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Integration site distribution of expressed proviruses
Miklík, Dalibor ; Hejnar, Jiří (advisor) ; Kejnovský, Eduard (referee) ; Indik, Stanislav (referee)
To establish efficient expression of their genes, retroviruses integrate proviral copies into the genomes of the cells they have infected. Epigenetic events, however, silence expression of the integrated proviruses. This silencing protects host cells from harmful viral spread, but also creates a reservoir of latent proviruses that subsequently hinders the cure of retroviral (e.g., HIV-1) infections. Furthermore, the silencing of retrovirus-derived integrative vectors complicates their application in transgenesis and gene therapy. The goal of this thesis is to describe the interaction between retroviral expression and host (epi)genomic environment at the site of proviral integration. To pursue the goal, we sought to define the (epi)genomic environment of the proviruses, which expression is not affected by the epigenetic silencing. Diverse retroviral vectors derived from avian sarcoma and leukosis virus (ASLV), murine leukemia virus (MLV), and human immunodeficiency virus type 1 (HIV-1) were used as model retroviral systems, and expression stability of the vectors in human cell lines was examined. In order to identify the features unique to integration sites of the active proviruses, we sorted the cells positive for the proviral expression, identified their proviral integration sites, and compared them to...
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Expression of selected membrane transporters in placentas of pregnant women diagnosed for preterm rupture of membranes
Michalská, Martina ; Čečková, Martina (advisor) ; Jirkovský, Eduard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Michalská Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Expression of selected membrane transporters in placentas of pregnant women diagnosed for preterm rupture of membranes Placenta is a key organ for pregnancy maintenance. One of its main functions is transport of compounds between mother and her fetus. The transplacental penetration is ensured due to membrane transporters that are present in the apical or basal side of trophoblast. Their expression level is affected by many physiological and pathological factors, among others it can be influenced by infection and inflamatory reaction. Inflammation is also one of the risk factors of preterm deliveries and it can be therefore assumed that these pathological states are accompanied by changes in expression of placental transporters. This study was performed using 51 placentas obtained from Faculty hospital in Hradec Králové from women who underwent preterm delivery and on 15 placentas delivered in term. The study employed quantitative RT-PCR approach. The mRNA expression of membrane transporters ABCB1, ABCG2, OATP1A2, OATP1B3, OATP2A1, OATP2B1, OATP3A1, OATP4A1 was assessed and the results were compared to...
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The effects of endocrine disruptors on the expression and the activity of cytochromes P450 2B in laboratory rat as a model organism
Měkotová, Barbora ; Dračínská, Helena (advisor) ; Levová, Kateřina (referee)
Endocrine disrupting chemicals are compounds that interfere with natural hormones and they are responsible for functional changes which may lead to damage of the endocrine system. Their presence in the environment is associated with a number of diseases whose extent is hard to predict. As endocrine disrupting chemicals, a wide range of exogenous and endogenous compounds is present in the environment. Important exogenous endocrine disrupting chemicals include benzo[a]pyrene (BaP) and 17α-ethinylestradiol (EE2); the female sex hormone 17β-estradiol (E2) can act like endogenous endocrine disruptor. In this thesis, the effect of these three compounds and their combinations on the expression and the activity of rat biotransformation enzymes cytochromes P450 2B is studied. The gene expression was determined by quantitative PCR, the expression of the protein itself was studied using Western blot method and consecutive immunodetection. The results show that CYP2B expression is almost unchanged after BaP premedication, whereas estrogenic compounds, E2, EE2, their combination and their combinations with BaP, significantly decrease the expression. The enzyme activity of CYP2B was also studied in rat liver microsomes using the marker substrate 7-pentoxyresorufin. EE2, E2 and their combination decrease the...
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Expression and activity of rat cytochromes P450 3A after exposure to benzo[a]pyrene and Sudan I
Ličko, Vojtech ; Dračínská, Helena (advisor) ; Ptáčková, Renata (referee)
The aim of this Bachelor thesis is the study of the effect of two carcinogenic compounds, benzo[a]pyrene and Sudan I, co-administered to rats individually or in combination, on the expression and the activity of important biotransformation enzymes cytochromes P450 of subfamily 3A in liver - a main organ of xenobiotic metabolism, in which the amount of CYP3A is especially high. Using the quantitative PCR method, the decrease of the gene expression of CYP3A1/2 in the livers of rats exposed to benzo[a]pyrene and Sudan I individually or in combination, was observed. Using the Western Blot method with a consecutive immunodetection, we found the decrease of the protein expression of CYP3A in the livers of rats treated with benzo[a]pyrene and Sudan I alone. Specific activity of CYP3A, determined by marker reaction of CYP3A, which is 6β-hydroxylation of testosterone, did endorse the previous results only in some of the premedicated groups of rats. It can be concluded that the exposure of rats to both studied compounds with carcinogenic potential resulted in a decrease in the expression of hepatic CYP3A in vivo. (In Czech) Keywords: cytochromes P450, benzo[a]pyrene, Sudan I, expression, enzyme activity
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MicroRNA expression in glucocorticoid-treated patients with systemic autoimmune
Uher, Martin ; Kuchařová, Monika (advisor) ; Nováková, Veronika (referee)
Rheumatoid arthritis is the most common joint disease of autoimmune origin. It is accompanied by inflammatory conditions that lead to irreversible changes in the joints, their deformities ending with permanent disability. Treatment of the disease involves routine regimens, surgical, as well as pharmacological treatment, which is necessary for advanced forms. Glucocorticoids play an important role in the therapeutic intervention in the course and progression of the disease. In spite of their anti-inflammatory effect, which is a key to improving the condition of the patient, they have a number of side effects in the long term- use. In this study, we have focused on the impact of these drugs on microRNA expression changes in arthritic patients treated with pulsed doses of glucocorticoids. MicroRNAs are nowadays widely studied due to their possible use as biomarkers in monitoring disease progression and the effect of treatment. MiRNA expression analysis was performed by quantitative real-time PCR array of 754 miRNAs with reverse transcription using stem-loop primers that allow amplification of short sequences that microRNAs are. Data analysis revealed 29 miRNAs differentially expressed at the significance level p ≤ 0.05, 14 miRNAs were at significance level p ≤ 0.025 (respectively 7 miRNAs at p ≤ 0.005...
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Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines.
Kouklíková, Etela ; Novotná, Eva (advisor) ; Wsól, Vladimír (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Etela Kouklíková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines Cyclin-dependent kinase inhibitors (CDKi) are considered as a suitable treatment especially in patients with wrong prognosis or advanced stage of cancer. It has only recently been discovered that CDKi are able to influence the activity of some enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. AKR and SDR enzymes belong to a group of carbonyl reducing enzymes that are involved in the metabolism of endobiotics and xenobiotics. An important group of drugs that are metabolized by these enzymes to less efficient compounds are anthracyclines. The aim of this diploma thesis was to find out whether purvalanol A, roscovitin, dinaciclib, AZD5438 and R547 can affect the expression of the most important anthracycline reductases (AKR1A1, AKR1B10, AKR1C3, AKR7A2 and CBR1) in human HepG2 and HL-60 cell lines. Expression of anthracycline reductases in cells exposed to CDKi was evaluated at mRNA level by RT-qPCR and at protein level by Western blotting. The...
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MicroRNA expression in glucocorticoid-treated patients with systemic autoimmune
Uher, Martin ; Kuchařová, Monika (advisor) ; Nováková, Veronika (referee)
Rheumatoid arthritis is the most common joint disease of autoimmune origin. It is accompanied by inflammatory conditions that lead to irreversible changes in the joints, their deformities ending with permanent disability. Treatment of the disease involves routine regimens, surgical, as well as pharmacological treatment, which is necessary for advanced forms. Glucocorticoids play an important role in the therapeutic intervention in the course and progression of the disease. In spite of their anti-inflammatory effect, which is a key to improving the condition of the patient, they have a number of side effects in the long term- use. In this study, we have focused on the impact of these drugs on microRNA expression changes in arthritic patients treated with pulsed doses of glucocorticoids. MicroRNAs are nowadays widely studied due to their possible use as biomarkers in monitoring disease progression and the effect of treatment. MiRNA expression analysis was performed by quantitative real-time PCR array of 754 miRNAs with reverse transcription using stem-loop primers that allow amplification of short sequences that microRNAs are. Data analysis revealed 29 miRNAs differentially expressed at the significance level p ≤ 0.05, 14 miRNAs were at significance level p ≤ 0.025 (respectively 7 miRNAs at p ≤ 0.005...
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Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines.
Kouklíková, Etela ; Novotná, Eva (advisor) ; Wsól, Vladimír (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Etela Kouklíková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines Cyclin-dependent kinase inhibitors (CDKi) are considered as a suitable treatment especially in patients with wrong prognosis or advanced stage of cancer. It has only recently been discovered that CDKi are able to influence the activity of some enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. AKR and SDR enzymes belong to a group of carbonyl reducing enzymes that are involved in the metabolism of endobiotics and xenobiotics. An important group of drugs that are metabolized by these enzymes to less efficient compounds are anthracyclines. The aim of this diploma thesis was to find out whether purvalanol A, roscovitin, dinaciclib, AZD5438 and R547 can affect the expression of the most important anthracycline reductases (AKR1A1, AKR1B10, AKR1C3, AKR7A2 and CBR1) in human HepG2 and HL-60 cell lines. Expression of anthracycline reductases in cells exposed to CDKi was evaluated at mRNA level by RT-qPCR and at protein level by Western blotting. The...
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The effect of selected endocrine disruptors on cytochromes P450 1B1 and 3A1/2
Holecová, Jana
Many exogenous and endogenous compounds are referred to as endocrine disruptors (EDCs), as they interfere with natural synthesis, signaling and metabolism of endogenous hormones. Common exogenous endocrine disruptors are benzo(a)pyrene (BaP) and 17α-ethinylestradiol (EE2). Endogenous endocrine disruptor 17β-estradiol (E2) is frequently present in the environment as well. In this thesis, the effect of the mentioned EDCs and their combinations on gene and protein expression of CYP1B1, 3A1 and 3A2 in rat liver, kidney and lung was determined. Protein expression was studied using Western blot method and specific antibodies; gene expression was assessed by quantitative PCR. Moreover, the effect of tested EDCs and their combinations on BaP metabolism and CYP3A specific activity (measured as testosterone 6β-hydroxylation) were studied in liver microsomal samples. It was confirmed, that BaP significantly increases CYP1B1 expression in rat liver and lung both alone and together with EE2 or E2. Pretreatment of rat with E2 and BaP increases the ability of BaP to induce CYP1B1 expression. On the contrary, EE2, E2 and their combination decrease the CYP1B1gene expression. The rate of BaP metabolites formed in liver microsomal samples increases in rats pretreated with BaP and its combinations. In liver, there was...
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