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The role of microRNAs in pathogenesis of placental insufficiency - related pregnancies
Fárková, Eva ; Hromadníková, Ilona (advisor) ; Korabečná, Marie (referee)
MicroRNAs (miRNAs) are small noncoding RNAs of length ranging from 18 to 25 nucleotides that regulate gene expression at posttranscriptional level. Expression of some miRNAs is tissue specific. This task assumed that placental insufficiency related pregnancy complications may be characterised by the dysregulation of microRNAs that are crutial for the regulation of cardiovascular system and cardiovascular diseases. The gene expresion of 32 cardiovascular miRNAs was studied in two types of biological material - placental tissue and maternal whole peripheral blood. Patient cohort of clinically manifested preeclampsia (PE), gestational hypertension (GH) and intrauterine growth restriction (IUGR) was compared with samples derived from normal pregnancies (FG). In total, 127 placental tissue samples (20 FG, GH 20, 20 IUGR , 67 PE) and 80 maternal whole peripheral blood samples (20 FG, GH 20, 20 IUGR , 20 PE) were processed. Before the research, two pilot studies focused on the selection of endogenous controls for data normalization of gene expression in both types of biological material were performed. Detection and quantification was carried out by quantitative real-time PCR. In a group of placental tissue several miRNAs showed elevated levels in GH (miR-1, miR-16, miR-17, miR-20a, miR-21, miR-23a,...
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Profiling of extracellular microRNA in acute myeloid leukemia before and after treatment
Štěrbová, Monika ; Korabečná, Marie (advisor) ; Beličková, Monika (referee)
Acute myeloid leukemia (AML) the most common acute leukemia in adults is characterized by various cytogenetic and molecular abnormalities. However, the genetic etiology of the disease is not yet fully understood. MicroRNAs (miRNAs) are small single- stranded noncoding RNAs that are negative regulators of gene expression. miRNAs influence processes of proliferation, differentiation and apoptosis. Deregulation of miRNAs expression can contribute to human disease. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as breast cancer, colorectal cancer and lung cancer. However, defining a plasma miRNA signature in AML that could serve as a biomarker for diagnosis has been conducted only once. We studied miRNA expression in plasma of 8 AML patients in first detection of the disease and repeatedly after achieving remission using TaqMan miRNA microarray for 750 human miRNA. The plasma expression level of 25 miRNA was down-regulated whilst that of 20 miRNA was up-regulated in the AML group at diagnosis when compared to healthy controls. The plasma expression level of 21 miRNA was down-regulated whilst that of 13 miRNA was up-regulated in the AML group in remission compared to healthy controls. Keywords acute myeloid leukemia (AML), biomarker, microRNA (miRNA), plasma, TaqMan Low...
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The use of DNA analysis in kinship studies and investigation of distant biological relationship of people
Červená, Tereza ; Šimková, Halina (advisor) ; Korabečná, Marie (referee)
AJ The thesis deals with the possibilities and problems of DNA analysis in its use in kinship studies and investigation of distant biological relationship of people. It presents a summary of useful DNA markers with a focus on linking autosomal markers and lineage markers - mtDNA and Y-STR; characterizes the typical heredity of individual types of markers and explains the basic principles of interpretation of their analysis. The research also deals with computer software exploited in statistical calculations; case studies, specific examples of the use of DNA analysis in kinship studies and investigation of distant biological relationship of people are also included at the final section. Key words DNA analysis, genealogy, kinship study, STR, SNP, DNA marker
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Forensic SNP identification of trichological material by High Resolution Melting analysis
Strobachová, Joanne ; Stenzl, Vlastimil (advisor) ; Korabečná, Marie (referee)
The analysis of mitochondrial DNA (mtDNA) from shed hairs has gained high importance in forensic casework since telogen hairs are one of the common type of evidence left at the crime scene. This type of evidence very often lack of nuclear DNA (nDNA), so the identification based on the STR analysis is not possible. The analysis of trichological material lacking nDNA is not a commonly used method of the forensic praxis because of the high price of the regularly used mtDNA analysis method, as well as the time demandingness. Based on this, the main goal of this work is to establish a relatively fast, easy and cheap method able to analyze trichological material lacking of nDNA. For these reasons we decided to analyze the mtDNA and identify the trichological material by analysis of chosen SNP's followed by High Resolution Melting. We have analyzed 12 series of different biological materials that belongs to one person (e.g. buccal cells, trichological material in anagen - head, axillary and pubic hair shafts) in order to confirm the identity of mtDNA isolated from different type of biological materials. We have analyzed several set's of buccal cells and head hair shafts in telogen from 17 specimens. From total number of 5 selected SNP's, this essay was able to optimize analysis of 3 SNP's. Due to the...
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Validation of High Resolution Melting (HRM) for the purpose of DNA diagnostics: mutation analysis of the cystic fibrosis gene and selected candidates genes of male intertility
Peldová, Petra ; Macek, Milan (advisor) ; Brdička, Radim (referee) ; Korabečná, Marie (referee)
During the last years we have observed a rapid development of molecular genetic diagnostics (DNA diagnostics). New methods and technologies are rapidly being introduced and the spectrum of genetic services is gradually extended. Since germline genetic tests might have lifelong influence health and quality of patient's life, all efforts should aim at improvement of the overall quality of provided diagnostic services. An increasing number of laboratories replace their "in-house" developed techniques by the commercial diagnostic assays, but they often modify manufacturer's instructions. Therefore, it is necessary to validate and verify all methods and techniques before their implementation into routine DNA diagnostics. In this thesis I have focused on evaluation and application of High Resolution Melting (HRM) in clinical diagnostic practice based on its comprehensive validation, according to the major international quality assurance standard ISO 15189. On the model of selected genes (BRCA1, MTHFR, CFTR) we have confirmed the high utility of HRM for mutation scanning of unknown variants, as well as genotyping of common variants. Concurrently, we have provided a list of methodical guidelines which could be applied for setting up HRM in other genetic laboratories and provided a diagnostic validation strategy for...
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Utilisation of molecular cytogenetic techniques in productive genetics
Paulasová, Petra ; Macek, Milan (advisor) ; Korabečná, Marie (referee) ; Vodička, Radek (referee)
Title.: Utilisation of molecular cytogenetic techniques in reproductive genetics Chromosomal abnormalities constitute one of the most important causes of birth defects, fertilization failure and/or human infertility. Approximately, 40-50% of human conceptuses are chromosomally abnormal, 6% of the abortions during the first trimester of gestation are directly linked to chromosomal abnormality, while at term 0.6% of livebirths present with such features. Most of these abnormalities originate from the gametogenesis and arise through disturbed meiotic processes. Each gamete is a final and original product of the meiosis carrying a unique chromosomal set. Therefore, cytogenetic examination of individual gametes represents an important scientific challenge for our undrstandidng of the formation, incidence and etiology of aforementioned chromosomal abnormalities. Nonetheless, it is very technically demanding to perform efficient chromosomal investigation on gametes, hence single cells. My Ph.D. thesis is focused on the development of new techniques for the detection of chromosomal abnormalities in gametes and embryos. We developed a PNA-based technique as an alternative to conventional FISH and PRINS-based methods for fast, efficient and robust in situ detection of chromosomal abnormalities in human...
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Forensic SNP identification of trichological material by High Resolution Melting analysis
Strobachová, Joanne ; Stenzl, Vlastimil (advisor) ; Korabečná, Marie (referee)
The analysis of mitochondrial DNA (mtDNA) from shed hairs has gained high importance in forensic casework since telogen hairs are one of the common type of evidence left at the crime scene. This type of evidence very often lack of nuclear DNA (nDNA), so the analysis of mtDNA is the only possibility. The analysis of trichological material lacking nDNA is not commonly done in the forensic practice because of the high price of the regularly used mtDNA analysis method, as well as the time demandingness. For these reasons we decided to analyze the mtDNA and identify the trichological material by analysis of chosen single based polymorphism's (SNP) followed by High Resolution Melting (HRM). In order to verify the robustness of the HRM analysis method on several types of biological material that belongs to one person, we tested trichological material in anagen and telogen. From total number of 5 selected SNP's, this paper was able to optimize analysis of 3 SNP's. Due to the analysis of SNP C14766T (applicable for the trichological material in anagen and telogen), SNPs C7028T and G3010A (applicable only for the trichological material in anagen). The mtDNA sequences of different biological materials did not show intra-individual differences. Protocol that could be used for the forensic practice with the use...
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Role of polymorphic DNA markers in personal identification and determination of selected phenotypic traits
Zidkova, Anastassiya ; Korabečná, Marie (advisor) ; Drábek, Jiří (referee) ; Hradil, Roman (referee)
Nowadays intensive research is conducted for application of genetic polymorphisms for degraded samples analysis, identification and kinship determination. Another area of research in forensic genetics is biogeographical and phenotypic traits (eye, hair and skin color) determination. First part of presented work dealt with population study on the Czech popu- lation using Investigator DIPplex (QIAGEN, Germany) marker set containing 30 autosomal insertion-deletion polymorphisms. Power of Discrimination (PD), which is the probability of random selection of two persons with different genotypes, was 99.9999999999% for the whole marker set. This part of study concluded that ana- lyzed marker set is suitable as an additional marker panel for identification and kinship determination in the Czech Republic. Second part of the presented study was devoted to population research of Cen- tral Croatia using Mentype Argus X-8 kit (QIAGEN, Germany) containing 8 short tandem repeat polymorhisms located on X choromosomes (X-STR) divided into 4 linkage groups. PD for the whole kit reached 99.9999% and 99.99999999% for males and females, respectively. This kit could be used in Central Croatian population for kinship analysis and for identification as an additional marker panel. The next part of the presented study was the...
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Association study with the ADA gene and detailed characterization of two chromosome aberrations in autism
Vážna, Alžběta ; Sedláček, Zdeněk (advisor) ; Ferák, Vladimír (referee) ; Korabečná, Marie (referee)
Autism affects up to 1/150 children and represents therefore a serious social problem. It is a complex disorder with a clearly documented genetic component, but so far unexplained aetiology, which is currently a subject of intensive research. In the field of genetics various gene and chromosome defects are examined, as well as other mechanisms, including epigenetics, which could play a role in pathogenesis. In our work we tried to replicate the finding of association between the ADA*2 risk allele and autism in a sample of 385 Czech children. Our sample was larger than those originally published. We also focused on individual endophenotypes (types of autism, degree of mental retardation and co-morbidity). Our results did not confirm the association of autism with the ADA*2 allele in the complete sample or in any of the subsets. Chromosomal changes represent another finding in autistic patients. We performed the analysis of a ring chromosome 17 and a chromosome X deletion in two patients. Our studies represent an up to now unimaginable link between classical cytogenetics and molecular genetics at the DNA sequence level. As the first in the world, we described the structure of a human ring chromosome. Characterization of the defects allowed us to speculate on the impact of the genes involved in the phenotype...
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