|
| |
|
|
Asymmetric Dimethylarginine and Its Relation to Atherogenesis
Široká, Romana ; Racek, Jaroslav (advisor) ; Teplan, Vladimír (referee) ; Hyánek, Josef (referee)
ASYMMETRIC DIMETHYLARGININE - COMPARSION OF CHROMATOGRAPHY AND IMMUNOMETRIC METHODS Objective:Asymmetric dimethylarginine (ADMA) is often discussed in connection with hyperhomocysteinemia and its toxic effect on vessel wall. ADMA concentration is usually measured by HPLC (High Performance Liquid Chromatography) after previous derivatisation. Recently, ELISA (Enzym Linked Immuno Assay) methods for ADMA determination were introduced and ELISA kits are commercially available. Method and Result:The aim of the study was to compare HPLC and ELISA methods for ADMA determination. For HPLC determination we used equipments from Thermo separation product (Florida, USA). After solid-phase extraction on polymer cation-exchange column and the following derivatisation with o-phthaldialdehyde the samples were separated using C18 column (mobile phase 8.7% acetonitril, 50 mmol/l phosphate buffer, pH 6.5) and a fluorescence detector. NG-monomethyl-L-arginine was used as an internal standard. ADMA® ELISA kit, based on a competitive principle, was obtained from DLD Diagnostika, Hamburg, Germany. ADMA was measured in EDTA plasma of 40 healthy blood donors and 40 hemodialysis patients with hyperhomocysteinemia. Conclusion: In spite of different principles both methods showed a very good correlation (r = 0.944, p<0.0001). ELISA...
|
|
|
Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies
Fornůsková, Daniela ; Zeman, Jiří (advisor) ; Hyánek, Josef (referee) ; Stiborová, Marie (referee)
Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...
|
|
|
Advanced lipidomics in selected clinical conditions
Staňková, Barbora ; Tvrzická, Eva (advisor) ; Hyánek, Josef (referee) ; Kazdová, Ludmila (referee)
Abnormalities of lipid metabolism are considered risk factors for cardiovascular, metabolic, nephrologic diseases amd some malignancies, as well. Nowadays, a lot of effort is devoted to study new risk factors and surrogate markers of conditions mentioned above to improve their prognosis and decrease mortality. The aim of this thesis was to provide a comprehensive survey of lipid metabolism, characteristics of different lipid compounds in health and diseases and of possibilities of utilization of selected lipid parameters in the diagnostics of pathological conditions listed above. Selected lioid parameters were observed in several studies, focused on specific pathological conditions. Besides conventional lipid analytes, the composition of fatty acids in plasma lipid pools was studied in healthy controls, and in the patients suffering from metabolic syndrome, chronic pancreatitis, and pancreatic cancer, as well. Markers of an oxidative stress (oxidatively modified LDL particles and conjugated dienes in precipitated LDL) were assessed in healthy controls, patients with metabolic syndrome, chronic pancreatitis, pancreatic cancer, and in the patients with different concentrations of plasma apoB-48, too. LDL particles subfraction were investigated in healthy controls, in the patients with different...
|
|
| |
|
|
Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies
Fornůsková, Daniela ; Zeman, Jiří (advisor) ; Hyánek, Josef (referee) ; Stiborová, Marie (referee)
Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...
|
|
|
název v anglickém jazyce není uveden
Belošovičová, Hana ; Calda, Pavel (advisor) ; Hyánek, Josef (referee) ; Kvasnička, Jan (referee)
Souhrn Cíl: Stanovit hladiny celkového plazmatického cysteinu, homocysteinu, cysteinylglycinu a glutathionu u normotenzních primigravid ve druhém a třetím trimestru a porovnat je s hladinami u těhotných s následným rozvojem preeklampsie. Metodika: Do studie bylo zařazeno 65 zdravých primipar. U každé těhotené byl nabrán vzorek krve v 19.-21. a 30.-32. týdnu těhotenství. Hladiny celkového plazmatického cysteinu, homocysteinu, cysteinylglycinu a glutathionu byly stanoveny HPLC metodou. Hladiny aminothiolů v druhém a třetím trimestru byly porovnány pomocí párového t testu. Hladiny cysteinu u těhotných byly porovnány s hladinami cysteinu u kontrolního souboru netěhotných žen. Výsledky: Hladina celkového plazmatického cysteinu u normotenzních těhotných byla signifikantně nižší ve třetím trimestru v porovnání strimestrem druhým (medián 176.1 |amol/l [163.0, 189.4] versus 187.4 |amol/l [178.7, 205.2], P < 0.001). Hladiny ostatních aminothiolů se nelišily. U třech z 65 těhotných došlo k rozvoji mírné preeklampsie. Hodnoty t-Cys ve třetím trimestru u těhotných s následným rozvojem preeklampsie vykazovaly nárůst nebo menší pokles než u normotenzních těhotných. Závěr: Celkový plazmatický cystein je signifikantně nižší ve třetím trimestru než v trimestru druhém. Tento pokles je pravděpodobně dán spotřebou cysteinu...
|
|
|
Asymmetric Dimethylarginine and Its Relation to Atherogenesis
Široká, Romana ; Racek, Jaroslav (advisor) ; Teplan, Vladimír (referee) ; Hyánek, Josef (referee)
ASYMMETRIC DIMETHYLARGININE - COMPARSION OF CHROMATOGRAPHY AND IMMUNOMETRIC METHODS Objective:Asymmetric dimethylarginine (ADMA) is often discussed in connection with hyperhomocysteinemia and its toxic effect on vessel wall. ADMA concentration is usually measured by HPLC (High Performance Liquid Chromatography) after previous derivatisation. Recently, ELISA (Enzym Linked Immuno Assay) methods for ADMA determination were introduced and ELISA kits are commercially available. Method and Result:The aim of the study was to compare HPLC and ELISA methods for ADMA determination. For HPLC determination we used equipments from Thermo separation product (Florida, USA). After solid-phase extraction on polymer cation-exchange column and the following derivatisation with o-phthaldialdehyde the samples were separated using C18 column (mobile phase 8.7% acetonitril, 50 mmol/l phosphate buffer, pH 6.5) and a fluorescence detector. NG-monomethyl-L-arginine was used as an internal standard. ADMA® ELISA kit, based on a competitive principle, was obtained from DLD Diagnostika, Hamburg, Germany. ADMA was measured in EDTA plasma of 40 healthy blood donors and 40 hemodialysis patients with hyperhomocysteinemia. Conclusion: In spite of different principles both methods showed a very good correlation (r = 0.944, p<0.0001). ELISA...
|
|
|
Monitoring compensation of patients with cystinuria by spectrophotometric method
Leitner, Martin ; Hyánek, Josef (referee) ; Chrastina, Petr (advisor)
The presented bachelor's thesis deals with the issue of cystinuria, hereditary metabolic disorder of transport of dibasic amino acids. The objective of the thesis was to select a spectrophotometric method of determining cystine in urine, to find the most suitable conditions for determining cystine in a clinical laboratory and to apply the method on samples of urine of patients with cystinuria. For determining cystine in urine, the method using coloured red and violet product of cystine reaction was selected after reduction to cystine with ninhydrin agent in acid environment. Absorption spectra of standard of cystine and urine in a patient with cystinuria were measured with the maximum at 560 nm. Interference of ornithine and other amino acids was excluded. Kinetics of reduction of cystine to cysteine was measured. The most suitable time of reduction is 5 minutes, in longer time cysteine is oxidized to cystine. Kinetics of creation of a coloured product was measured. The maximum absorption in heating the reaction mix with ninhydrin to 100řC was reached after 10 minutes, later only degradation processes and absorbance reduction takes place. Stability of a coloured product of reaction in light was tested. The product is stable for ten minutes, then absorbance drops by 8% after 45 minutes. Linearity of...
|
|
|
The study of mitochondrial biogenesis during fetal development
Pejznochová, Martina ; Zeman, Jiří (advisor) ; Hyánek, Josef (referee) ; Brdička, Radim (referee)
Postnatal adaptation of neonate to extrauterine life is among others dependent on effective mitochondrial biogenesis during fetal development. Therefore the study of mitochondrial biogenesis on molecular and biochemical level may improve the care of very premature neonates, especially critically ill premature neonates. This thesis has been worked out in The laboratory for study of mitochondrial disorders (Department of Pediatrics, 1st faculty of Medicine, Charles University in Prague). The thesis is based on molecular genetic analyses, which are focus on characterization of expression of gene involved in mitochondrial biogenesis including gene of oxidative phosphorylation system (OXPHOS) and on changes in mtDNA content during human fetal development. The results than enable : Efective analysis of the mRNA expression level by quantitative real-time PCR method in fetal tissues. Analysis of the changes in the mtDNA content in different fetal tissues To understand and to explain the tissue-specific differences in expression of the OXPHOS genes and of the genes involved in mtDNA transcription and in regulation of mtDNA content during second trimester of gestation.
|
guest ::
