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Novel Approaches in Electrochemical Determination of Xenobiotic Compounds and in Study of Their Interaction with DNA
Hájková, Andrea
Presented Ph.D. Thesis is focused on the development of analytical methods applicable for determination of selected xenobiotic compounds and for monitoring DNA damage they can induce. The main attention has been paid to the development and testing of non-toxic electrode materials for preparation of miniaturized electrochemical devices and novel electrochemical DNA biosensors. 2-Aminofluoren-9-one (2-AFN) was selected as a model environmental pollutant, which belongs to the group of hazardous genotoxic substances. Its carcinogenic and mutagenic effects may represent a risk to living and working environment. 2-AFN has one oxo group, where the cathodic reduction occurs, and one amino group, where the anodic oxidation occurs. The voltammetric behavior of 2-AFN in the negative potential region was investigated at a mercury meniscus modified silver solid amalgam electrode (m-AgSAE) representing a non-toxic and more mechanically robust alternative to mercury electrodes. This working electrode was subsequently used for the development of a newly designed miniaturized electrode system (MES), which has many benefits as the possibility of simple field measurements, easy portability, and the measurement in sample volume 100 µL. Moreover, a glassy carbon electrode (GCE) was used for further investigation of...
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DNA damage induced by occupational exposure to copper oxide nanoparticles
Rössner st., Pavel ; Pelcová, D. ; Elzeinová, Fatima ; Mikuška, Pavel ; Večeřa, Zbyněk ; Coufalík, Pavel ; Vlčková, Š. ; Fenclová, Z. ; Rössnerová, Andrea
Copper oxide nanoparticles (CuO NPs) have a widespread use in industry, chemistry, in production of electronic devices and as an antimicrobial agent. Although copper is an important biogenic element, CuO NPs are toxic with the ability to induce oxidative stress, apoptosis, cell cycle arrest or DNA damage. For humans, the inhalation route is the most common way of exposure to CuO NPs. In the body, CuO NPs may be either deposited in the lungs, or transported to other organs. Their presence usually causes oxidative stress or inflammatory responses, consequently leading to DNA damage. In this study, we investigated the effect of CuO NPs inhalation on DNA damage in a group of researches conducting animal exposure experiments. The subjects were exposed to various metal oxide nanoparticles, including CuO NPs, by inhalation for an average of 4.9 ± 0.4 years. The average mass concentration of Cu in the air during the experiment was 7.3 ± 3.2 ng/m3. Subjects not exposed to nanoparticles served as a control group. We applied micronucleus assay using Human Pan Centromeric probes to detect DNA damage and to distinguish between the frequency of centromere positive (CEN+) and centromere negative (CEN−) micronuclei (MN) in the binucleated cells. We\ndid not find differences between both groups for either mean MN frequency (10.38 ± 2.50 vs. 11.88 ± 3.01 MN/1000 binucleated cells), or CEN+/CEN- ratio (58%/42% vs. 55%/45%), for the exposed and controls, respectively. In conclusion, inhalation of CuO NPs at this low-level exposure had no effect on chromosomal losses and/or breaks.
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Role of skin stem cells in aging and regeneration
Hlaváčková, Tereza ; Nešporová, Kristina (advisor) ; Procházka, Jan (referee)
Skin is a natural and effective barrier of the body against the adverse effects of the external environment. Which, however, requires its constant regeneration and, in the event of damage, repair. Stem cells stored in the epidermis, hair follicles and dermis contribute to this. As with other stem cells, these are characterized by so-called stemness, ie the ability to self-renew and differentiate into other cell types, thus providing a source of cells for skin renewal. During aging, due to internal and external factors (mainly due to oxidative stress and DNA damage), the integrity and functionality of the skin barrier are lost. This process is related, among other things, to a reduction in the number and function of skin stem cells. Today several therapeutic approaches are being developed that use stem cells, but at the same time, it is clear that their origin also significantly affects their use. Therefore is necessary to have a good understanding of the specific properties of the function of skin stem cells to modulate the properties of the skin. This work aims to create a review of scientific literature, which covers the topic of skin stem cells, their role in the processes of regeneration and repair and their role in aging. The work will also address the issue of the skin or other types of stem...
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Senescent cells and their elimination by the immune system
Novotný, Ondřej ; Reiniš, Milan (advisor) ; Mrázková, Blanka (referee)
Cell senescence is a type of cell cycle arrest in which the spectrum of the expressed genes changes specifically, also a change in the shape, size and other properties of a cell occurs. Senescent cells secrete a specific set of substances that affect the surrounding tissue, immune system and themselves. All this due to the induction of signalling pathways, inherent to individual types of senescence. The senescent cells accumulate in the body both during pathological conditions and during the natural process of aging and tissue renewal, with varying intensity depending on the type of tissue and organism. The consequence of their presence in the body is often ambivalent - for example, they are an effective mechanism of defence against tumour growth, but at the same time they can be its cause. The positive elimination of senescent cells usually has a positive effect - the immune system is responsible for this in vivo. Studies mapping the natural rate of accumulation and elimination of senescent cells in individual organs, together with new immunotherapeutic elimination procedures, are an important tool for developing new approaches to treating a wide range of human diseases and potentially to prolong human life.
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Maintenance of chromosomes integrity in Giardia intestinails as a model organism.
Uzlíková, Magdalena ; Nohýnková, Eva (advisor) ; Lalle, Marco (referee) ; Stejskal, František (referee)
Giardia intestinalis is a protozoan causing diarrhea worldwide. Beside its medical importance, it is evolutionary distant protist with two nuclei within a cell adapted for parasitic life in the environment poor of oxygen. Its genome is small and compact in term of gene content and size. It is therefore an attractive model organism for studies of minimal requirements for cellular processes. Present work brings new partial information on different levels of chromosome integrity maintenance of this parasite. Our study presents characteristics of chromosome termini and their protection. We localized telomeres during all stages of the trophozoite cell cycle and determined the length of Giardia telomeres ranging from 0.5 to 2.5 kb, we proved an existence of an active telomerase enzyme synthesizing telomeric repeats in in this parasite, despite the fact that giardial telomerase is structurally divergent. Present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes. We described effects of commonly used drug for treatment of anaerobic infections, metronidazole, on DNA and cell cycle progression in susceptible and resistant cell lines. Incubation of cells with this drug causes phosphorylation of histone H2A in cell nuclei...
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Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
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New Strategies in Electrochemical Detection of DNA Damage Induced by Interaction with UV radiation
Přibylová, Monika ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
V této bakalářské práci byl zkoumán netradiční redoxní indikátor 5-nitro-1,10-fenantrolin jako možný ukazatel poškození DNA. Měření probíhalo elektrochemicky technikou diferenční pulzní voltametrie v tříelektrodovém zapojení. Jako pracovní elektroda byla použita elektroda z ultračistého grafitu, která byla modifikována nízkomolekulární DNA z lososích spermií. Poškození DNA probíhalo pomocí UVC záření o vlnové délce 254 nm. Byl studován rozdíl výšky voltametrických signálů 5-nitro-1,10-fenantrolinu mezi nepoškozenou a poškozenou DNA. Nově vyvinuté metody se ukázaly jako slibné pro detekci míry poškození DNA v závislosti na čase ozařování a v závislosti na vzdálenosti od zdroje ozařování. Poškození DNA bylo rozsáhlejší u obou studovaných faktorů v závislosti na jejich rostoucích hodnotách. Klíčová slova Elektrochemie DNA biosenzory Netradiční elektrodové materiály Poškození DNA Biologicky aktivní látky Záření
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Role of yeast WSS1 protease in DNA repair.
Adámek, Michael ; Grantz Šašková, Klára (advisor) ; Čáp, Michal (referee)
Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.
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DNA damage response in mammalian oocytes
Vachová, Veronika ; Šolc, Petr (advisor) ; Nevoral, Jan (referee)
During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...
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Ddi1-like proteins: a novel family of retroviral-like aspartyl proteases
Šmilauerová, Kristýna ; Grantz Šašková, Klára (advisor) ; Šmahel, Michal (referee)
Ubiquitin-proteasome system is one of the key pathways which maintain cell homeostasis. Its purpose is to degrade damaged, misfolded or unnecessary proteins. It is also involved in multiple other processes such as DNA damage repair, cell cycle control or signaling. The entire system consists of multiple components, which are mutually strictly regulated. Important part of this system is group of so called proteasome adaptor proteins. Their role is to recognize and bind targeted substrates and transport them to the proteasome for degradation. Ddi1-like (abbrev. from DNA damage-inducible protein 1) protein family, a group of proteins with retroviral aspartyl protease-like domain, belongs to proteasome adaptor proteins. Global biological role of this protein family is only partially understood the most studied member is Ddi1 protein from Saccharomyces cerevisiae, and it is thus a subject of active research. This thesis summarizes published information about this protein family, describes its general characteristics and known functions, situates them in the context of cell processes and thereby might suggest the course of further study.
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