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Mitochondrial permeability transition pore
Sereghy, Ursula ; Hansíková, Hana (advisor) ; Čapková, Markéta (referee)
The mitochondrial permeability transition pore (mPTP) is highly evolutionarily conserved channel found in the inner membrane of mitochondria. This pore is non-selectively permeable for molecules below 1,5 kD. Consequences of the pore opening due to an increase of Ca2+ or reactive oxygen species (ROS) and following depolarization of the membrane involve a disruption of the proton gradient, decrease in the production of ATP and prevalently a cell death. Death of a cell as a result of the mPTP opening is a physiopathological mechanism which follows ischemic diseases and neurodegenerative disorders such as Alzheimers and Huntingtons disease. Study of a structure and function of mPTP is essential for the research of mechanisms and progression of diseases, and it is crucial for the development of responding drugs and an overall decrease in the morbidity of the patients. This work compiles the course of the research into structure and function of the channel under physiological and pathological conditions and briefly puts down some of the experimental methods.
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Study of selected apects of protein modification by β-N-acetylglucosamine
Bittenglová, Kateřina ; Hansíková, Hana (advisor) ; Kohoutová, Michaela (referee)
Glycosylation O-linked β-N-acetylglucosamine (O-GlcNAc) is post-translational modification of proteins, regulated by β-N-acetylglucosaminyltransferase (OGT) and β-N-acetylglucosaminidase (OGA). This intracellular glycosylation differs from the other glycosylation types - it is dynamically regulated, similarly to phosphorylation, β-N-acetylglucosamine serves as a nutrient and stress sensor in cell. Chronically dysregulated O-linked glycosylation by GlcNAc is associated with pathology of various diseases, such as diabetes mellitus type II, oncological and neurodegenerative diseases. Expression of enzymes OGT and OGA is very sensitive for homeostasis of GlcNAc, which is the product of hexosamine biosynthetic pathway. Changes in expressions of these ezymes could be used as a potencial blood marker, e.g. in early stage of diabetes. The aim of this master thesis was to study changes in expression of genes encoding ezymes OGT and OGA in cohort of obese patients in comparison with healthy controls and also to compare the state before and after change of lifestyle (loosing weight). Analysed cohort comprised of 34 samples of isolated lymphocytes from peripheral blood from obese adolescent patients and 80 samples of adults patients. RNA was isolated by TriReagent, quantification of the expression of mRNA was...
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Dolichol content analysis by mass spectrometry in urine from patients with congenital disorders of glycosylation
Zdražilová, Lucie ; Hansíková, Hana (advisor) ; Kohoutová, Michaela (referee)
Dolichol is a membrane lipid, which carries monnosaccharides and glycans for N-linked protein glycosylation and glycosylphosphatidylinositol-anchor biosynthesis occuring in endoplasmic reticulum. Its structure is composed of isoprenoid units. Dolichol is present in all tissues and in most of the membrane organelles of eukaryotic cells. Recently some types of congenital disorders of glycosylation have been described as a consequence of dolichol biosynthesis and metabolism defects, which are not detectable by standard methods. The aim of this diploma thesis was to analyze dolichol content in urine and in different tissues from patients with deficiency in dolichol biosynthesis by mass spectrometry and to study the impact of these defects on energetic metabolism. Biological material for this study consisted of urine samples from 76 controls with age ranging from 1 months to 81 years, 6 patients with congenital disorders of glycosylation and 43 patients with suspicion of congenital disorder of glycosylation; samples of frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in NUS1 gene and controls. Urine samples were stored at -20 řC and tissue homogenates were stored in -80 řC until analysis. Lipid fraction after extraction was separated by liquid chromatography. Dolichols were...
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The role of mitochondria in the pathogenesis of Huntington's disease
Řeháková, Kateřina ; Hansíková, Hana (advisor) ; Kohoutová, Michaela (referee)
Huntington's disease is a neurodegenerative disease affecting the nervous system. It is caused by the mutation of the huntingtin protein coding gene. The mutation is manifested by the multiplication of CAG triplets. Huntingtin is present more in the cytoplasm. It interacts with many proteins and has roles also in transcription and cell transport. Huntingtin also participates in correct regulation of embryonic development and development of nerve tissue. Mutant huntingtin causes oxidative stress, mitochondrial biogenesis and OXPHOS disorders. Diagnosis of Huntington's disease is based on a laboratory examination of the presence of an allele predisposing to the disease. Clinical imaging is also an important part of diagnostics. The patient exhibits uncontrollable choreatic body movements and dementia. The aim of the thesis was to describe the main characteristics of Huntington's disease with the focus of HD on mitochondrial energy metabolism. In the practical part, the aim was to analyze mitochondrial respiration by high resolution polarography in a set of 3 fibroblast lines from patients with Huntington's disease. Polarographic analyses showed that respiration of HD fibroblasts was decreased in comparison with controls. We found that complex II of oxidative phosphorylation was most affected in HD...
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Congenital disorders of glycosylation: methods of glycans analysis
Bittenglová, Kateřina ; Hansíková, Hana (advisor) ; Veselá, Kateřina (referee)
Congenital disorders of glycosylation represent (CDG) a group with more than 100 types of metabolic disorders, which are caused by defects in biosynthesis and modification of glycoconjugates. CDG manifest by broad spectrum of clinical symptoms, from disorders of nervous system to disorders of digestion and excretion. Identification of the specific type of CDG is not possible without broad spectrum of biochemical and molecular-genetic methods. The goal of this bachelor thesis was to describe the most often used methods for analysis of glycans, which are components of glycoproteins or glycolipids, in the theoretic part. Isoelectric focusing of selected blood serum glycoproteins, (e.g. transferrin and apolipoprotein C-III) serve as screening methods. Measurement of enzymatic activity, mass spectroscopy, PDO and LLO analysis (protein derived oligosaccharide,lipid-links oligosaccharide) HPLC and CZE (capillary zone electrophoresis and high performance liquid chromagraphy) are performed in second level. Molecular-genetic methods are used to confirm the final diagnosis by identification of causal mutations in specific gene. The aim of practical part of this bachelor thesis was to analyse the expression of genes OGA (N-acetylglukosaminase) and OGT (N-acetylglukosaminyltransferase). This enzymes take part...
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The role of mixed function oxidases system with cytochrome P450 in metabolism of drugs and carcinogens
Mrízová, Iveta ; Stiborová, Marie (advisor) ; Levová, Kateřina (referee) ; Hansíková, Hana (referee)
6 Abstract Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activity. This antitumor agent binds to DNA and forms covalent DNA adducts. Enzymes, which are involved in its enzymatic activation, are cytochromes P450 (CYP) and peroxidases. To elucidate the effect of ellipticine on the expression and enzymatic activity of the individual components of the microsomal mixed function oxidase system in different tissues, we used rat model. Simultaneously, the effect of ellipticine and its cytotoxicity on different tumor cell lines was also investigated. Another part of the presented work was targeted on preparation of anti-peptide antibody against orphan cytochrome P450 2S1, which is highly expressed in many human tumours of the epithelial origin, for its detection in these tissues. For better understanding how CYP2S1 can contribute to the metabolism of xenobiotics, the protein was prepared by heterologous expression in E. coli. Furher, its role in metabolism of an antitumor drug ellipticine, a carcinogenic environmental pollutant benzo[a]pyrene (BaP) and its derivate BaP-7,8-dihydrodiol was examined. Utilizing a mouse model, the impact of pulmonary inflammation on the metabolism of an environmental carcinogen was...
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Biochemical and molecular studies of the congenital disorders of glycosylation
Ondrušková, Nina ; Hansíková, Hana (advisor) ; Stiborová, Marie (referee) ; Hřebíček, Martin (referee)
Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...
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Effect of biguanides on liver cells metabolism
Švecová, Eliška ; Kalous, Martin (advisor) ; Flachs, Pavel (referee) ; Hansíková, Hana (referee)
The extract from the plant Galega officinalis containing the guanidine derivative galegin has been used in the treatment of diabetes-associated complications since middle ages. Nevertheless, the positive effects of the treatment were often overweight by the adverse side effects. Some sixty years ago guanidin was replaced by the less toxic synthetic biguanide derivatives - metformin, phenphormin and buformin, the latter two being withdrawn due to the unacceptable risk of fatal lactate acidosis. Metformin is still widely used antidiabetics and belongs to the first choice drugs in the treatment of type 2 diabetes. Phenphormin is now gaining renewed attention with regard to its antineoplastic properties. Despite its long-term clinical use the mechanism of biguanides action is not fully understood yet. At present it is generally accepted that the core of its antihyperglycemic effect lays in the inhibition of hepatic gluconeogenesis. In contrast, there is less consensus regarding the particular metabolic pathway or target that are responsible for the metformin-induced attenuation of gluconeogenesis. For a long time, a hot candidate for metformin target in the cell was AMP-activated kinase (AMPK) but the metformin effect was proved also in mice carrying the dominant negative mutation of AMPK α subunit. Quite...
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