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Metabolic detoxication of carcinogenic aristolochic acids by cytochromes P450
Levová, Kateřina
Aristolochic acids (AA) are the major alkaloid of Aristolochia species. Carcinogenic and nephrotoxic plant alkaloid aristolochic acid I (AAI), the major toxic component of AA, causes the development of Aristolochic acid nephropathy (AAN). It is a unique type of rapidly progressive renal fibrosis which leads to a total renal failure. AAI also causes a similar type of kidney fibrosis with malignant transformation of the urothelium, Balkan endemic nephropathy (BEN). One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One possible explanation for these different responses could be individual differences in the activities of the enzymes catalyzing the biotransformation of AAI. Thus, the identification of enzymes principally involved in the metabolism of AAI, and detailed knowledge of their catalytic specificities is of a major importace. Aristolochic acid I is oxidized by demethylation to form AAIa. This metabolic pathway is known as detoxication. AAI metabolite AAIa was separated from AAI by HPLC. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification of AAI in rat and human liver. The efficiency of human recombinant CYPs to oxidize AAI was also tested. To find...
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Human F1Fo-ATPsynthase deficiency
Suldovská, Sabina ; Tesařová, Markéta (advisor) ; Černá, Leona (referee)
F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...
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Mitochondrial cytochrome c oxidase: cyanide inhibition and role of assembly factor Surf1 defect
Nůsková, Hana ; Kalous, Martin (referee) ; Drahota, Zdeněk (advisor)
The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....
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Gene Expression Regulation of Biotransformation Enzymes and Transporters in Placental Barrier
Stejskalová, Lucie ; Pávek, Petr (advisor) ; Modrianský, Martin (referee) ; Vondráček, Jan (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and toxicology Candidate Mgr. Lucie Stejskalová Supervisor Doc. PharmDr. Petr Pávek, Ph.D. Title of Doctoral Thesis Gene expression regulation of biotransformation enzymes and transporters in placental barrier Placenta is a unique organ facilitating the communication between mother and fetus. It serves as a respiratory, excretory, endocrine and metabolic organ during intrauterine development of the fetus and is necessary for maintenance of pregnancy and for the fetal protection. Syncytiotrophoblast is a single layer that represents the critical morphological and metabolic component of the placental metabolic and exchange barrier. The placental trophoblast contains multiple drug transporters and metabolizing enzymes that form placental metabolic barrier. The levels of the enzymes are not stable and fluctuate throughout pregnancy. Most of the enzymes are expressed at very low mRNA levels, however, in some cases there is absence of any relevant detectable protein or catalytic activity. Cytochrome CYP1A1 is the only placental xenobiotic-metabolizing enzyme of the cytochrome P450 superfamily for which significant expression and catalytic activity have been conclusively demonstrated in placental trophoblasts...
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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...
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Role reaktivních forem kyslíku a proteinové fosforylace na funkci spermií ryb
GAZO, Ievgeniia
Spermatozoa of externally fertilizing fish species after releasing into aqueous environment are particularly vulnerable to damage mainly due to alterations in composition of media surrounding sperm. Among factors affecting spermatozoa movement in external medium are water pollutants, temperature, pH and osmotic conditions. The goal of this thesis was to investigate the possible effects of oxidative stress on sperm performance and intracellular signaling, particularly the effect of pollutants occurring in water environment. In addition the molecular mechanisms of stress response and motility activation for spermatozoa of several freshwater fish species were analyzed. Our results show that xenobiotics, such as vinclozolin, induce a dose-dependent reduction in sterlet (Acipenser ruthenus) spermatozoa motility and velocity at environmentally relevant concentrations. Increased levels of lipid oxidation (LO) and protein carbonylation (CP), as well as changes in antioxidant activity of superoxide dismutase (SOD) indicate the development of an oxidative stress in spermatozoa exposed to xenobiotic. Moreover, increased DNA fragmentation as well as a reduction of the level of ATP was observed in spermatozoa incubated with xenobiotic in vitro. These results demonstrated that sterlet spermatozoa are highly susceptible to the presence of pollutants, which induce excessive ROS production even at low concentrations. Further studies were performed in order to evaluate the role of ROS production in fish sperm and protective properties of seminal plasma. The ROS were generated in carp (Cyprinus carpio L.) spermatozoa by in vitro incubation with xanthine - xanthine oxidase system (X-XO). A time- and dose-dependent reduction in spermatozoa motility and velocity was observed as well as increased LO, CP and DNA fragmentation. Moreover, it was shown that O-linked N-acetylglucosamine transferase and septin-8-A changed their phosphorylation state on tyrosine residue, and acid phosphatase activity decreased in response to oxidative stress. On the other hand, catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) in combination with seminal plasma can reduce oxidative stress in carp spermatozoa and improve sperm quality. Our next study was applied to determine how the protein phosphorylation pattern changed after motility activation in carp and sterlet spermatozoa, where phosphorylated proteins are located in spermatozoon and to identify proteins involved in sperm motility. It was shown that the pattern of protein phosphorylation and their localization differs significantly between two species. Phosphorylation on serine and tyrosine residues, as well as protein kinase A (PKA) and protein kinase C (PKC) substrates play an important role in spermatozoa motility activation and regulation in both species. Numerous signaling proteins involved in carp and sterlet spermatozoa movement were identified in this study, giving a better understanding of molecular mechanisms underlying sperm motility. As a conclusion, the results of this study provide new data on the effect of xenobiotics and oxidative stress on fish spermatozoa motility, DNA integrity, lipid and protein oxidation, antioxidant defense system and intracellular signaling. These data proved the toxicity of water pollutants and ROS for fish spermatozoa and proposed the use of CAT, SOD, or GTH in combination with seminal plasma to reduce oxidative stress in these cells. Moreover, we identified many spermatozoa proteins involved in stress response and motility. In practice, the data presented in this thesis could be useful for elaboration of suitable medium for cryopreservation and artificial propagation of freshwater fish species.
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Non-coding RNA in hepresvirus infection
Pelák, Ondřej ; Holá, Dana (referee) ; Drda Morávková, Alena (advisor)
MiRNAs are endogenous short RNAs that are well conserved in eukaryotic organisms and are thought to be a vital and evolutionarily ancient component of genetic regulation. Their key role is in post-transcriptional regulation of gene expression. Some viruses were able to utilize the capabilities of these tiny RNAs to regulate their own gen expression or to regulate host gene expression in use of avoidance from immunity answers to their presence. Among these viruses belongs also the Herpesviridae family. Members of this family are using miRNA in various ways, from regulation of their own gene expression to targeting host mRNA, which prevents translation of factors related to immune answers. This work is intent on biogenesis of miRNA, its mechanism, function and manner in which it is used by representatives from Herpesviridae family.
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Molecular mechanisms of Diamond-Blackfan anemia
Handrková, Helena ; Petrák, Jiří (advisor) ; Šebela, Marek (referee) ; Trka, Jan (referee)
Diamond-Blackfan anemia (DBA) is a rare congenital syndrome that presents with ane- mia and selective deficiency of erythroid precursors, while other blood lineages are usu- ally unaffected. Approximately half of the patients display additional somatic anoma- lies and growth retardation. The therapy is mostly symptomatic and is dominated by corticosteroids, other modalities include regular blood transfusions or hematopoietic stem cell transplantation. At the beginning of this work, only two DBA causal genes were known, RPS19 and RPS24, being mutated in approximately 1/4 of all DBA patients. The goals of this work were to study the consequences of the known DBA causal mutations on cellular level and to find novel DBA causal genes. To date, over a half of DBA patients have been reported to carry a mutation in one of nine known DBA causal genes, including RPS17, RPL11 and RPL5, that are reported in this dissertation. All confirmed DBA causal genes encode for ribosomal proteins (RPs) that were essential for ribosome assembly. We further hypothesized a non- ribosomal protein participating in this process might be involved in DBA pathogenesis, too. In one DBA patient, we identified a rare sequence variant in one such candidate, a protein arginine methyltransferase 3 (PRMT3). We reported that the patient PRMT3...
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Comparison of level total cholesterol in serum for residents from Pilsen and Czech Budweis provided similar eating habits
FRAITOVÁ, Lucie
Measuring cholesterol is considered as a screening test used primarily to determine cardiovascular risk and monitoring lipid-lowering therapy. Since this is a screening examination, there is a need for further laboratory tests - the lipid profile (HDL cholesterol, LDL cholesterol, triglycerides). Patients who get examined as a part of lipid-lowering therapy should follow a diet. These patiens need to be examined more frequently. Population of Czech Republic is considered at risk of early occurrence of atherosclerosis and its complications. Every year in the Czech Republic nearly 60 000 people die from cardiac and vascular disease. Cardiovascular disease increasingly affects younger age group. They are also a cause of disability of people of working age. One of the main indicators is high level of cholesterol. Accurate and precise measurement of blood cholesterol plays a role in the reduction of morbidity and mortality of people with cardio vascular disease. The aim of this thesis is to: 1) Measure the total cholesterol sample population of Plzeň and České Budějovice 2) Compare and find differences in serum total cholesterol in both locations In the theoretical part of the thesis I was interested in what foods increase cholesterol and what foods can reduce high cholesterol. Furthermore, the theoretical part describes diseases that may occur during hypercholesterolemia. The basic ones include atherosclerosis and its complications, which include heart disease or stroke. Preanalytical factor that may significantly affect laboratory tests are described in this part too. The practical part describes everything from an intake of biological material in the laboratory through the preparation (centrifugation, creating aliquots) to insertion of a secondary sample into the analytical device. Research for the thesis was conducted in the biochemical laboratory of synlab czech s.r.o. in České Budějovice. In this part I investigate ? under supervision ? a total of 100 samples of venous blood from people of Plzeň and České Budějovice. I determined the total cholesterol in serum. Cobas Integra 800 automatic biochemical analyzer was used for the examination of the biological material. In routine practice enzymatic methods are used to determine levels of cholesterol. Cholesterol esters are converted by cholesterol esterase into free cholesterol and fatty acids. Free cholesterol is oxidized by cholesterol oxidase to form cholestenone and hydrogen peroxide. The resultant hydrogen peroxide reacts with 4-aminoantipyrine and phenol to form a red color and water. The color intensity is directly proportional to the concentration of cholesterol and is measured by absorption spectrophotometry at 512 nm.Measured laboratory results were statistically processed and neatly sorted into tables and graphs using the computer program. Data from Plzeň and České Budějovice were sorted according to gender and evaluated using graphs. The data sets were then compared to each other and the difference in cholesterol levels was registered. All the results are in the table and graphs. Hypercholesterolemia was detected in 29 samples from Plzeň and 27 from České Budějovice.
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