National Repository of Grey Literature 42 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Vliv expozice těžkých kovů na expresi mRNA isoforem metalothioneinu u adherentních buněčných linií
Jordanová, Lucie
Metallothionein is a small cysteine-rich protein that plays an important role in metal homeostasis and protection against heavy metal toxicity, DNA damage and oxidative stress. This bachelor thesis was conducted to investigate the effect of heavy metal exposure on the mRNA expression of metallothionein isoforms in adherent cell lines. The effect of zinc exposure in the form of ZnSO4 on the mRNA expression of MT-1A, MT-1X and MT-2A isoforms in adherent breast cancer cell lines MCF-7, MDA-MB-231 and T-47D was investigated. Using MTT viability assay, the cell viability changes at different ZnSO4 concentrations and RNA integrity is verified using denaturing agarose gel. Polymerase chain reaction combined with reverse transcription qRT-PCR is used to study mRNA levels.
Preparation and characterisation of mRNA/DNA transfection vectors
Horák, Tomáš ; Chmelíková, Larisa (referee) ; Skopalík, Josef (advisor)
This diploma thesis deals with genetic engineering, especially the transfection of DNA into MSCs (Mesenchymal stromal cells) and dendritic cells. Both lipoplexes and metal magnetic nanoparticles were tested to introduce the vector into cells. The research was focused on finding more efficient methods of transfection. According to analysis on MADLS and gel electrophoresis, aspects playing an important role in conjugation and subsequent transfection were found. Conjugation occurs after only 4 minutes, as evidenced by an increase in zeta potential, but to achieve full conjugation it is necessary to incubate the sample for 20 minutes. Incomplete conjugation to iron nanoparticles resulted in strong carrier-carrier interactions, which formed an unwanted conglomerates. Encapsulation into liposomes with cationic surface treatment was without complications. The success rate of GFP-labeled protein expression after transfection by these methods was calculated to be 95%, resp. 91%. This result is due to low cytotoxicity. However, commercial tested kits on dendritic cells had a success rate below 5% with high cytotoxicity.
The effect of synthetic modified mRNAs induced proliferation on pancreatic beta cells
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a chronic disease caused by the loss of pancreatic beta cells due to autoimmune destruction or increased apoptosis. Beta-cell deficiency results in reduced insulin production, which plays an important role in glucose metabolism. The number of beta-cells in the body is one of the main factors that influence the development of this chronic disease. Therefore, it is necessary to find a way by which the number of beta-cells of the organism can be increased and thus the insulin production can be restored in a natural way without any need for the use of insulin infusions. However, the ability of beta-cells to divide decreases with age and is virtually nil in adulthood. The study of the cell cycle, especially the early and late cyclins and cyclin-dependent kinases, which act as cell cycle regulators, thus appears to be a promising way to restore natural insulin-producing tissues. In order to increase the number of beta cells entering the cell cycle, we focused on studying the effect of in vitro transcribed (IVT) mRNAs, encoding cyclins type D and cyclin dependent kinases 4 and 6 on stimulating cell division of isolated beta-cells. We found that transfection IVT mRNAs for type D cyclins in combination with cyclin-dependent kinases 4 and 6 significantly increased the proliferation of beta-cells...
Induction of beta-cell proliferation by synthetic modified mRNAs encoding cell cycle regulators
Ivanovská, Dana ; Koblas, Tomáš (advisor) ; Černá, Věra (referee)
Diabetes mellitus is a metabolic disease associated with a high blood glucose level over a prolonged period of time. Hyperglycemia is caused by the loss of pancreatic insulin producing beta cells. Diabetes mellitus II is linked with insulin resistence, which can indirectly lead to beta cell deficiency. It logically follows that the replacement or regeneration of beta cells could lead to a successful remission of diabetes. D type cyclins (D1, D2, D3) and cyclin-dependent kinases (Cdk) 4/6 appear to have the potential to induce beta cell proliferation. These proteins are responsible for driving cell mitotic entry. The aim of this bachelor thesis was to verify the possibility of inducing beta cell proliferation via D type and Cdk4/6 synthetic mRNA transfection. In vitro-synthesized mRNA induces short-therm protein overexpression. Cyclins harboring mutations are characterized by a higher protein stability and an increased half-life. The presence of D type cyclins and Cdk4/6 after cell transfection was detected using indirect immunofluorescence. Also a Western blot analysis with subsequent immunodetection was performed. Transfecting rat islet cells with various D type cyclins and Cdk4/6 mRNA combinations has shown to lead to a significant induction of beta cell proliferation. The levels of beta cell...
Post-transcriptional modification of mRNA molecules in viruses of the Poxviridae family
Jakešová, Kristýna ; Vopálenský, Václav (advisor) ; Šmahel, Michal (referee)
Post-transcriptional modifications of mRNA molecules in viruses belonging to the Poxviridae family, including the vaccinia virus, play a key role in regulating viral gene expression and also influence virus-host cell interactions. These modifications include the synthesis of a 7-methylguanosine cap, 2'-O-methylation, 3' polyadenylation of the mRNA strand, hydrolysis of the 7-methylguanosine cap, and 5' polyadenylation of the mRNA strand. Studying post-transcriptional or co-transcriptional mRNA modifications in Poxviridae viruses can contribute to a better understanding of viral pathogenesis and the development of new strategies for treating infections caused by viruses in this family. The aim of this bachelor's thesis is to summarize current knowledge of co-transcriptional or post-transcriptional mRNA modifications in Poxviridae viruses, with a special focus on vaccinia virus.
Preparation and characterisation of mRNA/DNA transfection vectors
Horák, Tomáš ; Chmelíková, Larisa (referee) ; Skopalík, Josef (advisor)
This diploma thesis deals with genetic engineering, especially the transfection of DNA into MSCs (Mesenchymal stromal cells) and dendritic cells. Both lipoplexes and metal magnetic nanoparticles were tested to introduce the vector into cells. The research was focused on finding more efficient methods of transfection. According to analysis on MADLS and gel electrophoresis, aspects playing an important role in conjugation and subsequent transfection were found. Conjugation occurs after only 4 minutes, as evidenced by an increase in zeta potential, but to achieve full conjugation it is necessary to incubate the sample for 20 minutes. Incomplete conjugation to iron nanoparticles resulted in strong carrier-carrier interactions, which formed an unwanted conglomerates. Encapsulation into liposomes with cationic surface treatment was without complications. The success rate of GFP-labeled protein expression after transfection by these methods was calculated to be 95%, resp. 91%. This result is due to low cytotoxicity. However, commercial tested kits on dendritic cells had a success rate below 5% with high cytotoxicity.
Effect of selected antineoplastic drugs on the gene expression of DNA repair proteins
Klčová, Silvia ; Jirkovská, Anna (advisor) ; Suchá, Simona (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Silvia Klčová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: Effect of selected antineoplastic drugs on the gene expression of DNA repair proteins. Every single cell of the human body is continuously exposed to a wide range of stress factors, with the consequence of damage to the DNA molecule. The resulting changes represent variety of alterations - from simple alkylation modifications of bases to the most unfavorable double-strand breaks (DSBs). However, the effect of cellular stress and subsequent genotoxic DNA damage is a double-edged sword. On the one hand, typical alterations in the genome can be triggered by mutagenic agents (such as components of tobacco smoke or ionizing radiation). Consequences of their action can accumulate and trigger loss of control over various steps of the cell cycle, which results in tumor cell transformation. On the other hand, however, inducing detrimental impact affecting the genome of tumor cells is one of the fundamental approaches in cytostatic treatment of cancer. Therefore, we focused our research on several antineoplastic drugs widely used in clinical practice (etoposide, daunorubicin, dexrazoxane) or undergoing clinical...
Characterization of pancreatic beta cells after their in vitro proliferation induced by synthetic modified mRNA
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Černá, Věra (referee)
The origin and development of type I. and II. diabetes mellitus is directly related to homeostasis of proliferation and apoptosis of pancreatic β-cells. Any imbalance that leads to a decrease in the number of β-cells consequently increases the pro- bability of developing this disease. Patients suffering from diabetes mellitus are de- pendent on partial or complete exogenous insulin replacement, as their pancreas is unable to meet the body's insulin needs. Therefore a need for restoration of normal β-cell mass in diabetic patients leads to the attempts to develop new therapeutic approaches that could expand remaining β-cells of the organism and restore phys- iological insulin production. A major obstacle in this regard is a low sensitivity of terminally differentiated β-cells to mitogenic stimuli that could induce the entry of β-cells into the cell cycle. Activation of β-cell proliferation is associated with the G0/G1/S cell cycle transi- tion, which is under the control of retinoblastoma protein (RB). In order to activate cell cycle entry RB must be phosphorylated. RB phosphorylation is provided by specific cell cycle regulators, particularly cyclin-dependent kinases 4 and 6, which associate with family D cyclins. In accordance with the aim of this Diploma thesis, the effect of these cell cycle...
Detection and quantification of maternal RNA localization during early development
Šimková, Kateřina ; Šindelka, Radek (advisor) ; Frolíková, Michaela (referee)
The asymmetric localization of maternal RNAs and proteins is a crucial mechanism for the body plan development in many animal species. These maternal factors are expressed during the oogenesis and they are used for the regulation of early developoment. In this diploma thesis, I addressed the role of asymmetrically localized RNAs along animal- vegetal axis in the early development of Ambystoma mexicanum. The second part of my thesis is focused on RNA localization in dorso-ventral and left-right axes. I identified the localization patterns of many known RNAs along the animal-vegetal axis using RT-qPCR and I also detected several genes, which can be involved in the dorso-ventral or lef-right patterning. Furthermore, we performed transcriptome wide analysis, which revealed changes classified into the following categories: RNAs relocalization, de novo synthesis before the onset of MBT and RNAs degradation during early development. Many vegetally localized genes in Xenopus laevis with the important role in the development of primordial germ cells have been previously described. Surprisingly, my results show that many of these genes are degraded during the early development of A. mexicnum. I believe that this degradation may be due to a different mechanism of PGC development in A. mexicanum and X. laevis....
The effect of cancerogenic azo dye Sudan I on expression of biotransformation enzymes
Hejduková, Žaneta ; Svášková, Dagmar (advisor) ; Dračínská, Helena (referee)
Sudan I is a widely used azo dye which has the ability to cause carcinomas in organs and tissues of experimental animals. During reactions catalyzed by microsomal monooxygenase enzyme systems or cytoplasmic biotransformation enzymes, Sudan I is oxidized to reactive metabolites that covalently bind to nucleic acids and cause their damage. Sudan I can also be metabolized by reduction, e. g. by a DT-diaphorase enzyme (NQO1). Reduction of Sudan I is considered to be a detoxification reaction. In this work, the in vivo action of Sudan I is examined in terms of its ability to induce an expression of the biotransformation enzyme DT-diaphorase in tissues of rats treated with the azo dye. The aim of this work was to quantify the degree of NQO1 induction at mRNA level. After the isolation of total RNA from organs of rats treated with Sudan I, the RNA was converted to cDNA by reverse transcription using random hexamers as primers. Using specific probes, the abundance of mRNA for the enzyme NQO1 in the organs of treated rats was quantified by "real-time" PCR, relatively to the control gene with a constant expression (β-actin). Through comparing thus determined amounts of mRNA in individual organs of treated and untreated rats, it has been found that Sudan I had caused a significant increase in the expression...

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