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Developmental disorders of the lens and anterior segment of the eye
Jedličková, Jana ; Ďuďáková, Ľubica (advisor) ; Šafka Brožková, Dana (referee) ; Cvekl, Ales (referee)
The anterior segment of the eye includes the eyelids, eyelashes, tear film, cornea, conjunctiva, iris, pupil, ciliary body, chamber angle, and lens. Cataracts and anterior segment dysgenesis are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance. The aim of this thesis was molecular genetic analysis of 51 patients from 23 families with congenital and early onset cataracts and/or anterior segment dysgenesis, using a modern as well as conventional methods, such as next-generation sequencing, direct sequencing, bioinformatic and functional analyses including the use of artificial splicing vectors. We identified 24 causal variants in the coding, non-coding and regulatory regions of the PAX6, FTL, FOXC1, PITX2, FOXE3, and CHRDL1 genes. We confirmed the role of the n.37C>T variant in MIR204 in the development of chorioretinal dystrophy variably associated with iris coloboma, early-onset cataracts and congenital glaucoma. Splicing assay confirmed pathogenic effect of two mutations on pre-mRNA splicing of the PAX6 gene. Establishing molecular genetics diagnosis improves patient counselling and their relatives in terms of the prognosis and risk of developing secondary glaucoma Key words: molecular-genetic diagnosis, anterior segment dysgenesis, next-generation sequencing,...
Molecular genetic analysis of rare ocular disorders in Roma population from the Czech Republic
Rysková, Natálie ; Lišková, Petra (advisor) ; Klímová, Aneta (referee)
The Roma are the largest and most widespread transnational ethnic community. The Czech Republic estimates their 2% representation in its population. Due to the high level of endogamy, the spectrum and frequency of hereditary diseases in the Roma population differ from the majority population. Hereditary eye diseases are one of the most common causes of blindness in younger adults and thus represent a real socio-economic burden. The aim of the thesis was to perform molecular genetic analysis in individuals of Roma origin suffering from hereditary diseases affecting vision, including dual impairments and syndromes, and to determinate the frequency of the detected pathogenic variants in this population. Molecular genetic analysis of 17 families was performed using direct and whole exome sequencing. Within the framework of an international collaboration, the degree of their mutual kinship was calculated using the PLINK program. The frequency of selected variants was determined in a control dataset comprising 156 Roma exomes and genomes. The spectrum of analyzed diseases included various retinal dystrophies, primary congenital glaucoma, Usher syndrome, neuronal ceroid lipofuscinosis, Noonan syndrome, nanophthalmos and congenital cataract, facial dysmorphism and neuropathy. The results of the thesis...
Sequence variants in metastatic colorectal cancer and their evolution during the disease course and treatment
Jandová, Eliška ; Boublíková, Ludmila (advisor) ; Vymetálková, Veronika (referee)
A deeper understanding of the molecular background of colorectal cancer (CRC) can help explain the development of the disease and its resistance to treatment, predict disease progression, and improve treatment prognosis. Some minimal molecular testing has been incorporated into standard clinical management to determine if a particular patient will benefit from a particular therapy, but more and more new genetic alterations are being discovered that appear to be associated with the development of resistance. Tissue biopsy of the tumor is the gold standard in terms of molecular testing, but there is an increasing demand for more non- invasive methods such as liquid biopsy. Using targeted next-generation sequencing, we analyzed sequence variants present in primary tumor, metastases, and cell-free tumor DNA - ctDNA of patients with metastatic CRC. The objectives were to analyze sequence variants of the primary tumor and identify possible pathogenic variants, to analyze differences between DNA of the primary tumor and metastases, to evaluate the use of ctDNA as a diagnostic tool, and to identify potential tumor-specific markers in ctDNA that can be used to monitor disease progression. Our results suggest the feasibility of using ctDNA for diagnostic purposes or even to monitor disease progression, but...
Identification of hereditary factors influencing the formation of a pancreatic carcinoma and other solid tumours
Král, Jan ; Janatová, Markéta (advisor) ; Hojný, Jan (referee) ; Šeborová, Karolína (referee)
Pancreatic adenocarcinoma (PDAC) has one of the worst prognoses out of all cancers worldwide. Endometrial carcinoma (EC) is the most common gynecological cancer. Genetic background of tumors is highly heterogenous and differs among populations. We have analyzed DNA of 226 PDAC patients and 527 EC patients using panel next- generation sequencing. Targeted genes were divided into main predisposition genes (11 for PDAC, 19 for EC) and other candidate genes. EC patients were categorized based on meeting the indication criteria for germline genetic testing. Two sets of population-matched controls were used (controls with negative cancer history, and general population controls). Germline pathogenic variants (PV) in main predisposition genes were identified in 18 (8.0%) PDAC patients. The most mutated gene was BRCA2 (50% of carriers). PDAC risk was significantly elevated in carriers of PV in BRCA1 (OR = 10.4, p = 0.04), BRCA2 (OR = 6.4, p = 0.0009), and CHEK2 (OR = 17.5, p = 0.003). Germline mutations in genes participating in homologous recombination processes were associated with improved overall survival of patients. Among EC patients there were 60 (11.4%) carriers of PV in main predisposition genes. Carriers of PV in Lynch syndrome (LS) genes had markedly elevated risk of developing EC (OR = 22.4, p...
Složení společenstev endoyfytických mikroorganismů osídlujících listy hemiparazitických rostlin
This bachelor´s thesis focuses on describing the community composition of endophytic microorganisms colonizing foliar parts of selected Central-Europaean species of parasitic plants and their potential hosts. The aim is to find specific differences in the structure and diversity of bacterial and fungal endophytes between plant species with parasitic and non-parasitic life strategy.
Pathogenetic mechanisms of immune dysregulation and hematopoietic disorders
Svatoň, Michael ; Froňková, Eva (advisor) ; Filipp, Dominik (referee) ; Čermák, Jaroslav (referee)
Pathogenetic mechanisms of immune dysregulation and hematopoietic disorders Abstract The aim of my work was to introduce whole exome sequencing (WES) and a routine algorithm for data analysis in pediatric patients with suspected inborn error of immunity (IEI) or hematopoiesis. A clear molecular diagnosis based on genetic testingwas achieved in 25% of patients. Additionally, novel mutations not previously described in the pathogenesis of IEI or hematopoietic failure were revealed in 9% of the patients. Validation experiments were designed to confirm the causality of the identified mutations in the pathogenesis of the disease, based on literature review and known mechanisms of immune system development and regulation in these patients. Thanks to WES, we were able to elucidate the cause of two new congenital diseases - megaloblastic anemia due to a homozygous mutation of the SLC19A1 gene and immune dysregulation syndrome with autoimmune and autoinflammatory manifestations due to an X-linked mutation of the TLR8 gene in monozygotic twins with chronic autoimmune hemolytic anemia (AIHA). In five additional patients, novel mutations in the STAT3, SPTA1, SAMD9 and XIAP genes were identified and their causality in the pathogenesis of hematopoietic failure or IEI was demonstrated. In one patient with an atypical...
Molecular genetic approaches used in preimplantation genetic analysis of human embryos in assisted reproduction
Limbergová, Jana ; Machoň, Ondřej (advisor) ; Šimoník, Ondřej (referee)
Preimplantation genetic testing is one of the main clinical procedures that take place in assisted reproduction centres around the world. It is sought after by couples for many different reasons. The causes of problems may relate to hereditary diseases that potential parents do not wish to pass on to their offspring, or may include the risk of transmission of chromosomal alterations. In addition, infertility may be caused by an increased risk of embryo aneuploidy, which correlates with mother's increased age and other factors that this bachelor thesis discusses. The bachelor thesis also focuses on up-to-date approaches to cytogenetic examinations for reproductive genetics and discusses recent clinical molecular methods that are used to improve the diagnosis and therapy of infertile couples. These are, for example, microarray methods, quantitative real-time PCR or next-generation sequencing and methods of whole-genome amplification. This work also summarizes methods that are gradually being abandoned, e.g. fluorescent in situ hybridization, and compares their advantages and disadvantages. Key words: preimplantation genetic testing, aneuploidy, structural rearrangements, monogenic diseases, biopsy, fluorescent in situ hybridization, microarray methods, comparative genomic hybridization, quantitative...
The role of WT1 gene and other molecular biological abnormalities in testicular germ cell tumors
Bakardjieva - Mihaylova, Violeta ; Boublíková, Ludmila (advisor) ; Chovanec, Michal (referee) ; Kiss, Igor (referee)
Testicular germinal tumors (TGCT) are relatively rare solid tumors in adults. Even so, they affect more than 700 men a year in the Czech Republic, mostly young patients aged 18- 45 years. A large number of patients are curable by a combination of surgery and chemotherapy, yet about 50 men a year in the Czech Republic succumb to this tumor, in the vast majority of cases due to the development of resistance to chemotherapy containing cisplatin. The rare occurrence and high curability are probably the cause of infrequent molecular and clinical studies carried out in these tumors, and our understanding of the biological processes leading to primary tumor development and the development of cisplatin resistance (CDDP) is still limited. At present, no specific molecular markers that could be used as prognostic or predictive factors and improve patient stratification or treatment tailoring are available in TGCT management. In this work, we studied the molecular-genetic background of TGCT development and CDDP resistance at several levels. To comprehensively study the development of cisplatin resistance, we prepared and analyzed CDDP-exposed TGCT cell lines. Long-term exposure to CDDP increased resistance 10-fold in the NCCIT cell line, while no significant resistance was achieved with Tera-2. The...
Studium celogenomové variability lidského cytomegaloviru.
Dvořák, Jan ; Tachezy, Ruth (advisor) ; Roubalová, Kateřina (referee)
This work is part of a project focused on the study of the variability of human cytomegalovirus (HCMV) among clinical isolates with the aim to map the geographical distribution of HCMV genotypes, reveal the relationships between genotypes and the severity of HCMV-associated diseases, and identify regions in the HCMV genome with a potential for use as diagnostic and therapeutic targets. Attention was paid to the development of the methodology for the preparation of the material for next-generation sequencing (NGS) from HCMV clinical isolates and evaluation of the obtained sequencing data. Blood and urine samples collected from hematopoietic stem cell transplantat recipients and congenitally infected children were analyzed. Samples suitable for NGS were sequenced by the Illumina platform and sequences were created by de novo assembly followed by mapping assembly. Urine samples in comparison to blood samples had higher yield of material for NGS. Of the samples positive for HCMV DNA (7 of 50) after amplification in the cell cultures, only one sample had high purity of the viral DNA (98%) while six samples had purity of less than 7%. The sample containing 98% of the viral DNA was fully sequenced and the sequence was compared to the sequences of other clinical isolates from Belgium in 11 polymorphic...
The effect of microbiota on pathogenesis of gut diseases
Galanová, Natalie ; Kolařík, Miroslav (advisor) ; Hudcovic, Tomáš (referee)
Gut microbiota is considered an important factor in the development of various diseases including inflammatory bowel disease (IBD, n = 127), Ulcerative colitis, Crohn's disease, and colorectal cancer (CRC, n = 64). A part of this thtesis is to prepare clinical material of different sorts (stool, biopsy) for sequencing on Illumina Miseq platform. This is achieved trough DNA isolation, amplification of 16S and internal transcribed spacer (ITS), normalization and ligation of sequencing adaptors. The aim of this project is to describe the differences between microbiota in healthy and diseased subjects in case of IBD or unimpaired and tumorous tissue for CRC patients. This research is also being based on cultivation, where a fresh stool samples (n = 3) are cultivated in a broad range of conditions, which enables us to obtain ecophysiological and species diversity of these samples by traditional and molecular methods. The cultivable fungi are also assigned reliable taxonomy by amplification of relevant genes (ITS1, β tubulin, second largest subunit of RNA polymerase II, RPB2) followed by both-sided Sanger sequencing. Selected species of fungi are processed into lysates, which are used for stimulation of mice macrofage cell line (RAW). Therefore the impact on immunity response is studied in vitro and...

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