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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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Iron containing cofactors in anaerobic parasite Giardia intestinalis
Pyrih, Jan ; Tachezy, Jan (advisor) ; Martásek, Pavel (referee) ; Truksa, Jaroslav (referee)
Iron is an essential element in nearly all organisms. It is present mainly as a component of iron sulfur (FeS) clusters or as a heme iron. These cofactors enable proteins to transfer electrons or diatomic gasses, signal sensing and enzyme catalysis. Numerous FeS and heme depending proteins are involved in photosynthesis and respiratory chain pathways, which are well described processes. However, there is still much to learn about more recently discovered pathways such as formation of FeS clusters in various cell compartments and about roles of novel FeS or heme proteins. Particularly, only limited information is available about how FeS clusters are assembled or how heme is used in anaerobic protists, in which cytochrome-dependent respiration and photosynthesis does not occur. We decided to focus on iron cofactors in anaerobic parasite Giardia intestinalis. This organism undergone dramatic reductive evolution that resulted in formation of one of the smallest eukaryotic genome and the most reduced form of mitochondria, the mitosome. We characterized some components of mitochondrial (ISC) and cytoplasmic (CIA) FeS assembly machineries. We have detected ISC components in mitosome by proteomic analysis. Furthermore we investigated the presence and subcellular localization of CIA proteins in Giardia. In...
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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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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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Inherited Disorders of Bilirubin Metabolism
Šlachtová, Lenka ; Martásek, Pavel (advisor) ; Baxová, Alice (referee)
Inherited disorders of bilirubin metabolism - hereditary hyperbilirubinemias - are metabolic disorders manifested in early childhood. Unconjugated hyperbilirubinemias result from the defect of the enzyme uridine diphosphoglucuronosyltransferase (UGT1A1). UGT1A1 mediates the conjugation of bilirubin with glucuronid acid in hepatocytes and its elimination to water soluble compound. In the next step of bilirubin degradation the transport of conjugated bilirubin from hepatocyte into the bile occure. It is caused by the ATP dependent transporters ABCC2, ATP1B1 and OATP1B3. Mutations in the genes coding the bilirubin transporters results in conjugated hyperbilirubinemia Dubin-Johnson or Rotor syndrome. This study is focused on unconjugated hyperbilirubinemia in adolescents including the non-typical manifestations and the defects of ABCC2 transporter and their phenotype in humans.
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Lung Biotrauma
Vobruba, Václav ; Martásek, Pavel (advisor) ; Straňák, Zbyněk (referee) ; Pachl, Jan (referee)
The aim of this study was to investigate longitudinal changes of the pulmonary inflammatory process as a result of mechanical stress due to mechanical ventilation (MV). The concentrations of IL-8, TNF-α, MIP-1β, nitrites/nitrates and inducible nitric oxide synthases (iNOS) were investigated indicate in bronchoalveolar lavage (BAL). 23 piglets were divided into three groups. Group I: animals breathing spontaneously; group II: MV (TV=7 ml/kg, PEEP= 5 cmH2O); group III: MV (TV=15 ml/kg ; PEEP= 0 cmH20). The focus of the study was the influence of CMV on the hemodynamics, pulmonary function, changes in certain chemokine and cytokine levels, and the inducible NOS and nitrite/nitrate production in BAL. A significant increase in heart rate was found in Group III during the 3rd hour of the experiment - both in relation the the initial levels and to the levels of the other groups (p = 0.01 and 0.008 respectively). During the same time period, a significant drop in blood pressure readings was detected in this group as well. A significant increase of the CVP levels was found in Group III starting already from the 1st hour of the experiment. CMV with high tidal volumes lead to a sinificant decrease in lung compliance in Group III already from the 1st hour of the experiment (p < 0.001). Concentrations of...
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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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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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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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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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