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Study of extracellular placental specific microRNAs in maternal circulation and their utilization in clinical diagnostics of pregnancy-related complications
Kotlabová, Kateřina ; Hromadníková, Ilona (advisor) ; Černá, Marie (referee) ; Korabečná, Marie (referee)
Objectiees: Our etudy inieetigated the poeeible utliiaton of extracellular microRNAAe ae noiel non-iniaeiie biomarkere for diaignoetce of preignancy-related complicatone euch ae igeetatonal hyperteneion (GH), preeclampeia (PE) and fetal igrowth reetricton (FGR). Firet, we identifed appropriate preignancy-aeeociated (placenta epeciifc) microRNAAe in maternal circulaton in preignanciee with normal couree of igeetaton. Then, we quantifed eelected extracellular C19MC microRNAAe in maternal circulaton oiertme in normally proigreeeinig preignanciee. Subeequently, we compared C19MC microRNAA expreeeion proiflee in maternal circulaton between preignanciee with clinically eetabliehed preignancy-related complicatone (PE, FGR, GH) and igeetatonal-aige-matched controle. Finally, monitorinig of eelected placenta epeciifc C19MC microRNAAe in maternal circulaton within the ifret trimeeter of igeetaton wae performed with the aim to identfy extracellular C19MC microRNAAe able to diferentate between normal preignanciee and thoee at riek of eubeequent deielopment of preignancy- related complicatone. Reeultes: The leiele and expreeeion proiflee of extracellular placental epeciifc microRNAAe in circulaton of non-preignant indiiiduale and preignant women were performed ueinig real- tme RT-PCR. The eelecton of...
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Postpartal expression profile of cardiovascular microRNAs with regard to occurrence of pregnancy-related complications - study on mothers 3-10 years after the delivery
Marvanová, Veronika ; Hromadníková, Ilona (advisor) ; Korabečná, Marie (referee)
The aim of this study was to investigate gene expression of cardiovascular miRNAs in peripheral blood of mothers after delivery. MiRNAs are small non-coding RNAs, which significantly modulate posttranscriptional adjustments of mRNA and thus regulate gene expression across biological processess. Dysregulation of miRNAs is associated with many pathological phenomena, thanks that we can use them for diagnosis and potentionaly we can treat these diseases by the manipulation of miRNA gene expression. We examined gene expression of circulating miRNAs associated with cardiovascular diseases, and we investigated, how the expression profile depends on pregnancy course and manifestation of pregnancy-related complications. For this purpose we examined material from 221 mothers 3-10 years after delivery. A group with identical pregnancy-related complication was always compared with a group of mothers after physiological pregnancy. Gene expression of 29 cardiovascular miRNAs in peripheral blood was studied using reverse transcription and quantitative real-time PCR. It was confirmed, that the expression profile of miRNAs differed between pregnancy-related complications and physiological controls. We also confirmed, that the profile of gene expression discovered at mothers 3-10 years after delivery was different...
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The significance of extracellular DNA in osteoclastogenesis from peripheral blood precursors - in vitro study
Jelínková, Ivana ; Daňková, Pavlína (advisor) ; Korabečná, Marie (referee)
Introduction: Extracellular DNA (ecDNA) is a common component of blood plasma. Increased levels of ecDNA in plasma can be found in some autoimmune diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis or celiac disease which are associated with inflammatory processes. These diseases are also associated with an increased risk of osteoporosis. Bone is a dynamic structure undergoing constant modelling caused by osteoblasts, osteocytes and osteoclasts. Shifting their equilibrium can lead to pathological conditions such as osteoporosis. In this thesis we focused on elucidating whether ecDNA, an inflammatory agent with proven immunoregulatory effects can alter differentiation potential of monocytes and alternatively lead to osteoclastogenesis via TLR9. Material and methods: We obtained monocytes from peripheral blood of healthy donors and cultivated them with four types of ODNs control (CO), stimulatory (ST), inhibitory (INH, telomeric (TLM) with phosphodiester (-pO) or phosphorothioate (-pS) backbone for two weeks to establish their effect on differentiation potential of monocytes into osteoclasts. Osteoclastogenesis was evaluated by number of yielded osteoclasts observed on a light microscope. To establish the effect of ODNs on osteoclast activity samples were analysed by qPCR for...
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Postnatal screening of cardiovascular microRNAs in children descending from pregnancy-related complications
Semencová, Andrea ; Hromadníková, Ilona (advisor) ; Korabečná, Marie (referee)
Children descending from pregnancy complicated by gestational hypertension, preeclampsia or fetal growth restriction have a lifelong increased risk of development of cardiovascular disease. This study investigates the expression profile of 29 cardiovascular and cerebrovascular microRNAs in children at the age of 3 to 11 years. MicroRNAs are short non-coding RNA molecules affecting gene expression by posttranscriptional modifications of mRNA, which affects biological processes. Abnormal microRNA levels can lead to pathological conditions of the individual. This study explores the relationship between dysregulated microRNA levels in whole peripheral venous blood of children and the presence of complications during pregnancy. Furthermore, this study looks at expression profile specificities depending on the presence of pathology in the child's cardiovascular system, as found in a clinical examination consisting of BMI assessment and evaluation, blood pressure testing and ultrasound examination of the heart. Dysregulated profile was present in children with gestational hypertension in miR-1-3p, miR-17-5p, miR-20a-5p, miR-21-5p, miR-26a-5p, miR-29a-3p, miR-126-3p, miR-133a-3p, miR-181a-5p, miR-199a-5p, miR-342-3p and miR-499-5p. In children with preeclampsia, dysregulation was found with miR-1-3p and...
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MicroRNAs in AML pathogenesis
Koutová, Linda ; Korabečná, Marie (advisor) ; Zemanová, Zuzana (referee) ; Kotyza, Jaromír (referee)
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...
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Analysis of cell-free nucleic acids in urine of urological patients.
Šantorová, Šárka ; Korabečná, Marie (advisor) ; Brdička, Radim (referee) ; Drábek, Jiří (referee)
The two studies follow free nucleic acids in urine in search for biomarkers to distinguish urinary bladder cancer patients from controls. Bladder cancer forms 4 % of newly diagnosed oncological diseases in the Czech Republic. Nowadays, there is no accredited non-invasive method for its diagnosis, which is sufficiently accurate. Urine supernatant, which is washing the bladder mucosa and which does not contain cell debris, seems to be an appropriate source of biomarkers for non-invasive diagnosis. miRNAs, as a non-invasive biomarker of urinary bladder cancer, were studied in one of the studies. miRNAs are short noncoding RNA, which block the process of translation. miRNAs occur in all body fluids and are relatively stable. A study with three phases was assessed to find a suitable miRNA marker. 109 individuals were examined in total (36 controls and 73 bladder cancer patients). The analysis of miRNAs was based on RT-PCR (Reverse Transcription Polymerase Chain Reaction). In the first phase, the urine of 59 individuals was analyzed on TaqMan array card with 381 miRNAs. In the second phase, the results of the first phase were confirmed on the same cohort by a single miRNA assay. In the third phase, a new cohort was used (23 controls and 27 bladder cancer patients), analyzed by a single miRNA assay again....
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Analysis of cell-free nucleic acids and its potential clinical application.
Pazourková, Eva ; Korabečná, Marie (advisor) ; Drábek, Jiří (referee) ; Vodička, Radek (referee)
This work presents the results ofour research of cell-free nucleic acids (cfNA). The first part shows changes in methylation patterns of immune response genes promoters that are detectable in plasma during the hemodialysis sessions and also differences in methylation between patients and healthy subjects. Alterations include genes that play their role in the regulation of hematopoiesis and these changes are in close relation with the need of anemia therapy. In the other plasma cfNA study we detected miRNA signatures in patients with acute myeloid leukemia at diagnosis (6 highly abundant miRNAs found) and in remission achieved after standard chemotherapy (trend to n01malization, lower levels ofthese miRNAs). Another part of work presents data from the study of potential non-invasive biomarker of bladder cancer. The amounts of cfDNA in urine are higher in patients than in healthy subjects and there were found 5 down-regulated miRNAs. Simultaneously it was established set of 30 miRNAs that are constantly present in urine supematants independently on sex, age and healthy status of subjects. The last part presents analysis ofcell-free fetal DNA. We analyzed differences between a new quantification method - droplet digital PCR and real-time PCR which is used routinely nowadays. Slightly more precise was...
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Selection of appropriate INDEL polymorphisms on the X chromosome to detect the fetal fraction of free circulating DNA during pregnancy with female fetuses
Veselá, Barbora ; Korabečná, Marie (advisor) ; Šimková, Halina (referee)
The analysis of cell-free fetal DNA (cffDNA) in maternal plasma became an important component of non-invasive prenatal diagnostics in recent years. Detection of Y chromosome sequences in free circulating DNA (cfDNA) indicates the presence of a male fetus; the absence of the Y-chromosomal signal confirms the female gender. The aim of this work is to confirm the utility of insertion-deletion polymorphisms (INDEL) for determination of the female sex of the fetus using the analysis of cffDNA by Digital Droplet PCR (ddPCR). In the thesis, X chromosomal INDEL polymorphisms with a suitable allelic frequencies were selected from databases to allow the determination of the presence of the paternal X chromosome in cffDNA and to lead to the confirmation of the female sex of the fetus by a positive amplification signal and to rapid determination of the fetal fraction size of the circulating DNA. Molecular genetic examination of these polymorphisms was established using ddPCR method. A population study was carried out to verify the utility of the proposed polymorphisms with regard to non-invasive prenatal diagnosis. We examined X chromosomal INDEL polymorphisms: rs2307932, rs16397, rs16637, rs3048996, rs16680 using the ddPCR methodology. In order to obtain population data, we performed tests from the buccal...
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Postpartal expression profile of cardiovascular microRNAs with regard to occurrence of pregnancy-related complications - study on mothers 3-10 years after the delivery
Marvanová, Veronika ; Hromadníková, Ilona (advisor) ; Korabečná, Marie (referee)
The aim of this study was to investigate gene expression of cardiovascular miRNAs in peripheral blood of mothers after delivery. MiRNAs are small non-coding RNAs, which significantly modulate posttranscriptional adjustments of mRNA and thus regulate gene expression across biological processess. Dysregulation of miRNAs is associated with many pathological phenomena, thanks that we can use them for diagnosis and potentionaly we can treat these diseases by the manipulation of miRNA gene expression. We examined gene expression of circulating miRNAs associated with cardiovascular diseases, and we investigated, how the expression profile depends on pregnancy course and manifestation of pregnancy-related complications. For this purpose we examined material from 221 mothers 3-10 years after delivery. A group with identical pregnancy-related complication was always compared with a group of mothers after physiological pregnancy. Gene expression of 29 cardiovascular miRNAs in peripheral blood was studied using reverse transcription and quantitative real-time PCR. It was confirmed, that the expression profile of miRNAs differed between pregnancy-related complications and physiological controls. We also confirmed, that the profile of gene expression discovered at mothers 3-10 years after delivery was different...
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