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Role of genetic factors responsible for development of pancreatic cancer
Borecká, Marianna ; Janatová, Markéta (advisor) ; Václavíková, Radka (referee) ; Živný, Jan (referee)
Cancer is a major health problem, worldwide, and is the second-most frequent cause of death (www.uzis.cz). Research is urgently necessary to reduce cancer incidence and the costs associated with cancer management, to develop more efficient and effective risk prediction strategies and personalised patient treatment. Germinal mutations in genes that predispose individuals to hereditary cancer syndromes are clinically important and can be used to classify carriers being at high risk of cancer development. Identification of these mutations can influence the prognosis and treatment of probands and can be used to include their family members into high-risk groups with corresponding preventive strategies. This study is focused on the currently underestimated description of newly identified genetic factors among the Czech population that predispose individuals to develop pancreatic ductal adenocarcinoma (PDAC). In 2017, the incidence of PDAC in the Czech Republic was 21 cases per 100,000 persons, and PDAC was the fourth-most frequent cause of death among all cancer diseases (www.svod.cz). Using a variety of screening techniques, which included high-resolution melting (HRM) analysis, Sanger sequencing of whole genes, and next-generation sequencing (NGS), with a CZECANCA panel that was generated in our...
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Pathophysiology of inflammatory bowel disease. Relation to primary scklerosing cholangitis, liver transplantation and carcinogenesis.
Bajer, Lukáš ; Drastich, Pavel (advisor) ; Živný, Jan (referee) ; Procházka, Vlastimil (referee)
Inflammatory bowel disease (IBD) represents a group of multifactorial illnesses with increasing incidence worldwide. Crohn's disease (CD) and ulcerative colitis (UC) are the two most thoroughly defined phenotypes of IBD. IBD associated with primary sclerosing cholangitis (PSC) - a progressive biliary disease leading to cirrhosis and liver failure - is considered as specific IBD phenotype (also referred to as 'PSC - IBD') due to its clinical and pathophysiological characteristics. The aim of the experimental part of this thesis was to define specific features of PSC - IBD in the key areas of IBD pathogenesis. These are: microbiota composition, gut - barrier failure, genetic predisposition and aberrant cellular and antibody immune response. Furthermore, the other goals were to describe relation of IBD status and activity to liver transplantation (LTx) and carcinogenesis based on thorough analysis of clinical data in patients under surveillance at the liver transplantation unit. Using the next-generation parallel sequencing technology, we discovered specific bacterial and mycobial features of gut microbiota composition in PSC - IBD which significantly differed from UC and healthy controls recruited from Czech general population. Moreover, we identified numerous seral biomarkers distinguishing CD, UC...
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The role of Kit ligands in hematopoiesis of Danio rerio
Oltová, Jana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...
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Role of Rnf207b in zebrafish hematopoiesis
Vondráková, Zuzana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee)
Hematopoiesis is the process of proliferation, differentiation and self-renewal of hematopoietic stem cells. Regulation of hematopoiesis is a complex process, which takes place on many different levels and is directed by many signals. RNF207 is one of the perspective genes chosen based on a screen in chicken model, where obtained data show its role in hematopoiesis. The aim of this work was to confirm the role of rnf207b as a new regulator of hematopoiesis in Danio rerio and to find out on which level of hematopoiesis is active. Danio rerio is an excellent model to study the function of genes in vivo, thanks to the easy manipulation of genetic expression and wide range of phenotypes during the development. To study the effect of rnf207b in hematopoiesis of Danio rerio we performed the knock-down of this gene by microinjection of morpholino oligonucleotides into one cell stage embryos. In these injected fish, we saw the effect in both thrombocyte and erythroid lineage, suggesting that rnf207b could be a regulator at the hierarchical level of progenitors or even more upstream. The results of developmental and tissue specific expression analysis then show that expression of rnf207b begins as early as 18 hpf, at the time of primitive hematopoiesis. Although rnf207b is expressed in the kidney (an...
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Analysis of quantitative and qualitative genetic features in the pathogenesis of hereditary solid tumors.
Zemánková, Petra ; Kleibl, Zdeněk (advisor) ; Živný, Jan (referee) ; Tichý, Boris (referee)
Cancer the second most common causes of death in the Czech Republic. Carriers of mutations in genes predisposing to hereditary cancers represent a small but clinically significant group of high risk individuals. Today, dozens of predisposing genes for hereditary tumor syndromes are known and targeted next generation sequencing (NGS) has become a standard approach for their analysis. NGS allows rapid acceleration diagnostics of causal mutation in high-risk individuals. To identify mutations in genes predisposing to hereditary cancers, we designed a panel NGS analysis including subsequent bioinformatics analysis allowing a reliable identification of single nucleotide variants, insertions/deletions, and large intragenic rearrangements. The bioinformatics procedures described in this thesis were used for panel NGS validation, but also for identification of alterations associating with so far undescribed hereditary tumor types. Bioinformatics analyzes have become the basis for the unified processing of large datasets from the CZECANCA consortium and enable the construction of a population-specific database of genotypes that serve to improve clinical diagnostics of cancer predisposition in Czech patients. The versatility of NGS also allows its use for RNA (cDNA-based) analyzes of splicing variants in the...
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Pathobiochemistry of lysosomal storage disorders: Study of Fabry disease and generation of cellular models of X-linked disorders.
Rybová, Jitka ; Ledvinová, Jana (advisor) ; Entlicher, Gustav (referee) ; Živný, Jan (referee)
Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...
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Characterization of hematopoietic cells in patients with mature B-cell malignancies
Maswabi, Bokang Calvin ; Živný, Jan (advisor) ; Otáhal, Pavel (referee) ; Alberich Jorda, Meritxell (referee)
(English) Using flow cytometry we analyzed absolute and relative proportions of hematopoietic stem and progenitors cells (HSPC) populations including hematopoietic stem cells (HSC), multipotent progenitors (MPP), multilymphoid progenitors (MLP) and pro B cells from bone marrow of patients with mature B cell malignancies and in healthy controls. We found lower absolute and relative numbers of MLP and higher relative numbers of HSC were observed in patients when compared to age-matched controls irrespective of bone marrow (BM) involvement. On the other hand significantly decreased absolute numbers of MPP were observed only in patients who had their BM infiltrated by disease. We also confirmed published data showing increasing absolute and relative percentages of MLP with increasing age, decreasing relative percentages of HSC with increasing age, and decreasing absolute and relative pro B cell frequencies with increasing age in healthy subjects. While decreased absolute and relative pro B cell numbers were also found in patient samples as age increased, no significant correlations were detected in patients HSC, MPP or MLP populations. Age-related sub-analysis of PTs samples demonstrated that most of the disease associated changes in HSPC frequencies were observable more prominently in the elderly (>45...
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Haematopoiesis in Sea lamprey
Kovář, Martin ; Bartůněk, Petr (advisor) ; Živný, Jan (referee)
To find out if the haematopoietic system is common feature of vertebrates, we decided to examine haematopoiesis in a sea lamprey (Petromyzon marinus). All blood cells arises from the haematopoietic stem cells in higher vertebrates. We assume that this is common for the higher vertebrates and a jawless vertebrates, but nobody was interested in the jawless haematopoiesis since 1970. Using a reverse genetic, we identify homologues of important hematopoietic of higher vertebrates in transcriptome of the sea lamprey with emphasis on important receptors or transcription factors, because they can be used as the specific markers of different blood cells and their progenitors. Then we use those sequences for cloning, expression measurements and other work. We picked up sea lamprey as model organism because its unique phylogenetic position, important foe evo-devo studies, but also because lack of elementary knowledge about sea lamprey haematopoiesis. Key words: Petromyzon marinus, haematopoiesis, HSC, evo-devo
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Functional analysis of the population-specific checkpoint kinase gene CHEK2 sequence variants
Stolařová, Lenka ; Kleibl, Zdeněk (advisor) ; Živný, Jan (referee)
CHEK2 gene codes for serin/threonine kinase Chk2 (Checkpoint kinase 2). In response to genomic DNA damage, Chk2 phosphorylates its substrates (proteins Cdc25C, BRCA1 or p53), whose activation leads either to cell cycle arrest, DNA damage repair or induction of apoptosis. Germline mutations in CHEK2 gene increase risk of cancer development. Analysis of high risk breast cancer patients in Czech Republic reveals rare CHEK2 mutations (mainly missense) with yet unknown clinical significance. This work focuses on functional impact of these variants and analysis of kinase activity of variant isoforms of Chk2 kinase. For this purpose, recombinant constructs were expressed in bacterial cells of E. coli. Enzymatic activity of Chk2 kinase isoforms in crude cell lysates was measured by the phosphorylation of Chk2 arteficial substrate spectrophotometrically. Results of in vitro kinase assay were correlated to the results of in silico prediction software. The results show that from 15 analyzed mutations (together with one in frame deletion), kinase activity was abrogated in all variants affecting the kinase domain of Chk2, in concordance with in silico predictions. The same result has been found for a FHA domain variant p.R145Q. No significant changes in kinase activity were observed in case of two FHA domain variants...
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