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Fetal microchimerism in gynecologic malignancies.
Pírková, Petra ; Hromadníková, Ilona (advisor) ; Daňková, Pavlína (referee)
The existence of fetal microchimerism has been demonstrated many years ago. This phenomenon is associated with observation of two or more genetically different populations of cells present in one person. Fetal microchimerism originates naturally during pregnancy, by bidirectional transfer of the cells through placenta from fetus to mother (fetal microchimerism) and from mother to fetus (maternal microchimerism). In some cases fetal cells persisted in mother for decades after pregnancy. In my thesis I showed the presence of fetal microchimerism in tissues of endometrial cancer, breast cancer and ovarian cancer and in control, nonmalignant tissues. I worked with deep-frozen tissues, native tissues and cell cultures created from native tissues. I planed also the analysis of paraffin-embedded tissues; however this type of material showed to be unusable for fetal cells detection. On the contrary, native and deep-frozen tumor and control tissues are suitable for this type of research and fetal microchimerism was observed in part of samples. For detection and amplification of DNA extracted from tissues and cell cultures I used quantitative real-time PCR and SRY gene located on the Y chromosome as a marker of fetal cells. I detected the presence of male fetal cells. Fetal genome was found in both tumor and...
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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...
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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
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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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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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Signification of activatory and inhibitory ligands on leukemia cells to NK stimulations.
Imryšková, Zuzana ; Hromadníková, Ilona (advisor) ; Bezouška, Karel (referee)
In last decades, with expansion of immunological and biological methods are developed new diagnostical and treatment processes, which enable stratification of patients into sanative groups and trend to individual therapy. Absolutely transparent are effects relevant to leukemia. Present treatment procedures enable not only longer survivance of patients, but often their stable sanation. In present time is in progress intesive research imunotherapy NK cells, which could be able to finish minimal residual disease after chemotherapeutical treatment, which is evoke by persistant malignant cells. Next advantage of this treatment procedure is elimination of system disease in cosequence of exactly pointed cure. In this work he attended in vitro testing to possibility of utilization imunotherapeutic treatment by NK cells acute and chronic myeloid leukemia and chronic lymfoblastic leukemia. Using flow cytometry methods we detected activation and inhibitory ligands which are recognized by NK cells on the cell surface of leukemia blasts. These are members of MHC complex HLA-E, molecules derived from MHC class I (MICA, MICB), UL16-binding proteins (ULBP-1, ULBP -2, ULBP -3, ULBP -4) and also Hsp70 protein according to the newest observation. We also atended to detection of expression inducible heat shock...
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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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The role of human heat shock protein HSP70, HSP60 and mycobacterium bovis HSP65 in pathogenesis of graft versus host disease and rheumatoid/juvenile idiopathic arthritis
Nguyen, Thi Thu Hien ; Hromadníková, Ilona (advisor) ; Tučková, Ludmila (referee) ; Stříž, Ilja (referee)
We investigated IgG antibodies against Mycobacterium bovis heat shock protein 65 (M. bovis Hsp65) fragments produced by cleavage with cyanogen bromide (CNBr) in 10 healthy controls, 11 patients with juvenile idiopathic arthritis (JIA) and 10 children with various malignant and non-malignant diseases before allogeneic stem cell transplantation (SCT) by using Western blotting (WB). CNBr cleaved M. bovis Hsp65 to three larger fragments: Pl-163, molecular weight (Mw) 17.1 kDa; P191-285, Mw 10.4 kDa and P290-534, Mw 25.3 kDa. Humoral reactivity against M. bovis Hsp65 derived fragments differed on a case-by-case basis. The sera either of JIA patients ar those befare SCT reacted with individual M. bovis Hsp65 fragments more frequently when compared with healthy controls. While IgG anti-Hsp65 antibody levels showed no significant differences between the small studied cohorts, significantly higher levels of antibodies against M. bovis Hsp65 epitopes were observed in patients befare SCT and JIA patients when compared with healthy controls. Comparing WB reflectance densities (DRl and DR2), significantly elevated antibodies against Pl-163 (DRl: p=0.014; DR2: p=0.022) and P290-534 (DRl: p=0.009; DR2: p=0.003) epitopes were found in patients befare SCT. Similarly, significantly increased DRl and DR2 values of antibodies...
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Heat Shock Proteins - Focus on Therapeutic Approaches in Oncology
Rutkowski, Radoslaw ; Hromadníková, Ilona (advisor)
Since the discovery of heat shock proteins in 1960s and their immunogenic properties 20 years later in 1980s they have been under rigorous investigation with great hopes for advent of immunotherapy in oncology as well as other pathologies. So are the HSPs a breakthrough in therapy of cancers or will they be just another form of palliative treatment of these notorious and deadly diseases? HSPs is a family of proteins expressed virtually by all living organisms. These proteins have a number of functions that are crucial to organism's intracellular and extracellular homeostasis. Due to the significance of HSPs, over the eons of the evolution, they have been highly conserved in our genetic code. The human HSPs shares almost 50 percent homology with simple unicellular prokaryotes to well over 95 percent homology with higher species of living organisms. Despite of the fact that HSPs have been knows for approximately 50 years and despite of the intensive resears in the field, currently Russia is the only place where HSP immunotherapy has been approved as of 2008 for treatment in renal cell carcinoma. The HSPs immunotherapy has proven benefitial as an adjuvant treatment in some stages of a selected cancers. The advantage of HPSs immunotherapy is that it has less side effects compared to standart chemotherapy...
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Non-invasive prenatal diagnosis based on the presence of extracellular fetal nucleic acids in maternal circulation
Liberko, Marián ; Hromadníková, Ilona (advisor)
The discovery of circulating cell-free fetal DNA in maternal plasma/serum, in 1997, and the demonstration of the relative easiness and reliability with which it can be detected have opened up a new possibilities for NIPD. NIPD offers diagnostics of certain abnormalities, with minimalization of the risk of abortion when compared to standard invasive procedures such as chorious villi sampling and amniocenthesis. Compared to fetal cells in maternal circulation, cffDNA in maternal plasma could be detected earlier, more frequently and in greater amounts than the cellular species. The clearance of cffDNA from maternal circulation is very rapid and therefore it is a suitable target for prenatal diagnostics without being biased by any previous pregnancies. The detection of fetal derived Y-chromosomal sequences in maternal plasma has potential for the application in the prenatal diagnostics of X-linked disorders. Besides Y-chromosome, the first genetic locus which was examinated was RHD gene. Its presence proves that the fetus is RhD-positive. Other groups have focused on the detecion of chromosomal aneuploidies (mainly trisomy 21), and paternally inherited mutations causing Huntington disease, myotonic dystrophy, achondroplasia. There have also been provided studies demonstrating and/or excluding the presense of...
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