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Association of genetic polymorphism of oxidative stress with diabetes mellitus type 1 and 2
Kloboučková, Lucie ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Daňková, Pavlína (referee)
Diabetes mellitus is a chronic autoimmune disease in which the immune system attacks the insulin-secreting ß-cells in the pancreas. It leads to an absolute deficiency of insulin. Chronic hyperglycemia induces increased production of reactive oxygen species, which leads to a decrease of natural antioxidant level in blood, and it contributes to genesis of diabetes complications (e.g. vascular or pulmonic). Moreover, the oxidative stress results in onset of pancreas inflammations and the damage of its ß-cells. Aims: Our aim was to assess whether or not certain genotypes or their combinations occur with higher frequency among groups of patients of type 1 diabetes (T1D) and type 2 diabetes and in a control group of healthy individuals. Methods: The study included groups of 40 T1D patients, 40 T2D patients and 45 healthy individuals. The polymorphisms of genes involved in the oxidative stress response were analyzedby using RFLP, PCR with TaqMan probes and allele specific PCR. The target genes involved superoxide dismutase SOD1 and SOD3 genes; glutathione-S-transferase GSTM1, GSTT1, GSTP1 genes; glutathioneperoxidase gene GPX1 and catalase gene CAT. The levels of plasma malondialdehyde were measured by using liquid chromatography. Results: Statistically significant differences were found in the...
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Epigenetic regulation of HLA class II genes in relation to senescence of organism
Říhová, Adéla ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Slavčev, Antonij (referee)
Introduction: Glycoproteins of the major histocompatibility complex (MHC) are an irreplaceable part of immune response regulation and immune homeostasis maintenance. The regulation of the expression plays an important role in adaptive immune response. Recently, DNA methylation in regulatory areas, crucial for DNA availability to transcription factors, is one of the most researched mechanisms of this type of regulation. The DNA methylation is, among others, related to the aging processes. Increased predisposition age-related immunosenescence in higher age could result from the changes in methylation status of regulatory areas of MHC class II genes. Aims: The aim of this thesis is to analyze the methylation status of regulatory areas of DQB1 gene and to compare the differences between generations and specific alleles. The differences in the levels of DQB1 gene mRNA transcription between generations and specific alleles is also compared. Methods: Both DNA and RNA were isolated from blood samples obtained from donors of three different age groups. DNA was genotypized and modified by bisulfite conversion. The regulatory areas of DQB1 genes were then amplified and subcloned into bacteria. The positive clones were selected and subjected to DNA methylation analysis. RNA was reverse transcribed into cDNA...
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Epigenetic regulation of DQB1 gene in patients with type 1 diabetes mellitus
Gécová, Dominika ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Holá, Dana (referee)
Background: Type 1 diabetes mellitus is a multifactorial disease caused by beta cell destruction of Langerhans pancreatic islets. From the genetic aspect the main predisposition lays on HLA class II genes (40 - 50%), molecules of which present exogenous peptides to CD4+ T lymphocytes. Enviromental factors play a crucial role in the etiopathogenesis of T1DM. Through epigenetic regulation (e.g. DNA methylation) the genetic and enviromental factors communicate. The level of methylation in the regulatory regions can significantly affect expression of these genes. Aims: The aim of the diploma thesis was to define methylation profile of HLA DQB1 alleles in type 1 diabetes mellitus patients and determine their expression. Methods: The genotyping of HLA class II genes (HLA-DRB1, HLA-DQA1, HLA-DQB1) was performed using sequence specific primers. DNA was treated with sodium bisulfite, regulatory region of HLA DQB1 was amplified and cloned into E.coli, strain DH5α/XL1-Blue. Positive clones were sent for sequencing and results analyzed. RNA was transcribed to cDNA by reverse transcription and the level of expression was analyzed by quantitative PCR. Results: Statistically significant differences were found in total methylation of DQB1*0201 and *0302 alleles in the B section of DQB1 gene. Difference in...
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Regulation of gene expression of HSP70 genes and its dependence on the genotype of HSP70 genes.
Ambrož, Antonín ; Novota, Peter (advisor) ; Kotrbová - Kozak, Anna Katarzyna (referee)
The topic of the presented thesis is the regulation of gene expression level of the three HSP70 genes in mononuclear cells. We investigated the dependence of expression regulation (induction) abiliy on selected point mutations, so-called SNPs (single nucleotide polymorphism) in the observed genes. The mononuclear cells were obtained from peripheral blood samples of healthy individuals. In order to analyze their gene expression, we selected individuals who were homozygous for at least one of the monitored point mutations. Taking into account the chosen criteria for healthy individuals we based on interviews with these individuals and their personal history. We determined the polymorphisms observed in two cell stress-inducible HSP70-1 (HSPA1A) and HSP70-2 (HSPA1B) genes and in one constitutively expressed gene HSP70-Hom (HSPA1L). Further, we have analyzed HSP70s gene expression regulation and the relation between the expression regulation and studied polymorphisms. We determined the degree of regulation of a gene expression in the studied genes in relation to two SNPs -110A/C (rs1008438), +190G /C (rs1043618) gene HSP70-1, and two SNPs +1267A/G (rs1061581), +2074G /C (rs539689 ) of the HSP70-2 gene, and the mutation of one five-nucleotide (rs9281590) HSP70-2 gene, and one SNP +2437T/C (rs2227956) of...
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Epigenetic regulation of HLA class II genes and their role in autoimmune diseases.
Čepek, Pavel ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Horníková, Lenka (referee)
Abstract Background: Type 1 diabetes (T1D) is a multifactorial autoimmune disease. Its incidence in Europe is continuously rising. The highest T1D risk is associated with HLA (human leukocyte antigen) class II genes. HLA class II molecules play a key role in regulation of immune response. They contribute to the selection of T cell repertoire by presenting antigenic peptides to the CD4+ T lymphocytes. HLA class II expression is controlled by regulatory module that is situated 150 - 300 base pairs upstream of the transcription- initiation site in all HLA class II genes. Polymorphisms in this region are linked to some autoimmune diseases. There were identified several promoter alleles (named QAP) in the HLA DQA1 gene promoter region. Most of the polymorphisms appear to be conserved within haplotype. Individual QAP alleles may have a different promoter strength by which they influence expression of HLA DQA1 gene alleles. Promoter strength can be modulated by DNA methylation. Aims:Our aim was to define methylation profile of HLA DQA1 promoters and determine the mRNA expression of individual alleles of HLA DQA1 gene in T1D patients. The mRNA expression level of HLA DQA1 gene alleles was determined using quantitative PCR. Methods: 30 diabetic pacients (age range 21 to 76 years), were included in this pilot...
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Prediction of type 1 diabetes mellitus by expression profile of peripheral leukocytes
Šornová, Veronika ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Daňková, Pavlína (referee)
Background: Type of 1 diabetes (T1D) is an autoimmune disease in which the cells of immune system attack the β-cells of pancreas. Consequently, destroyed β-cells do not produce insulin to reduce blood sugar levels. This disease is very complex, the pathogenesis is contributed by both genetic factors and environmental factors. In recent years, the number of individuals with T1D is increasing worldwide. Aims: The aim of this thesis was to investigate whether it is possible to predict T1D based on the expression profile of BACH2, CDC20, IGLL3P, EIF3A and TXNDC5 genes , which are involved in the development of immune system cells and insulin production. Another aim was to compare the expression of selected genes in children, in which the first detection of the disease may be done, and adults who suffer from prolonged T1D. The final goal was to compare the expression of individual selected genes in the HLA risk alleles DR04, DR03, DQA*05:01 a DQB*03:02. Methods: The DNA and RNA of patients with T1D and healthy individuals was isolated from blood. DNA was used to HLA genotyped. Isolated RNA was reverse transcribed into cDNA and then used in real-time PCR to determine the relative levels of gene expression. Conclusion: Significant results were obtained when the expression of BACH2, CDC20 and TXNDC5 genes...
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Association of genetic polymorphism of oxidative stress with diabetes mellitus type 1 and 2
Kloboučková, Lucie ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Daňková, Pavlína (referee)
Diabetes mellitus is a chronic autoimmune disease in which the immune system attacks the insulin-secreting ß-cells in the pancreas. It leads to an absolute deficiency of insulin. Chronic hyperglycemia induces increased production of reactive oxygen species, which leads to a decrease of natural antioxidant level in blood, and it contributes to genesis of diabetes complications (e.g. vascular or pulmonic). Moreover, the oxidative stress results in onset of pancreas inflammations and the damage of its ß-cells. Aims: Our aim was to assess whether or not certain genotypes or their combinations occur with higher frequency among groups of patients of type 1 diabetes (T1D) and type 2 diabetes and in a control group of healthy individuals. Methods: The study included groups of 40 T1D patients, 40 T2D patients and 45 healthy individuals. The polymorphisms of genes involved in the oxidative stress response were analyzedby using RFLP, PCR with TaqMan probes and allele specific PCR. The target genes involved superoxide dismutase SOD1 and SOD3 genes; glutathione-S-transferase GSTM1, GSTT1, GSTP1 genes; glutathioneperoxidase gene GPX1 and catalase gene CAT. The levels of plasma malondialdehyde were measured by using liquid chromatography. Results: Statistically significant differences were found in the...
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Epigenetic regulation of HLA class II genes in relation to senescence of organism
Říhová, Adéla ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Slavčev, Antonij (referee)
Introduction: Glycoproteins of the major histocompatibility complex (MHC) are an irreplaceable part of immune response regulation and immune homeostasis maintenance. The regulation of the expression plays an important role in adaptive immune response. Recently, DNA methylation in regulatory areas, crucial for DNA availability to transcription factors, is one of the most researched mechanisms of this type of regulation. The DNA methylation is, among others, related to the aging processes. Increased predisposition age-related immunosenescence in higher age could result from the changes in methylation status of regulatory areas of MHC class II genes. Aims: The aim of this thesis is to analyze the methylation status of regulatory areas of DQB1 gene and to compare the differences between generations and specific alleles. The differences in the levels of DQB1 gene mRNA transcription between generations and specific alleles is also compared. Methods: Both DNA and RNA were isolated from blood samples obtained from donors of three different age groups. DNA was genotypized and modified by bisulfite conversion. The regulatory areas of DQB1 genes were then amplified and subcloned into bacteria. The positive clones were selected and subjected to DNA methylation analysis. RNA was reverse transcribed into cDNA...
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Epigenetic regulation of DQB1 gene in patients with type 1 diabetes mellitus
Gécová, Dominika ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Holá, Dana (referee)
Background: Type 1 diabetes mellitus is a multifactorial disease caused by beta cell destruction of Langerhans pancreatic islets. From the genetic aspect the main predisposition lays on HLA class II genes (40 - 50%), molecules of which present exogenous peptides to CD4+ T lymphocytes. Enviromental factors play a crucial role in the etiopathogenesis of T1DM. Through epigenetic regulation (e.g. DNA methylation) the genetic and enviromental factors communicate. The level of methylation in the regulatory regions can significantly affect expression of these genes. Aims: The aim of the diploma thesis was to define methylation profile of HLA DQB1 alleles in type 1 diabetes mellitus patients and determine their expression. Methods: The genotyping of HLA class II genes (HLA-DRB1, HLA-DQA1, HLA-DQB1) was performed using sequence specific primers. DNA was treated with sodium bisulfite, regulatory region of HLA DQB1 was amplified and cloned into E.coli, strain DH5α/XL1-Blue. Positive clones were sent for sequencing and results analyzed. RNA was transcribed to cDNA by reverse transcription and the level of expression was analyzed by quantitative PCR. Results: Statistically significant differences were found in total methylation of DQB1*0201 and *0302 alleles in the B section of DQB1 gene. Difference in...
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Epigenetic regulation of HLA class II genes and its modification during the lifetime
Lamborová, Věra ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Černý, Jan (referee)
Background: The major histocompatibility complex (MHC) molecules play an important role in the immune response regulation and in the maintenance of the immune homeostasis. Regulation of their expression is therefore a key factor influencing the adaptive immune response. DNA methylation of gene regulatory regions is one of the mechanisms of gene expression control that affects the accessibility of DNA to transcription factors. Ageing is connected with changes in DNA methylation and increased predisposition to autoimmune diseases in older age could be associated with changes in MHC class II genes methylation. Aims: The aim of this diploma thesis is to analyze the methylation profile of DQA1 and DQB1 genes regulatory regions and to compare its differences between the generations and between individual alleles. The next aim is to compare DQA1 mRNA expression between the generations and between single alleles. Methods: DNA and RNA were isolated from blood of three age group donors. DNA was converted by the bisulfite treatment and regulatory regions of HLA class II genes were amplified and cloned into bacteria. Positive clones were sequenced and then analyzed. RNA was reverse transcribed and its expression level was determined by real-time PCR. Results: Statistically significant differences were found by...
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