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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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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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Processing of electrochemical metallothionein signals from Brdicka reaction
Dvořáček, Jiří ; Hynek, David (referee) ; Valla, Martin (advisor)
This thesis deals with the Brdička electrochemical reactions and the possibilities of description and processing of metallothionein. The first part deals only marginally, an introduction to the subject, a separate occurrence of this reaction, the discovery of the properties and functions of metallothionein in the human body, describe the possibilities and applications of cluster analysis on the measured electrochemical signal of metallothionein Brdička reactions and detection of the selected peak. The second part is based on requests made program to evaluate response Brdiča working on the basis of two detection signals from within.
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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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Vliv taurinu na biochemické ukazatele v orgánech laboratorních potkanů
Mrázková, Martina
The aim of this dissertation was to valorize the effect of various levels of taurine on the antioxidant status in rats body, monitor serum taurine and individual aminoacids in selectedorgans. This dissertation valorize the influence of taurine in the rats liver and brain after its application into feed mixtures. The effect of taurine was monitored on the ratio of GSH / GSSG,concentration of metallothionein and taurine. To this experiment were used 48 Wistar male rats, which were divided into eight groups (n = 6). During four weeks feed mixtures were given to these rats enriched with taurin eat a dose of 0, 1000, 1500, 2000 2500, 3000, 3500 and 4000 mg / kg / diet. Animals accepted diet and drinking water ad libitum. Average consumption of feed mixtures were 35 grams per day. Application taurine in creased concentration of taurine in the organism, which has an positive effect on antioxidant mechanisms of the organism. In deed we can not confirm that the addition of taurine in to the diet increases the concentrations of metallothionein and glutathione in the organism. In the brain was found increasing ratio of GSH / GSSG and reduced concentration of metallothionein. The other way around in the liver was reduction ratio of GSH / GSSG and increasing concentration of metallothionein.
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Charakterizace metalothioneinu pomocí kapilární elektroforézy
Drncová, Marie
Bachelor thesis is focused on the study of metallothionein (MT), a low-molecular mass, cysteine-rich protein, whose presence was confirmed across various organisms such as bacteria, fungi, plants, invertebrates and vertebrates. So far, the four isoforms have been described in mammals. The ability of MT to bind metal ions to its structure results in its functions in a number of physiological and pathological processes of living systems such as the influence of metal-ion homeostasis, preservation of the redox ho-meostasis, detoxification and transport of metal ions and regulation of gene expression. Connection between MT and neurodegenerative diseases, apoptosis and cancer is incre-asingly discussed. This work was aimed to create a comprehensive overview of bio-chemical, analytical and functional characteristics of MT focusing on neurodegenerative diseases. The experimental part was devoted to the optimization of the method of sepa-ration of partial isoforms and qualitative and quantitative analysis of binding properties of MT to selected metal ions using capillary electrophoresis.
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Izolace, separace a studium významných kov-vazných proteinů
Skaličková, Sylvie
The metal binding proteins are important group of biomolecules effects in living organisms. They represent series of vital important functions such as homeostasis of essential metals, they are involved in heavy metals detoxification, helps antioxidant system with free radical neutralization to prevent oxidation stress. Lot of them belongs to group of important enzymes catalyzing biochemical reactions. Last, but not least they are part of innate immunity system against pathogenes and could be utilized as a carrier cages in target drug therapy due to their unique transporter properties. A number of methods have been developed to study and observe the interactions between metalloproteins and specific biomolecules. First of all, the liquid chromatography enables isolation of required proteins from biological matrices. Further experiments utilized immunochemical, spectrophotometrical and electrochemical methods to clarify their properties and behaviour in various environments. The aim of the thesis was optimized analytical methods for separation and studying of the metal binding proteins.
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