebendem Gewebe und der reinen Zirrhose sowie zwischen ethyltoxischer und viraler Genese der o.g. Krankheitsbilder die mikroRNAs unterschiedlich stark exprimiert werden. Außerdem konnten aussichtsreiche Modelle berechnet werden, die eine Differenzierung zwischen Tumor und tumorumgebendem Gewebe mit 87,5 %, eine Unterscheidung von tumorumgebender Zirrhose von einer reinen Zirrhose mit 94,3 % bzw. 96,3 % und eine Differenzierung zwischen Tumorgewebe und reiner Zirrhose mit 91,9 % bzw. 92,1% ermöglichen. Die weitere Analyse zeigte, dass die Modelle ebenfalls dazu geeignet sind, die Lebererkrankungen nach der Ätiologie zu differenzieren. Die dargestellten Methoden sind in der Beachtung der mit großen Potenzial[24,30] versehenen mikroRNAs und der laborchemisch-klinischen Parameter neuartige Verfahren, die sowohl für die weitere Grundlagenforschung als auch für die Ergänzung der derzeit etablierten diagnostischen und allokativen Verfahren wichtige Erkenntnisse liefern.

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...

Die Lebertransplantation bietet gegenwärtig die beste Therapiemöglichkeit des Hepatozellulären Karzinoms und der Leberzirrhose im Endstadium. Für die positiven Resultate ist die rechtzeitige Diagnose- und Indikationsstellung entscheidend. Bei einem Japanese Integrated System (JIS) Score von 0 liegt die 5-Jahres-Überlebensrate nach einer Transplantation bei 73 %, bei einem Anstieg des Scores auf den Wert 3, sinkt sie auf 13 %[74]. Die Sensitivität der gegenwärtigen Diagnostik aus bildgebenden Verfahren und der Bestimmung des AFP zur Detektion eines HCC schwankt zwischen 20 % und 94 %[47,48,69]. Der für die Listung zur Transplantation entscheidende MELD-Score vernachlässigt ebenso wie die Milan-Kriterien die genetische und ätiologische Komponente dieser Tumorerkrankung, welche aber maßgeblich das rezidivfreie Überleben bestimmen[16–18,70]. Im Rahmen dieser Dissertation wurden Gewebeproben aus explantierten Lebern auf mikroRNA-Expression untersucht. Des Weiteren wurde zum ersten Mal analysiert, ob mit Hilfe binär-logistischen Regression und der Entscheidungsbaumklassifikation Algorithmen aus mikroRNA-Profilen und laborchemisch-klinischen Parametern zur Detektion und ätiologischen Differenzierung des HCC und der Leberzirrhosen entwickelt werden können. In der vorliegenden Arbeit konnte dargestellt werden, dass zwischen dem HCC-Gewebe und dem tumorumgebenden Gewebe, zwischen tumorumgebendem Gewebe und der reinen Zirrhose sowie zwischen ethyltoxischer und viraler Genese der o.g. Krankheitsbilder die mikroRNAs unterschiedlich stark exprimiert werden. Außerdem konnten aussichtsreiche Modelle berechnet werden, die eine Differenzierung zwischen Tumor und tumorumgebendem Gewebe mit 87,5 %, eine Unterscheidung von tumorumgebender Zirrhose von einer reinen Zirrhose mit 94,3 % bzw. 96,3 % und eine Differenzierung zwischen Tumorgewebe und reiner Zirrhose mit 91,9 % bzw. 92,1% ermöglichen. Die weitere Analyse zeigte, dass die Modelle ebenfalls dazu geeignet sind, die Lebererkrankungen nach der Ätiologie zu differenzieren. Die dargestellten Methoden sind in der Beachtung der mit großen Potenzial[24,30] versehenen mikroRNAs und der laborchemisch-klinischen Parameter neuartige Verfahren, die sowohl für die weitere Grundlagenforschung als auch für die Ergänzung der derzeit etablierten diagnostischen und allokativen Verfahren wichtige Erkenntnisse liefern.

Rheumatoid arthritis is the most common joint disease of autoimmune origin. It is accompanied by inflammatory conditions that lead to irreversible changes in the joints, their deformities ending with permanent disability. Treatment of the disease involves routine regimens, surgical, as well as pharmacological treatment, which is necessary for advanced forms. Glucocorticoids play an important role in the therapeutic intervention in the course and progression of the disease. In spite of their anti-inflammatory effect, which is a key to improving the condition of the patient, they have a number of side effects in the long term- use. In this study, we have focused on the impact of these drugs on microRNA expression changes in arthritic patients treated with pulsed doses of glucocorticoids. MicroRNAs are nowadays widely studied due to their possible use as biomarkers in monitoring disease progression and the effect of treatment. MiRNA expression analysis was performed by quantitative real-time PCR array of 754 miRNAs with reverse transcription using stem-loop primers that allow amplification of short sequences that microRNAs are. Data analysis revealed 29 miRNAs differentially expressed at the significance level p ≤ 0.05, 14 miRNAs were at significance level p ≤ 0.025 (respectively 7 miRNAs at p ≤ 0.005...

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...

Lymphomas are heterogenous class of diseases characterized by proliferation of a malignant lymphocyte clone. MicroRNA miR-155 was found to be a key molecule in immune response, namely in inflammation and germinal reaction of B cells. On the other hand, miR-155 can drive lymphoproliferation in mouse and its levels were found to be elevated in certain lymphoma types in human. MiR-155 down-regulates expression of its target gene PU.1, a hematopoietic transcription factor important for B cell differentiation. Expression of the gene encoding miR-155, known as MIR155HG, is controled by several transcription factors, among them MYB, a member of an oncogenic E-box protein family. Levels of MYB itself are controled by microRNA miR-150. In this study, we measured levels of miR-155, PU.1, MYB and miR-150 in lymph nodes of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL, N=20), diffuse large B-cell lymphoma (DLBCL, N=24), follicular lymphoma (FL, N=29), Hodgkin lymphoma (HL, N=25), marginal zone lymphoma (MZL, N=13), and mantle cell lymphoma (MCL, N=10). We also measured levels of these molecules in lymph nodes with the finding of strong inflammation (N=4). We found that patients of all the diagnoses except of MCL display heterogeneously elevated levels of miR-155 and correspondingly...

Hand osteoarthritis (OA) is a degenerative joint disease that causes pain, functional limitation and negatively affects the patients' quality of life. The most severe subtype of this disease is erosive OA. Erosive hand OA is characterized by an abrupt onset, inflammation and is linked to worse outcomes than non-erosive hand OA. Current methods do not allow early diagnosis or to distinguish between patients with different forms at disease onset. This could be changed by the utilization of biomarkers in clinical practice. Biomarkers are molecules released into circulation that reflect biological processes. The main goal of this study was to analyze the levels of circulating biomarkers with the aim to differentiate patients from healthy subjects and patients with erosive OA from patients with non-erosive disease. Serum concentrations of seven biomarkers and the expression of plasma microRNAs were determined. Patients with hand OA showed altered cartilage metabolism, increased levels of adiponectin, decreased levels of clusterin and a dysregulated expression of several microRNAs in comparison to the healthy population. Patients with erosive OA had lower levels of clusterin and decreased expression of miR-151-3p than those with the non-erosive form of the disease. These findings suggest the potential...

Markéta Sypecká CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, because of complications from CLL, but most patients live 5-10 years. However, every stage of this disease has significantly higher level of miR-155, which is known as oncomiR. Micro RNAs represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets. We hypothesized that by gene editing of CLL B - cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Method we use for gene editing is CRISPR/Cas9, which enables to delete sequence of mature miR-155 in genome of leukemic B-cells. Methods: CRISPR/Cas9, nucleofection, qRT-PCR, FACS Results:We achieved to isolate clone that bears one allelic deletion (miR-155-/+) in sequence for mature...

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,...

Acute myeloid leukemia (AML) is a very heterogeneous disease associated with cytogenetic aberrations and genetic mutations. Many of these changes have been revealed and their detection became usual part of the diagnostic process today. However, changes of expression profiles of small, noncoding RNAs, so called microRNAs (miRNAs), are less known and not used for diagnostics yet. These RNAs, 19-24 nucleotides long, take part in the regulation of expression of different genes through complementary base pairing to the 3'non- translated region (3'UTR) of the target messenger RNA (mRNA). They can influence key processes of the cell, like differentiation, proliferation or apoptosis. The changes in expression of different miRNAs are known from different types of cancers. In solid tumors, they are usually detected from bioptic samples; but also plasma samples are now in the center of attention as so called liquid biopsies providing the information about molecular genetic events in the organism. Many studies have revealed deregulated miRNAs in the bone marrow, full blood or isolated progenitor cells (CD34+) of AML patients, only four of them have analyzed plasma samples. We focused on the plasma samples and we targeted on such miRNAs, which levels differ at AML diagnosis and after the chemotherapy. Out of...