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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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Analysis of gene expresion via next generation sequencing techniques
Bláhová, Monika ; Krylov, Vladimír (advisor) ; Žárský, Vojtěch (referee)
1 Abstract The main task of this work is create review today's available methods for gene expression analysis, introduce advantages and disadvantages of this metods and compare them. Nowadays sequencing is one of the most usable molecular methods. Sequencing methods are divided to three groups, a first generation sequencing, a second generation sequencing and a third generation sequencing. The most useful are the second generation sequencing. However, the third generation sequencing have a big potencial too. It is not necessary amplificate samples using PCR thanks them. Amount of data raises rapidly, thanks decreasing costs and increasing efficiency. Demands on data starage, computers output are growing. And softwares for data analysis are much clever than ever before.
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Laccase activity profiling in Trametes versicolor cultures degrading endocrine-disrupting compound Delor 103
Plačková, Martina
In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...
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Study of effect of carcinogenic benzo[a]pyrene on cytochromes P450 and cytochrome b5 expression in organs of the rat
Blecha, Tomáš ; Moserová, Michaela (advisor) ; Dračínská, Helena (referee)
Polycyclic aromatic hydrocarbons are a large group of organic compounds which consist solely of carbon and hydrogen atoms, two or more fused aromatic rings. They represent group of persistent organic pollutants (POPs) present in all components and fields of the environment. Benzo[a]pyrene is characteristic compound of polycyclic aromatic hydrocarbons formed by five fused aromatic rings. Benzo[a]pyrene is procarcinogen with genotoxic effects, which is metabolically activated by variety of enzyme systems such as cytochrome P450 and epoxide hydrolase to reactive metabolite of BaP-7,8-diol-9,10-epoxide (BPDE) and 9-hydroxy-4,5-epoxy-BaP. These reactive metabolites can form covalent DNA adducts. In the present work, we studied the influence of carcinogenic benzo[a]pyrene on the expression of cytochrome P450 (CYP) 1A1, 1A2 and cytochrome b5 in livers, kidneys and lungs of laboratory rats. Total RNA was isolated and afterwards converted into cDNA with the participation of random primers. Using polymerase chain reaction in real time (RT-PCR) the relatively gene expression of CYP1A1, CYP1A2 and cytochrome b5 was quantified in the organs of rats treated with BaP and control (untreated) animals to a reference gene (β-actin). It was found that benzo[a]pyrene significantly increases expression of CYP1A1 and...
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Control of gene expression regarding cell pluripotency
Křtěnová, Petra ; Krylov, Vladimír (advisor) ; Láníková, Lucie (referee)
ESCs have been of interest to many research teams since their derivation from the mouse blastocyst 30 years ago. The main reason for studying ESCs is their ability to differentiate into almost all cell types. This feature is known as "pluripotency". The pluripotent state in ESCs is maintained by the control of the gene expression. To maintain their undifferentiated state it is necessary to repress the differentiation genes. This process is controlled primarily by pluripotent transcriptional factors, especially by OCT4, SOX2 and NANOG. Silencing of the differentiation genes is also influenced by the chromatin remodelling complexes. The regulation of the gene expression leading to the cell pluripotency also takes place at the post- transcriptional level via miRNA, lncRNA, hnRNPs or proteins which stabilize pluripotent factors and protect them from degradation. The aim of this thesis is to summarize mechanisms by which pluripotency is maintained in ESC.
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Localization matters: function of paxillin and phopholipids in the cell nucleus
Marášek, Pavel ; Hozák, Pavel (advisor) ; Půta, František (referee) ; Žárský, Viktor (referee)
(English) Both paxillin and PIP2 are well known components of the cell, although of a distinct origin. Focal adhesion protein paxillin spreads the signals from extracellular matrix via integrins and growth factor receptors to affect cellular motility and migration (Schaller, 2001). PIP2, a major structural component of cytoplasmic membrane, is utilized by phospholipase C to generate second messenger molecules (Hokin and Hokin 1953; Streb et al. 1983). Both molecules were recently shown to be localized in the nucleus. Their original functions have been well established, but together with other research colleagues we are now shedding more light on completely different functions of these biological molecules and moreover, in the different compartments than they were primarily believed to function in. Here, we introduce paxillin as an important factor of the cell nucleus, where it regulates transcription of two important growth-related genes, IGF2 and H19. It does not affect the allelic expression of these imprinted genes, it rather regulates long-range chromosomal interactions between H19 or IGF2 promoter, and the shared distal enhacer on an active allele. In detail, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, activating IGF2 gene transcription, while it restrains...
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Regulatory mechanisms of ornithin transcarbamylase and beta-glucocerebrosidase gene expression and their relevance to diagnostics
Lukšan, Ondřej ; Jirsa, Milan (advisor) ; Kožich, Viktor (referee) ; Kříž, Vítězslav (referee)
5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...
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Expression of WT1 and its splicing variants in myeloid leukemias
Lopotová, Tereza ; Moravcová, Jana (advisor) ; Zemanová, Zuzana (referee) ; Rázga, Filip (referee)
Myeloid leukemias include malignant diseases characterized by clonal expansion of the myeloid cell lineage. While in case of chronic myeloid leukemia (CML), the main cause of the disease has already been identified - t(9;22) and the aktivity of the fusion product of the translocation BCR-ABL, acute myeloid leukemia (AML) has been associated with plenty of different translocations and mutations. The aim of this work was to contribute to the improvement of monitoring of patients with myeloid leukemias via detailed study of the panleukemic marker Wilms tumor gene 1 (wt1) expression. Prognostic value of wt1 expression has been proved for AML patients, however, it has not yet been confirmed for CML patients. Expression of different wt1 variants (more then 36 protein products) is known very poorly in both, AML and CML as well as in normal hematopoiesis. Most of the study is focused on CML, only limited parts are dedicated to AML. In the first part of the work, we clearly proved prognostic value of total wt1 mRNA expression for CML patients. Statistical evaluations revealed critical wt1 values which enable to specify prognosis of patients responding non-optimally to imatinib. Bcr-abl looses much of its prognostic value in these patients. Further, we have designed and optimized PCRs for selected wt1...
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The Heme Catabolic Pathway in Chronic Hepatitis C
Subhanová, Iva ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Kráslová, Ivana (referee)
This thesis focuses on the importance of the heme catabolic pathway in chronic hepatitis C (HCV). The aim is mainly to investigate, whether expresion/activity of key enzymes of the heme catabolic pathway, heme oxygenase (HMOX) and biliverdin reductase (BLVRA) in the liver and blood (study A) or promoter variations of HMOX1 and UDP- glucuronosyltransferase (UGT1A1) (study B) may be associated with the progression of fibrosis and may also predict antiviral treatment outcome in patients chronically infected with HCV. We set up a new sensitive method to quantify HMOX activity by reduction gas chromatography. We developed and extensively validated RealTime PCR assay for HMOX and BLVRA expression in the liver and peripheral blood leucocytes (PBL). The (GT)n and (TA)n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis. No association was detected between either expression of HMOX/BLVRA or the HMOX1/ UGT1A1 promoter variants and the individual histological stages of liver disease in the HCV positive patients. A marked difference in BLVRA expression in PBL between the sustained responders (SVR) and patients with treatment failure (NVR) was detected before antiviral treatment and during the follow-up. Our data suggests, that BLVRA basal expression...
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Role onkogenní mikroRNA-155 a proto-onkogenu MYB u chronické lymfatické leukémie
Vargová, Karina ; Stopka, Tomáš (advisor) ; Móciková, Heidi (referee) ; Trka, Jan (referee)
(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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