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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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The impact of mutant huntingtin on oxidative stress in primary fibroblasts isolated from a new Huntington's disease knock in porcine model
Sekáč, Dávid ; Ellederová, Zdeňka (advisor) ; Hanzlíková, Hana (referee)
Huntington's chorea is a dominantly inherited disease caused by trinucleotide (Cytosine-Adenine -Guanine) expansion in a gene coding huntingtin protein. Carriers of these mutation show symptoms associated with motor impairment, a cognitive and psychiatric disturbance, which is called Huntington's disease (HD). The major sign of HD is striatal atrophy in the middle age of life. Since it is known that huntingtin protein participates in a lot of cellular processes, such as transcriptional regulation and metabolism, these processes change by its mutation. One of the features observed in HD pathogenesis is the presence of oxidative stress. The aim of the work was to monitor the molecular changes preceding the HD manifestation in the knock-in minipig model. As a material for monitoring molecular changes leading to this condition, primary fibroblasts were used. Whereas, the oxidative stress arises from an imbalance between oxidants and antioxidants, level of reactive species and lipid peroxidation together with expression of antioxidant response associated genes was measured. At the same time, expression of metabolic and DNA repair related genes was monitored. Although the differences in oxidative stress level or the expression of antioxidative response genes were not detected, the changes in the...
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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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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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Structure and molecular mechanisms of DNA repair by Nei glycosylase
Landová, Barbora ; Šilhán, Jan (advisor) ; Lux, Vanda (referee)
Abasic sites (Ap site, from apurinic/apyrimidinic) are one of the most common lesions generated in DNA by spontaneous base loss or DNA repair processes. There are two equilibrating forms of an Ap site - ring-open aldehyde and cyclic hemiacetal. Ring- opened aldehydes are reactive electrophilic groups capable of formation covalent adduct with nucleophilic sites in DNA. DNA interstrand cross-link (ICL) resulting from the Ap sites is formed spontaneously as a covalent bond between ring-open aldehyde and amin group of adenin residue in the opposite strand of double stranded DNA. ICLs block DNA replication and transcription. The formation of Ap site derived ICL is relatively long process taking several hours. We assume that the ring-opening of an abasic site is the rate-limiting step in the formation of the thermodynamic ICL. However, formation, stability and DNA repair of Ap-ICL are still poorly understood processes. Here, I have set up mechanistic in vitro experiments to reveal and calculate the probability of Ap-ICl formation in vivo. In more detail, I study the rates of formation of Ap-ICLs in the sequence context of neighbouring nucleotides of freshly formed covalent bond of ICL. I focus on sequence preference, the influence of AT/ GC rich regions and the length of oligonucleotides. I have...
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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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The Role of DNA Repair in the Onset and Therapy of Ovarian Cancer
Tomášová, Kristýna ; Vodička, Pavel (advisor) ; Čáp, Michal (referee)
DNA repair and DNA damage response are very important biological systems, inevitable to maintain genomic stability and fidelity of the genetic information, for the onset of ovarian cancer. Further, DNA repair is also substantially involved in the response to the therapy, since many chemotherapeutics act as DNA damaging agents. This literary analysis is intended to survay the relevance of DNA repair to ovarian carcinogenesis. Special emphasis is placed on repair defects, as it is inextricably associated with the onset of cancer and treatment outcome. Apart from well-known alternations in ovarian cancer susceptibility genes, such as BRCA1 and BRCA2 involved in homologous recombination repair, ample space will be dedicated to less common gene mutations across different repair pathways. Research confirms that abnormalities in the proteins responsible for homologous recombination repair are the leading cause of ovarian cancer. The majority of authors also suggested that targeting DNA repair pathways, especially base excision repair, can improve chemotherapy efficiency in a synergic manner. The same applies to nucleotide excision repair, which repairs platinum-DNA adducts and thus contibutes to platinum drugs resistance emerging. By way of contrast, mismatch repair in ovarian cancer is rather poorly...
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