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DNA Microarrays and Bioinformatics in Biomedical Research
Ivánek, Robert ; Forejt, Jiří (advisor) ; Martásek, Pavel (referee) ; Beneš, Ivan (referee)
Abstr€gt DNA microanays ť€present a relatively novel tecbnique for qualitative and quartitative analysis of nucleic acids. In various modificďions tbis tecbniqueďows expressionmalysis' comparative genomic Mridizatiotr, micronNe profiling, analysis of geire regulation md deteďon ďpolymorpbism or mutations.In this thesisI presentan overview ďvarious types of microarays and theř applióations in biomedical reserch. I documett their usefulnessful severalprojecb aimedatidentificatioaof diseasecausinggenes(Mucopolysaoetaidosis lII€, ATP syntbasedefieielrcy),at moleculaÍchaae*erizatim ď h'man disws using cell lines (clear cell renal carcinoma,acutemyeloid leukaenia) or meute modďs (meiotic ďlonoiug of unsynapsedcbronatin). My work de'monsbtes that oompetentapplication of microarray techiqge4. bve pobqtiel to speed rrp biomedicď re"searcáod already bocane one of its frrndmental analyticalapproaches. ' t
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Quantification of liver function using breath test with 13C labeled methacetin
Hendrichová, Miluše ; Horák, Jiří (advisor) ; Ehrmann, Jiří (referee) ; Martásek, Pavel (referee) ; Moťovská, Zuzana (referee)
Kvantifikace jaterních funkcí pomocí dechového testu s 13 C-methacetinem MUDr. Miluše HENDRICHOVÁ SUMMARY Efforts to evaluace and quantify liver functions has accompanied hepatology over the last 50 years at least. Quantification of liver function was hindered by multiple blood samploing, the low specificity of monitored parameters and the risk of allergic reactions when using conventional chromoexcretory tests. The introduction of breath tests using the non-radioactive isotope 13C allows non-invasive and highly accurate measurement of liver function. Especially 13C.methacetin is a very suitable substrate for evaluation demethylační and oxidative capacity of hepatocytes. Using the breath test with 13 C-methacetin is noninvasive, easy for patients and the results are reproducible. The aim of this thesis is to introduce the use of breath test with 13 C-methacetin into clinical practice as one of standard items in the care of patients with chronic liver diseases. In the first study we are evaluating liver function using breath test with 13 C-methacetin in patients with liver cirrhosis. The results show that the breath test reliably distinguishes patients with liver cirrhosis from patients without liver damage. Using ROC curves we demonstrate that the most advantageous time of the breath test that best predicts...
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Disorders of iron metabolism in skin and chronic liver diseases
Krátká, Karolína ; Horák, Jiří (advisor) ; Ehrmann, Jiří (referee) ; Lata, Jan (referee) ; Martásek, Pavel (referee)
Iron is one of the important biogenic trace elements and its role in the mammalian body is indispensable. In nature there is another element with similar characteristics. Iron is part of a series of compounds that provide key functions such as cellular respiration and oxygen transport to tissues. It is also important for cell proliferation and differentiation, the regulation of gene expression and applies also in the immune system. Given that the effects of iron accumulation in genetic hemochromatosis have been investigated in detail, in recent years, increasing attention and concern about the consequences of iron accumulation also in other diseases. Because the results of previous studies are inconclusive and often mutually contradictory, the aim of this work to analyze and clarify the relationship between HFE gene mutations and iron metabolism in the pathogenesis and progression of some skin and chronic liver disease among genetic hemochromatosis.
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The role of TGFß and study of prognostic factors of patients with MDS and AML
Provazníková, Dana ; Fuchs, Ota (advisor) ; Pohlreich, Petr (referee) ; Martásek, Pavel (referee)
We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...
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Molecular basis of endothelial sysfunction: endothelial nitric oxide synthase and heme oxygenase 1 genetic variations
Král, Aleš ; Martásek, Pavel (advisor) ; Baxová, Alice (referee) ; Schneider, Bohdan (referee)
Endothelial dysfunction is a pathologic state characterized by an altered equilibrium among vasodilatory and antithrombotic mediators and vasoconstrictive and prothrombotic mediators produced by the vascular endothelium. Multiple factors induce impaired production or increased consumption nitric oxide (NO), the key mediator of vascular homeostasis, produced by the nitric oxide synthase enzymes (NOS). Endothelial dysfunction represents one of the initial steps in the development of atherosclerosis, a chronic inflammatory disease of the vascular wall. The inducible enzyme heme oxygenase 1 (HO-1) represents one of the main cellular defense mechanisms against increased oxidative stress and decreased NO bioavailability accompanying endothelial dysfunction and atherosclerosis. We studied the genetic determinants of endothelial dysfunction and atherosclerosis by evaluating the association of the G894T endothelial NOS (eNOS) polymorphism and the HO-1 (GT)n promoter polymorphism with coronary artery atherosclerosis severity and risk profile and their evolution during hypolipidaemic treatment. In addition, we searched for genetic variations in exons 25 and 26 of eNOS gene, encoding the C-terminal part of the protein, deemed crucial for proper enzyme function and the 3'- untranslated region crucial for eNOS...
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Effects of antidepressants and depressive disorders on mitochondrial functions
Hroudová, Jana ; Fišar, Zdeněk (advisor) ; Martásek, Pavel (referee) ; Kuča, Kamil (referee)
Mood disorders are serious diseases. Nevertheless, their pathophysiology is not sufficiently clarified. Biological markers that would facilitate the diagnosis or successful prediction of pharmacotherapy are still being sought. The aim of the study was to find out whether mitochondrial functions are affected by antidepressants, mood stabilizers and depression. Our research is based on recent hypotheses of mood disorders, the advanced monoamine hypothesis, the neurotrophic hypothesis, and the mitochondrial dysfunction hypothesis. We assume that impaired function of mitochondria leads to neuronal damage and can be related to the origin of mood disorders. Effects of antidepressants and mood stabilizers on mitochondrial functions can be related to their therapeutic or side effects. In vitro effects of pharmacologically different antidepressants and mood stabilizers on the activities of mitochondrial enzymes were measured in mitochondria isolated from pig brains (in vitro model). Activity of monoamine oxidase (MAO) isoforms was determined radiochemically, activities of other mitochondrial enzymes were measured spectrophotometrically. Overall activity of the system of oxidative phosphorylation was measured electrochemically using high- resolution respirometry. Methods were modified to measure the same...
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Molecular pathology of selected porphyria with skin manifestation
Sameh Anwar Hussein Farrag, Mohamed ; Martásek, Pavel (advisor) ; Baxová, Alice (referee) ; Raman, C. S. (referee)
Porphyria is a group of inherited metabolic disorders due to enzymatic defect of the heme biosynthesis resulting in the overproduction of the heme precursors' porphyrins in different body organs. The enzymes of the heme biosynthesis are encoded by corresponding genes in which any defect in any of these genes lead to a specific type of porphyria. Numerous mutations were detected in these genes leading to impairment in the enzyme function and therefore developing of the clinical manifestations of porphyria. The aim of the present work was to investigate the UROD gene in patients with porphyria cutanea tarda (PCT) and hepatoerythropoietic protoporphyria (HEP) as well as the FECH gene in patients with erythropoietic protoporphyria (EPP) on a molecular level. We identified numerous mutations in the FECH and the UROD genes in three different populations, Czech, Slovak, and Egyptian. We described the novel mutations in the UROD gene in HEP Arabic patients from Egypt as well in the FECH gene in patients with EPP of Czech and Slovak origin. We expressed mutatted UROD protein in prokaryotic system and found 19 % of the wild-type enzymatic activity. Moreover, the current study presents for the first time the frequency of the low expression allele IVS3-48c in the FECH gene in healthy controls from the Czech...
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Cytochrome P450 oxidoreductase: Structurally functional study. Molecular pathology of Antley-Bixler syndrome.
Tomková, Mária ; Martásek, Pavel (advisor) ; Mazura, Ivan (referee) ; Králová, Jarmila (referee)
NADPH-P450 oxidoreductase (POR) is a membrane bound flavoprotein that donates electrons to a wide spectrum of heme-containing proteins, among which are several steroidogenic and many xenobiotics-metabolizing enzymes. Given the important role of POR protein in drug metabolism and pharmacogenomics, there is a particular need to understand the contributions of POR genetic variants to these processes. Mutations in POR gene cause a disorder called POR deficiency, which manifests with a wide phenotypic spectrum ranging from disordered steroidogenesis to skeletal malformation, namely, Antley-Bixler syndrome (ABS). The aim of the present work was to investigate the POR gene in patients suspected to have POR deficiency syndrome from Czech Republic and to perform genotyping in Czech and Jewish control populations. We analyzed 644 alleles in unrelated individuals from the general Czech population and 1128 alleles in Jewish population, where 330 alleles were of Askhenazi and 798 of Sephardic Jews. We have also studied the impact of selected new genetic variants on POR activity and identified fourteen amino acid variations, two of which we have studied in detail to establish their influence on POR activity. Using the available human POR three-dimensional structure, we then modelled the newly identified variants...
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Molecular pathology of selected inherited hyperbilirubinemias
Šlachtová, Lenka ; Martásek, Pavel (advisor) ; Schneider, Bohdan (referee) ; Králová, Jarmila (referee)
Inherited hyperbilirubinemias are a group of metabolic disorders, characterized by increased levels of total serum bilirubin or its conjugated fraction. Most of these hyperbilirubinemias are inherited autosomal recessively and are manifested in young age. Increased bilirubin reflects the genetic disturbances in one of the enzymes of heme degradation pathway, the defect of bilirubin conjugation (UGT1A1 gene) or its transport (ABCC2, OATP1B1, OATP1B3). All of these proteins are involved not only in elimination of bilirubin, but various substrates; therefore the performed studies have a great pharmacogenomics impact. We have studied the molecular pathology of hereditary hyperbilirubinemias in Caucasian and Roma population and to compare the clinical and biochemical results with the molecular genetic data. We described the impact of compound defect of c.-3279T>G and g.175492_175493insTA on total serum bilirubin and calculated the linkage disequlibrium of these two variants in promoter region of UGT1A1 gene. We also verified, that the population distribution of both variants is in concordance with the literature. In our second study, we have described the rare conjugated hyperbilirubinemia Dubin-Johnson type among 7 Roma families. We have found a novel variant NG_011798.1:c.[1013_1014delTG] together with...
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Genetic and clinical aspects of the restless legs syndrome
Pavlíčková, Jana ; Kemlink, David (advisor) ; Seeman, Pavel (referee) ; Martásek, Pavel (referee)
Introduction: The Restless Legs Syndrome (RLS) is a frequent neurological disorder with a prevalence ranging from 5 - 10%. RLS is characterized by an urge to move the lower extremities during the night, thus RLS causes sleep disturbance. It presents as both idiopathic and secondary form. Idiopathic RLS is associated with common genetic variants in MEIS1, BTBD9, PTPRD and MAP2K5/SCOR1. Recently, multiple sclerosis (MS) was identified as a common cause for secondary RLS, the prevalence of RLS in patients with MS ranges from 13.3 to 37.5%. The aim of our study was to analyse the clinical and genetic aspects of this disorder, especially in patients with multiple sclerosis. In the clinical part, we evaluated the prevalence of RLS among Czech patients with MS and we compared the extent of brain damage between patients with and without RLS using magnetic resonance imaging (MRI). In the genetic part, we further analysed the impact of known genetic variants (MEIS1, BTBD9, MAP2K5/SCOR1, PTPRD) for RLS in other European populations and in patients with MS. Methods: Clinical part: Each patient with MS underwent a semi-structured interview. A patient was considered to be affected by RLS if he/she met all four standard criteria at life- long interval. Lesion load (LL - T2), brain atrophy - T1 and brain...
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