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Epigenetic regulation of HLA genes asociated with celiac disease
Hudec, Michael ; Černá, Marie (advisor) ; Hušáková, Markéta (referee)
Introduction: HLA class II system presents one of the most important mechanism in immune system, which is able to recognise pathogens and damaged cells. Some HLA class II alleles are associated with autoimmune diseases, for example celiac disease, which is typical by chronic inflammation of small intestine and other following symptoms. The risk HLA class II variants are DQ2 and DQ8. Epigenetic mechanisms that regulates gene expression, especially methylation of cytosine in promoter region of DQ2 and/or DQ8 alleles, could have influence on development of T lymphocytes in the thymus, where T-lymphocytes develop and pass a few stages in, and only the survival clones can be part of function immune system. Aim: The aim of this study is to compare methylation level of promoter regions of HLA DQ2 and DQ8 alleles between celiac patients and healthy controls. Another goal is to compare expression level of DQ2 and DQ8 variants between these two groups. Methods: DNA and RNA were isolated from full blood of two sets of donors. DNA was converted by bisulphite conversion and then amplified by Nested PCR. The PCR product was cloned to bacteria. Than positive colonies were selected. Subsequent methylation analysis was performed. RNA was converted to cDNA by Reverse transcription. Relative expression was analyzed...
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Immunological markers for type 1 diabetes prediction
Včeláková, Jana ; Štechová, Kateřina (advisor) ; Černá, Marie (referee) ; Kverka, Miloslav (referee)
5 Abstract Type 1 diabetes (T1D) is an organ specific autoimmune disorder characterised by the immune-mediated destruction of insulin-producing pancreatic beta cells. Beta-cell destruction is mediated primarily by cellular components of the immune system, especially auto-reactive T cells. Nowadays, a goal of many studies is built up the best system for identification of individuals in prediabetes stage and to treat them to preserve sufficient amount of insulin producing beta cells. We identified several candidate pathways and proteins which could be important in pathology of T1D, like an antiviral responses and differentiation of Th17 pathways. We observed differences in dendritic cells count and in their cytokines production. Our data support the notion that the establishment of proinflammatory environment in genetically predisposed individuals along with the involvement of non-specific immune mechanisms is critical for the initiation of autoimmune, destructive insulitis. Nonetheless, patient's autoantibody profile reflects the type of cellular immune response and should be take in a count as well. This finding may be useful in design of immunointervention studies to prevent T1D. Considering the heterogeneity of the clinical course of this disease and perhaps different mechanisms of molecular pathology,...
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Gene expression of specific parts of CNS in a rat model for transgenerational transmission of drug addiction.
Radilová, Kateřina ; Černá, Marie (advisor) ; Rothová, Olga (referee)
Methamphetamine (MA) is a drug often abused by drug-addicted women and also is the mostly used drug in the Czech Republic. This drug crosses the placental and hematoencephalic barriers easily and therefore it can negatively affect not just mother herself, but also the development of the fetus. Even though there have been several published studies evincive negative effects of MA to posterity of drug-addicted mothers, the most critical term during pregnancy for the fetus remains unknown. In this study are examined CNS structures, which are directly influenced by methamphetamine action. Their function may lead to drug addiction through pleasurable feelings, such as reward feeling evoking. In the next chapter are analysed neurotransmitters and their transporters, which are related to methamphetamine effects. Studying their structure, localization or development during life may help to bring near the drug functioning in different life periods. Methamphetamine itself and its effects are closer described in the last part of this study. The problem of methamphetamine exposed fetus is outlined there - prenatal and postnatal so far known differences, but also sex differences in methamphetamine pharma- cokinetics.
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Prolactin and circulating monocytes - their significance and function in pathogenesis T1DM. Study in vitro
Bencúrová, Dominika ; Daňková, Pavlína (advisor) ; Černá, Marie (referee)
Introduction: Type 1 diabetes mellitus (T1DM) is characterized by an absolute deficiency of the insulin-producing beta cells of the islets of Langerhans in the pancreas. Among mechanisms that lead to pathogenesis of T1DM, innate immunity including key cells monocytes are involved. Based on expression of CD14 and CD16 surface markers, monocytes are classified into three subtypes with different functions. In addition to other markers, monocytes express on their surface prolactin receptor (PRLR) and toll-like receptors (TLR), which induce inflammatory responses, and produce extrapituitary hormone prolactin (PRL) that affects immune response. The aim of thesis was to study an effect of exogenous prolactin on the immune responses of monocytes and to try to detect its possible role in the pathogenesis of T1DM. Material and Methods: In vitro cultivation and stimulation of monocytes derived from 10 patients with T1DM and 10 healthy controls. As stimulating agents were used PRL and/or lipopolysacharide (LPS). For determination of mRNA levels of the studied cytokines (TNF-α, IL-6, FOS, IRF-1), total RNA isolated from monocytes acquired by immunomagnetic separation has been quantified by using Real Time PCR. The expression of surface markers (CD14, CD16, PRLR) was detected by flow cytometry. For detection of...
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The study of epigenetic regulation of HLA class II genes at the level of histone modification
Černoch, Marek ; Černá, Marie (advisor) ; Hirsch, Ivan (referee)
Introduction: The epigenetic modifications can significantly affect and alter the gene activity by regulating their expression, having direct impact on various processes in human body. Epigenetic processes are involved in ethiopathogenesis of many diseases. From this point of view, MHC genes are very important as they were linked to many autoimmune disorders, for example type 1 diabetes mellitus. In general autoimmune diseases appear to be connected to certain MHC class II genes. Aims: The aim of this thesis is to determine the relationship between expression levels and histone modifications present in the promoter area of MHC class II gene, DQA1. Moreover, we also analyze and compare the DQA1 gene mRNA expression depending on the QAP promoter allele. Methods: We isolated both nucleic acids (DNA and RNA) and leukocytes from peripheral blood samples collected from voluntary donors. DNA was utilized for genotypization of individuals. RNA was subjected to reverse transcription and the quantitative PCR was performed in order to determine the level of expression. Leukocytes were used for chromatin immunoprecipitation, which was evaluated using quantitative PCR. Results: The expression level of QAP allele 3.1 was found to be higher than for the rest of the alleles Allele 4.1A showed, on the other hand,...
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Epigenetics mechanisms
Šornová, Veronika ; Černá, Marie (advisor) ; Koc, Michal (referee)
Epigenetics is the study of heritable changes in gene activities that are not caused by changes in the DNA sequence. Epigenetic mechanisms can be employed at many levels, from transcription to translation. They include DNA methylation, histone modification, and with it connected chromatin modification, and RNA interference. The result is the change of chromatin conformation leading to decrease or increase of certain gene expression, X-chromosome inactivation or gene imprinting. Epigenetic regulation plays important role in etiopatogenesis of multifactorial diseases. Genetic predisposing factors (in autoimmune diseases there are genes of major histocompatibility complex) and environmental factors, which affect our genome just through epigenetic modifications, are involved in their manifestation. Key words: Epigenetic mechanisms, DNA methylation, histone modification, RNA interference, genomic imprinting, X-chromosome inactivation, multifactorial disease.
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Genetic backround and new biochemical markers in pathological pregnancy
Germanová, Anna ; Kalousová, Marta (advisor) ; Černá, Marie (referee) ; Bílek, Radovan (referee)
Receptor for advanced glycation end products (RAGE), its soluble form (sRAGE) and glyoxalase 1 (GLO 1) are important part of pathogenesis of many chronic diseases. The aim of this thesis was to elucidate role of sRAGE, four chosen RAGE polymorphisms and one GLO 1 polymorphism in physiologic pregnancy and in pregnancy with complications. Serum sRAGE levels were determined in healthy pregnant women (N=120) and in pregnancies complicated with threatening preterm labor (N=99), preeclampsia (N=35), intrauterine growth restriction (IUGR) (N=22) and intrahepatic cholestasis of pregnancy (ICP) (N=14). Four RAGE polymorphisms (RAGE -429T/C, -374A/T, RAGE Gly82Ser (557G/A), RAGE 2184A/G) and one glyoxalase 1 polymorphism GLO 1 Glu111Ala (419A/C) were studied in the same population of healthy pregnant women and women with pathological pregnancy. Serum sRAGE levels are low in comparison to non-pregnant controls, but they vary during the physiologic pregnancy. Serum sRAGE levels are low in the 1st trimester, increased in the 2nd trimester and again decreased in the 3rd trimester. Women with premature labor have significantly decreased serum sRAGE levels in comparison to women with threatening premature labor and in comparison to healthy pregnant women. sRAGE correlates negatively with leukocyte count in preterm...
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