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Humoral response to heat shock proteins in patients with juvenile idiopathic arthritis and in relation to complications following transplantation of hematopoietic stem cells
Zlacká, Denisa ; Hromadníková, Ilona (advisor) ; Říhová, Blanka (referee) ; Posová, Helena (referee)
ln 0\If worlu w~ aimN to.rudy the ml~ of ll! H>p60. U bol'i< Hsp6) md .u=;- indocibl~ ll! Hsp70 in pathogmesis of juvmil~ idiopathi<: anhriti, (JlA) md !haom:ot<>id anhriti, (RA), ln tbe.rudy of hUlllOflll =pons.r ~gainsl ll! Hsp60, M. 00.1:. H'p65 md rh Hsp70 w= found sigoificml rl«.·atM l~..,ls of mTi~, against -iodurib~ Hsp70 in a tot:tl cohon ofp:lTi..u, "~Ih J1'. whm COII1pMM with br:ollhy indi\idWS. 1k pr~\':oIroc~ of mTi-Hsp70 antibodies is much high<f in JlA pati<n15 wbro COIllp:If~d "ith br:olthy controls suggesTing tbeir possiblr rol~ in patbological mrclwlism oftbe ~ ln Ibr study of ~ ~,ioo of ioduribl~ Hsp70 W~ dtt<-crN high mrrubrnJt a~,ion of Hsp70 00 fibrob1asl-liU .yn,,,"i:ol c~Us dtri"N ftom syno\i. aff..,Tnl by allloimmunr inflamm:llion. W~ 5Cfffnr<l tbe 1""""""" of Hsp =q>IOI'S 1ikr TI1t2 2I!d TI.R4. CDl4. CDM. CD40 :md CD91 :os w~U ., thriI associaTion "ith inducibl~ Hsp71l OD RA <krj\"N fibrobbst-hl:~ syno,-i:Il cdls. 80th .ynovi.>! cdl, 2I!d skin fibroblasts aprrsstd bigh ~",l, of cdl.ruďac~ CD9!. bo\\'~""-_ DO Ol lov, b",ls of otbrr rtttplOf1i W~ sprrul:IlN tIw indociblr &p70 rd~2Sr<I from infIamrd S)'OO''ia1 1~ mighl br c:tpturt<l 0010 tbe ""U ,UĎ""" of 'yn<l\ia! ""U, from tbe txtrac~llular sp:Ic~ \;" CD91 fm'Jlt()[ ln tbe ,rudy of htm10ral respon= agairul tbe brat sbock...
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The use of cell-free nucleic acids in maternal plasma for non-invasive prenatal diagnosis of monogenic diseases, placental insufficiency-related complications and Down syndrome
Veselovská, Lenka ; Hromadníková, Ilona (advisor) ; Schierová, Michaela (referee)
Since the discovery of cell-free fetal DNA in peripheral blood of pregnant women, cell-free nucleic acids in maternal plasma are explored in relation to non-invasive prenatal diagnosis of various fetal conditions and pregnancy complications. Non-invasive prenatal diagnosis of monogenic diseases represented by TSC1-linked tuberous sclerosis could be achieved by detection of paternally-inherited mutant allele in the pool of maternal alleles in plasma. Reliability of detection of mutant allele could be improved by simultaneous mutation haplotype analysis or detection of universal fetal marker. None of the 3 methods (allele- specific real-time PCR, SNaPshot minisequencing and quantitative fluorescent PCR) evaluated using artificial mixtures and maternal plasma samples reliably and accurately detected low-frequency allele distinguished by point mutation, SNP or microsatellite in TSC1 gene or in its close proximity. We developed a strategy for prediction of proportion of informative couples for panel of SNPs of interest that can be applied to any monogenic disease. Exploiting differential methylation of promoters of genes RASSF1A, HLCS and OLIG2 in maternal and fetal genome, we failed to establish functional fetal marker. MicroRNAs of placental origin released into plasma could serve as biomarkers of...
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Detection of extracellular microRNAs in maternal circulation - diagnosis and prognosis of pregnancy related complications
Ondráčková, Markéta ; Hromadníková, Ilona (advisor) ; Daňková, Pavlína (referee)
MicroRNAs (miRNAs) are small noncoding RNAs of length 18 to 25 nucleotides that regulate gene expression posttranscriptionally. Expression of some miRNAs is tissue specific. I assumed that pregnancy induced complications associated with placental insufficiency could be characterized by a unique profil of placental-specific miRNAs in maternal circulation. I measured concentration and gene expression of selected miRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) in the plasma of patients with preeclampsia (PE), fetal growth restriction (FGR) and gestational hypertension (GH). The control group consisted of patients with a normal course of pregnancy (FG). I processed 168 plasma samples, the representation of individual diagnosis were as follows: PE 63, FGR 27, GH 23, FG 55. Detection and quantification was carried out by quantitative real-time PCR. I identified three miRNAs with elevated levels in a group of preeclamptic patients: miR-517*, miR-520a* and miR-525. The severity of the PE, which was characterized by a form of the disease (mild or severe PE) and term (before or after the 34th week of pregnancy), did not have a statistically significant effect on the levels of miRNAs. More than a quarter of patients had PE superimposed on previous hypertension. Previous history of...
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Relation between cardiovascular microRNAs and pregnancy-related complications
Dalecká, Linda ; Hromadníková, Ilona (advisor) ; Daňková, Pavlína (referee)
During the pregnancy, there are many complications, which can affect a fetal development and eventually, its or mother's life. Pre-eclampsia is one of these complications. It is a hypertensive disorder, which appears during the pregnancy. Another example of these complications is an intrauterine growth restriction. It is a condition, when the fetus is not able to reach its genetical growth potential. Both of those pathological disorders are accompanied by changes in microRNA gene expression in placenta, for example miR-16, miR-21, miR-210. Knowledge about these changes in gene expression could represent unique instruments in the field of noninvasive prenatal diagnosis of these disorders. This could be benefitial due to the fact, that both of these disorders are responsible for increasing maternal and perinatal morbidity and mortality. This work focused on microRNAs, their expression in aformentioned disorders and benefits, which could bring in diagnostics. In this work these two disorders, characteristics of microRNAs and their biogenesi are described. MicroRNAs are interesting for us as potential biomarkers for noninvasive prenatal diagnostics becouse they are present in body fluids, such as plasma, breast milk or amniotic fluid and they are not subject of rapid degradation. Key words:...
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Study of molecular markers correlating with prognosis and result of therapy colorectal carcinoma.
Protivová, Martina ; Souček, Pavel (advisor) ; Hromadníková, Ilona (referee)
Colorectal cancer is one of the most common cancer in this country and abroad. A major problem in the treatment of this disease is interindividual variability in response to treatment, since a large proportion of patients show resistance or adverse toxicity to the drug. The cause of this variability can be an individual's genetic makeup. From this perspective a need to find molecular markers for prognosis of the disease and markers through which we can predict response to therapy is growing. The main aim of this study was to find differences in gene expression between healthy and tumor tissue from patients well and poorly responding to treatment based on 5-fluorouracil and compare the results to clinical data. This study aimed to evaluate gene expression of 14 potential molecular markers involved in 5-FU pathways involving metabolism, transport, and objectives of the drug. Patients selection for the study was based on 5-FU regimens treatement. Expression was evaluated in two independent sets consisting of patients with indicated palliative or adjuvant chemotherapy. For each patient malignant and paired nonmalignant tumor tissue was available. Gene expression in these samples was measured using real-time RT-PCR with relative quantification using the preamplified cDNA. In the first phase the gene...
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Heat shock proteins - - their role in diagnosis and prognosis of pregnancy related complications
Dvořáková, Lenka ; Hromadníková, Ilona (advisor) ; Růžičková, Šárka (referee)
Heat shock proteins increase their gene expression after exposure of cells or organisms to some forms of stress, which may be high temperature, infection, inflammation, hypoxia, lack of nutrients and water. Stressful situations for the body are also pregnancy-related complications associated with placental insufficiency - preeclampsia and IUGR, as well as other pregnancy-related complications such as fetal growth restriction and gestational hypertension. Therefore, I examined whether the occurrence of pregnancy-related complications (preeclampsia, fetal growth retardation, gestational hypertension) affect the gene expression of heat shock proteins. Five hsp systems was detected, namely Hsp27, Hsp60, Hsp70, Hsp90 and HspBP1 in placental tissue samples and whole maternal peripheral blood. Samples came from women with physiological pregnancy and from women with certain pregnancy-related complications (PE, FGR, GH). RNA was isolated from the samples. Detection of hsp expression was performed by using real-time RT-PCR and the comparative Ct method. Changes in gene expression between the test samples and reference sample were examined. To assess the difference between physiological pregnancies and pregnancies with selected pregnancy- related complications, an analysis of variance (ANOVA) was used....
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Non-invasive prenatal diagnosis based on the presence of extracellular fetal nucleic acids in maternal circulation
Vullum, Kristin ; Hromadníková, Ilona (advisor)
Prenatal diagnosis is now part of established obstetric practice in many countries. However, conventional methods of obtaining fetal tissues for genetic analysis, including amniocentesis and chorionic villus sampling, are invasive and constitute a finite risk to the unborn fetus. Approximately one procent end with abortion because of the procedure, even though the fetus is healthy. Other methods also exist, that without risks, can give information about certain conditions that the fetus might have. But a certain diagnosis can not be given without several different examination methods by a specialist. It has been a long-sought goal in human genetics to develop methods of obtaining fetal genetic materials for analysis, without putting a risk on the mother and fetus. Research in this field have been intensified the last years, mostly because of technologically improvements that have given us new techniques and new valuable information. Based on analysis of fetal nucleic-DNA in maternal blood, pregnant women are able to get a diagnosis that is totally risk free for the fetus. Unfortunately, with new technology there will always be new problems that need to be discussed and solved before they can be used in clinical practice. The ideal situation would be that there was some kind of world wide policy for the...
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Role of heat shock proteins in the pathogenesis of leukaemia
Kopřivová, Olga ; Hromadníková, Ilona (advisor) ; Černá, Marie (referee)
(Abstract) Some of heat shock proteins (Hsp), for example the inducible form Hsp70, are expressed on the surface of tumour cells. High Hsp expression is reflected in tumour cell features, such as ability to progression, to metastasize and resistance to apoptosis. The question is whether Hsp gene expression correlates with surface expression. The aim of this master thesis is to compare surface and gene expression of Hsp70 and observe the gene expression of some other Hsp proteins (Hsp27, Hsp60, Hsp90 and HspBP1) in leukaemia. The research was carried out on cell lines obtained from leukaemic blasts of patients with acute myeloid leukaemia: UoC-M1, HL-60, OCI/AML3, THP-1, HU-3 and TF-1 that had been cultivated in vitro. Hsp70 surface expression was detected using flow cytometry, and gene expression of each Hsp was studied using real-time RT-PCR. It was found out that high surface expression of Hsp70 did not correlate with gene expression in consequence of negative feedback applied in Hsp expression regulation. Hsp27 gene expression was increased compared to negative (healthy) control on all tumour cell lines, with the highest increase on the THP-1 line. Hsp60 gene expression was increased compared to negative (healthy) control on all tumour cell lines and there were not remarkable differences in...
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