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Extracellular microRNAs and their role in pathologies especially in the field of gynecology and obstetrics.
Štěrbová, Monika ; Hromadníková, Ilona (advisor) ; Balušíková, Kamila (referee)
microRNAs (miRNAs) represent a relatively newly discovered group of RNA molecules and they serve to regulate gene expression. In spite of processes of differentiation, proliferation and apoptosis, miRNAs influence the whole biological systems, such as embryogenesis, oncogenesis, and immunity. There have been a number of experiments in recent years concerning diagnoses and predictions of complications during pregnancy, and tumour growth. Extracellular miRNA molecules participating in circulation of patients are used in the non-invasive diagnostics. RNA molecules usually get into the extracellular fluid during the apoptosis process. I chose four diseases, which extracellular miRNA have diagnostic potential - preeclampsia, intrauterine growth retardation, gestational diabetes mellitus and breast cancer - for my work. An aberrant expression of different levels of various extracellular miRNAs has been reported in these diseases but the clinical use of microRNAs in the diagnosis and prediction of those still requires further research and optimization. Keywords: breast cancer, extracellular nucleic acids, fetal growth retardation, gestational diabetes mellitus, microRNA, PCR, preeclampsia
The Study of Leukaemia Specific Diagnostic and Prognostic Markers
Kouba, Daniel ; Hromadníková, Ilona (advisor)
In my theses I concentrated on examination of specific prognostic markers of acute myeloid leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia. Today by AML diagnose the cytogenetic abnormalities are considered the main prognostic marker. Patients, whose prognostic group it is not possible to identify with certainty just on the bases of cytogenetic examination, can be stratified by proving the presence of specific gene mutations or high expression levels of of some genes. The most important and most frequently identified prognostic marker by AML is mutation of FLT3-LM. Protein coded by this gene is also a hypothetical aim of the treatment. The crucial diagnostic marker of CML is the gene bcr-abl, which, in most of the cases, is a product of t(22,9). The discovery of inhibitors of tyroxine kinase bcr-abl brought about a substantial change into the aproach of the treatment as well as into the prognose of the ill. Through examination of immunoglobulin heavy-chain gene mutation status it is possible to divide patients with CLL into two subgroups with different pathogenesis and advancement of the illness. Other prognostic data can be acquired from cytogenetic examination and from essay of the level of CD38 and ZAP-70.
Study of placental specific microRNA expression in pacients with spontaneous preterm birth and preterm prelabor rupture of membranes
Vintrová, Iva ; Hromadníková, Ilona (advisor) ; Daňková, Pavlína (referee)
MicroRNAs (miRNAs) are small non-coding RNAs with a length of 18 to 25 nucleotides playing a pivotal role in post-transcriptional regulation of gene expression. There are miRNAs whose expression is limited to a certain tissue type and diseases which are characterized by a unique miRNA expression profile. I assumed spontaneous preterm birth (PTB) and preterm prelabor rupture of membranes (PPROM) would be characterized by a unique miRNA expression profile. I observed the gene expression of 15 placental specific miRNAs (miR-512-5p, miR-515-5p, miR-516b-5p, miR-517-5p, miR-518b, miR-518f-5p, miR-519a-5p, miR-519d-3p, miR-519e- 5p, miR-520a-5p, miR-520h, miR-524-5p, miR-525-5p, miR-526a and miR-526b-5p) in placental tissue of pacients with PTB, PPROM and women with term in labor pregnancies (FG). PTB group consisted of 24 pacients, PPROM group of 75 pacients and FG group of 20 pacients. Quantitative real-time PCR was used to quantify gene expression. In the group of PTB pregnancies I identified 3 significantly upregulated miRNAs (miR-516b-5p, miR-519d-3p and miR-524-5p) and 4 miRNAs (miR-518b, miR-519a-5p, miR-520h and miR-526a) with a trend to upregulation compared to controls (FG). In the group of PPROM pregnancies I identified 3 miRNAs (miR-519d-3p, miR-520h and miR-256b-5p) with a trend to...
Postpartum expression of cardiovascular disease-associated microRNAs - comparison of expression levels between plasma, plasma exosomes and whole peripheral venous blood
Ševčíková, Adéla ; Hromadníková, Ilona (advisor) ; Korabečná, Marie (referee)
MicroRNA (miRNA) are short non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Many miRNAs are involved in the pathogenesis of cardiovascular diseases, which is associated with altered gene expression. This work compares miRNA gene expression profiles among various biological sources - whole peripheral venous blood (whole PB), plasma and plasma exosomes. For all tested groups combined, the expression levels of miRNA were maximal in whole PB and lowered in plasma and plasma exosomes, and the expression levels of miRNA were higher in plasma than in plasma exosomes, except miR-126-3p, which had a higher level detected in plasma exosomes compared to plasma. This work also compares expression levels of cardiovascular miRNA between women with anamnesis of gestational diabetes mellitus (GDM) and physiological gravidity 3-11 years postpartum in whole PB, plasma and plasma exosomes. In whole PB, 12 of 29 tested miRNAs were up-regulated in women with prior exposure to GDM. MiR-181a-5p was up-regulated in plasma exosomes and miR-499a-5p in plasma in women with prior exposure to GDM. The changes in whole peripheral venous blood seem to reflect the complex systemic response to the changes that occurred during the onset of GDM. Women with aberrant epigenetic profiles may...
Study of extracellular nucleic acids in maternal circulation in the cases of pathological and physiological pregnancies
Žejšková, Lenka ; Hromadníková, Ilona (advisor) ; Černá, Marie (referee) ; Dvořák, Michal (referee)
In 1997, Prof. Dennis Lo discovered the presence of cell-free fetal DNA (cffDNA) in the maternal plasma and serum of pregnant women. This finding started the development of new non-invasive prenatal diagnosis methods, which are currently in the forefront of the advanced care of mother and fetus. Non-invasive genetic tests based on the detection of paternally inherited alleles, including determination of fetal sex in cases at risk of X-linked disorders or congenital adrenal hyperplasia and RHD or RHCE genotyping in alloimunized pregnancies, were quickly introduced into routine practice. This thesis focuses on the basic characteristics of cffDNA and fetal cells in maternal circulation and its usage for non-invasive prenatal diagnosis, especially in cases of placental insufficiency related complications, e.g. preeclampsia and IUGR. This severe disorder is characterized by placental dysfunction with an abnormal invasion of trophoblasts and a defect in the transformation of maternal spiral arteries, leading to placental ischemia followed by increased apoptosis of trophoblast associated with an elevated concentration of cell-free nucleic acids in maternal circulation. Until recently, cffDNA quantification studies were mostly done using amplification of SRY or DYS-14 genes localized on chromosome Y, and...
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...
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...
Heat shock proteins - their role in diagnosis and prognosis of pregnancy-related complications
Dvořáková, Lenka ; Hromadníková, Ilona (advisor) ; Holáň, Vladimír (referee) ; Lipoldová, Marie (referee)
Heat shock proteins provide a universal stress response to cells, for example, exposed to high temperature, heavy metals, extreme pH, infection, inflammation, oxygen radicals, or in case of lack of nutrients and oxygen. We assumed that stress conditions associated with pregnancy-related complications will result in a change in gene expression profile of heat shock proteins we selected for the study. Heat shock protein mRNA levels (Hsp27, Hsp60, Hsp70, Hsp90α and HspBP1) were tested in placental tissue, whole peripheral venous blood and maternal plasma in women with the following pregnancy-related complications - preeclampsia (PE), fetal growth restriction (FGR), gestational hypertension (GH), spontaneous preterm birth (PTB), preterm premature rupture of membranes (PPROM) and normal pregnancies. We also investigated whether the severity of the disease had any impact on hsp gene expression in particular biological samples. In placental tissue, overexpression of Hsp27, Hsp90α and HspBP1 was found in patients with mild preeclampsia (that does not require immediate termination of pregnancy if properly treated), and in women with late onset of preeclampsia with clinical manifestation after 34th week of gestation. Concerning preterm birth, overexpression of Hsp27 and Hsp60 was observed in both groups (PTB...
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...
First-trimester screening of pregnancy-related complications using plasma exosomal C19MC microRNAs
Špačková, Kamila ; Hromadníková, Ilona (advisor) ; Daňková, Pavlína (referee)
Pregnancy-related complications such as gestational hypertension, preeclampsia, fetal growth restriction, gestational diabetes mellitus, spontaneous preterm birth, and preterm premature rupture of membranes may have severe consequences for both the mother and the child. The development of reliable early screening methods for pregnancy-related complications has therefore been a long-term goal of obstetrics. New possibilities for prenatal diagnostics have opened with the discovery of circulating microRNAs in maternal plasma. MicroRNAs are short, noncoding, 21 to 23 nucleotides long, single-strand RNAs whose main function is to regulate gene expression. During pregnancy, both common and unique miRNAs are expressed by the placenta, amongst them the miRNAs of the C19MC cluster. Several C19MC miRNAs have been shown to display a different expression profile associated with certain pregnancy-related complications. This thesis identifies the plasma exosomal profiles of six C19MC miRNAs (miR-516-5p, miR-517-5p, miR-518b, miR-520a-5p, miR-520h, and miR-525-5p) in patients in their first trimester of gestation who later developed pregnancy-related complications, and compares them with profiles in patients with normal pregnancies.

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