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Use of fluorescence methods for the study of protein interactions
Johaníková, Klára ; Bezděková,, Jaroslava (referee) ; Pavelicová, Kristýna (advisor)
The diploma thesis "Use of fluorescence methods for the study of protein interactions" is focused on the use of fluorescence methods for the study of protein interactions using electromigration methods and Förster resonance energy transfer (FRET). The aim of this work was to create a bioconjugate of metallothionein (MT) protein with quantum dots (QDs) and commercial dyes. FRET was subsequently studied between these conjugates. QDs were synthesized under UV light and conjugation with MT was performed via a carbodiimide zerolengthcrooss-linker (EDC / sulfo-NHS), which serves to activate carboxyl groups and allows bioconjugation of the ligand by covalent bonding. Due to the high proportion of cysteines in MT, this protein has a very high affinity for metals. It is also involved in scavenging free radicals and there are studies that show that MT is overexpressed in cancer cells. Attention was also paid to the study of MT dimerization, which leads to an understanding of oxidative dimerization of MT and thus can contribute to understanding the formation of free radicals in the body and to deepen the knowledge about neurodegenerative disorders such as Parkinson's or Alzheimer's disease or amyotrophic lateral sclerosis. The formation of the MT dimer was confirmed by energy transfer between the donor (QDs) and the acceptor (commercial dye-cyanine) through the physical phenomenon of FRET and MALDI-TOF-MS.
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Difference pulse voltametry for detection of metallothionein
Postránecká, Tereza ; Prášek, Jan (referee) ; Provazník, Ivo (advisor)
Metallothionein (MT) is a protein with a characteristic amino acid composition which thanks to its thiol groups can bind metal ions and be involved in the homeostasis of these ions in organism. The protein has its significance in carcinogenesis, could be a suitable marker for certain types of tumors. MT has many specific functions in organisms. It can be studied using several techniques. In common use electroanalysis is used as method for determining the concentration of MT. More specifically, this work is focused on differential pulse voltammetry using Brdička reaction.
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Study of metallothionein conjugates by electromigration methods
Johaníková, Klára ; Zemánková, Kristýna (referee) ; Vaculovičová, Markéta (advisor)
The bachelor thesis deals with the study of conjugates of metallothionein (MT) with fluorescent nanoparticles using electromigration methods. Particular attention is paid to the preparation and characterization of quantum dots (QDs) and their bioconjugates with the metallothionein protein, whose separation conditions (concentration and suitable electrolyte pH) have been carefully optimized. For the preparation of QDs, the action of UV radiation for one type of quantum dots and the thermal action of high temperature for the second type of species were used. Conjugation of QDs to the protein was performed via a carbodiimide-zero-length crosslinker (EDC / sulfo-NHS), which activates carboxyl groups and allows bioconjugation of the ligand by covalent bonding. The resulting bioconjugates were studied by capillary electrophoresis with absorption detection ( 214).
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On-chip and capillary electrophoresis for cancer research
Hájková, Tereza ; Babula, Petr (referee) ; Provazník, Ivo (advisor)
The thesis deals with cancer disease and its genesis. The prostate cancer is the second most dangerous disease in male population. It is very important to detect it in early stage. This thesis deals with the proteins which are or could be used as potential cancer markers. For example in current commonly used the prostate-specific antigen and newly the metallothionein. Metallothionein is a protein occuring in fauna and flora in four basic isoforms. There is a hypothesis that the isoform content or/and their ratio may have a diagnostic implication and could be used in clinical practise. For this reason they are studied and developed methods for specific and sensitive analysis of individual isoforms. In this work, capillary electrophoresis is used for the separation of the isoforms. The influence of the parameters such as type of background electrolyte, its pH and separation voltage etc. on the final separation are discussed.
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Potenciální vliv tkáňově specifických transkripčních faktorů na expresi metalothioneinů
Michálková, Nikola
Metallothioneins represent a group of intracellular proteins capable of binding metal ions, and their function includes the regulation of cellular levels of these metal ions. Transcription factors are central regulators of gene expression and play an important role in regulating the expression of metallothionein genes, which represents the basic mechanism for the cellular response to changes in metal ion levels. Although transcription factors that bind to the promoter regions of metallothioneins are known, their role in inducing the expression of metallothionein genes is far from clear. The subject of this thesis was the induction of gene expression of metallothioneins by zinc ions in HUH7 (hepatocellular carcinoma) and T47D (breast cancer) cell lines, and the subsequent analysis of gene expression of selected transcription factors. The selection of transcription factors was based on TF ChIP-seq analysis of ENCODE database. For the experimental part, the transcription factors FOXA1, FOXA2, HNF4A, and HNF4G were chosen, whose high levels are typical for liver tissue. Furthermore, gene expression of the ubiquitinated transcription factors JUND, NR3C1, RXRA, STAT3, SP1, and MTF1 was monitored. Statistically significant changes in gene expression (RT-qPCR) after short-term exposure (4-6 h) to 100 μM ZnSO4 were observed for FOXA1, JUND, and MTF1. In the case of FOXA1, a decrease in gene expression was observed in both tumor lines compared to the control, whereas an increase in expression was observed in JUND and MTF1 after ZnSO4 treatment. In the case of MTF1, a significant increase was observed only in the HUH7 tumor line. The results show that, in addition to the validated zinc-sensitive transcription factor MTF1, the transcription factors FOXA1 and JUND could also be involved in the regulation of metallothionein gene expression. FOXA1 could represent a potential repressor and inducer of JUND metallothionein expression in response to increased exposure to zinc ions.
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Study the optimal condition to knockout metallothionein by CRISPR-Cas9 in neuroblastoma
Májková, Klára
Cisplatin-based treatment strategies are commonly employed for the treatment of Neuroblastoma (Nbl), an embryonal tumour that most commonly affects infants and children under the age of five. However, the efficacy of these strategies has been found to be lower than 50 % due to the frequent chemoresistance developed by the tumour cells. Among others, this resistance has been linked to the increased expression of metallothionein (MTs) by the Nbl cells. Human MT-3, a protein primarily expressed in central nervous system cells, plays a vital role in metal detoxification and the maintenance of homeostasis during oxidative stress. Therefore, knockout of MT-3 using CRISPR/Cas9 could make the cells sensitive to cytotoxic drugs. This study is aimed to optimize the conditions for successful knockout of MT-3 from Nbl cells. The MT3-CRISPR/Cas9 plasmid was transfected into Nbl using Lipofectamine 2000 in three different plasmid concentrations (250 ng/μL, 500 ng/μL, and 1000 ng/μL). The optimal conditions were achieved using 0.75 μl of Lipofectamine, 1000 ng/μl of plasmid, and 0.5 ng/μl of puromycin.
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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.
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Use of fluorescence methods for the study of protein interactions
Johaníková, Klára ; Bezděková,, Jaroslava (referee) ; Pavelicová, Kristýna (advisor)
The diploma thesis "Use of fluorescence methods for the study of protein interactions" is focused on the use of fluorescence methods for the study of protein interactions using electromigration methods and Förster resonance energy transfer (FRET). The aim of this work was to create a bioconjugate of metallothionein (MT) protein with quantum dots (QDs) and commercial dyes. FRET was subsequently studied between these conjugates. QDs were synthesized under UV light and conjugation with MT was performed via a carbodiimide zerolengthcrooss-linker (EDC / sulfo-NHS), which serves to activate carboxyl groups and allows bioconjugation of the ligand by covalent bonding. Due to the high proportion of cysteines in MT, this protein has a very high affinity for metals. It is also involved in scavenging free radicals and there are studies that show that MT is overexpressed in cancer cells. Attention was also paid to the study of MT dimerization, which leads to an understanding of oxidative dimerization of MT and thus can contribute to understanding the formation of free radicals in the body and to deepen the knowledge about neurodegenerative disorders such as Parkinson's or Alzheimer's disease or amyotrophic lateral sclerosis. The formation of the MT dimer was confirmed by energy transfer between the donor (QDs) and the acceptor (commercial dye-cyanine) through the physical phenomenon of FRET and MALDI-TOF-MS.
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