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Sperm DNA damage: detection methods used in clinical practice
Tušlová, Magdaléna ; Postlerová, Pavla (advisor) ; Koubek, Pavel (referee)
The current routine semen analysis, known as a spermiogram, is not able to provide a complete picture of male fertility as it does not routinely include an assessment of DNA integrity. This parameter is closely related to difficulties during conception and increased risk of miscarriage. This paper discusses the causes of sperm DNA damage, the detection of this damage by various methods and concludes with a discussion of the possibilities of incorporating these tests into clinical practice to improve the assessment and treatment of male fertility. Mechanisms of sperm DNA fragmentation include impaired spermatogenesis, oxidative stress and impaired apoptosis, which can be influenced by genetic, anatomical and external factors. The main part of this thesis was devoted to a review of methods for the detection of sperm DNA fragmentation, including sperm chromatin structure analysis (SCSA), sperm chromatin dispersion test (SCD), TUNEL method, single-strand DNA break recognition test (ISNT), comet assay and chromatin staining. Although some of these methods are frequently used, challenges remain in their standardization and full integration into clinical practice. However, the possibility of their routine use could bring significant advances in the diagnosis and treatment of male infertility. Among the...
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Reorganization of in vitro reconstituted actin-based networks
Sabó, Ján ; Lánský, Zdeněk (advisor) ; Varga, Vladimír (referee) ; Cifra, Michal (referee)
The cytoskeleton provides living organisms with machinery to move. On the molecular scale, the same cytoskeletal components undergo constant reorganization to contribute to distinct cellular processes, such as the navigation of neuronal growth cones in brain development or the creation of chiral actin flows during cellular division. During brain development, neuronal growth cones navigate by employing cytoskeletal crosstalk between actin filament networks and microtubules. Crosstalk in the growth cones between these cytoskeletal components was linked with known microtubule polymerase human cytoskeleton associated protein 5 (CKAP5; homolog of XMAP215, MSPS, Zyg9), while the details of the direct molecular mechanism of CKAP5-provided remodeling of both microtubules and actin networks are lacking. Therefore, we used total internal reflection fluorescence (TIRF) microscopy together with state- of-the-art in vitro reconstituted assays combining microtubules, actin filament networks, and recombinant proteins to understand cytoskeleton-provided molecular mechanisms underlying cellular motion. In this thesis, we show that CKAP5 alone bundles both supposedly randomly oriented and parallel actin filaments, crosslinks actin filaments to microtubules regardless of their polarity, positions prevailing actin...
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Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2
Kalábová, Dana ; Obšilová, Veronika (advisor) ; Pavlíček, Jiří (referee) ; Žáková, Lenka (referee)
Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2 Abstract Caspase-2 is a protease standing apically in the cascade of reactions leading to apoptosis. Properly functional apoptosis eliminates damaged cells, autoreactive lymphocytes or redundant groups of cells in ontogeny. The process of caspase-2 activation must be precisely regulated. One of the described ways of caspase-2 regulation causing its inhibition is posttranslational modification phosphorylation with subsequent binding of the regulatory scaffold protein 14-3-3. The aim of this dissertation is to explain the molecular mechanism of this regulation. To understand the interaction between the proteins, it was necessary to first identify the phosphorylation sites in the caspase-2 molecule recognized by the 14-3-3 protein and then describe the detailed structure of the binding complex. The structure was characterized by a number of biochemical and biophysical methods, such as analytical ultracentrifugation, native electrophoresis in TBE buffer, polarization-fluorescence assay, hydrogen/deuterium exchange coupled to mass spectrometry, or crystallization; and the results led to stimulating conclusions. Activation of caspase-2 begins with its binding to adaptor proteins, cleavage and dimerization of the catalytic subunits. The...
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Water scarcity in the Czech Republic and its implementation in highschools
Kalinová, Petra ; Janský, Bohumír (advisor) ; Šobr, Miroslav (referee)
Water scarcity is one of the current issues not only in the Czech Republic. Given the fact that drought has hit the territory of the Czech Republic several times recently and also climate change, it is essential for our country to be able to prepare for repeated drought periods. Possibilities for dealing with the drought in the Czech Republic, including examples of concrete measures, as well as other basic information regarding drought in the Czech Republic, are presented in this bachelor's thesis. However, the main goal of this bachelor's thesis is to create a project day on the topic of Water scarcity in the Czech Republic. The creation of the project day is part of the PERUN project, which is focused on climate change and drought and its impact on the territory of the Czech Republic. The project day was verified in practice in the form of a pretest-posttest design, thus confirming its effectiveness and testing the quality of the materials used. In the vast majority of cases, positive trend of increasing correct answers amongst students is visible, and thus the participating students deepened their knowledge regarding this issue. Key Words: water scarcity, Czechia, high schools, tuition
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The study of the impact of the mutual interaction of selected antibacterial drugs in vitro on activity against enterococci
Netíková, Nina ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Nina Netíková Supervisor: RNDr. Klára Konečná, Ph.D. Title of diploma thesis: The study of the impact of the mutual interaction of selected antibacterial drugs in vitro on activity against enterococci Background: The point of the experimental part of this thesis was to evaluate the effect of the mutual interaction of two selected, clinically approved antibiotics in combination on two selected bacterial strains of genus Enterococcus. The effect of the combination was evaluated using the FIC index. Based on the calculation of the FIC index, the mutual interaction was further classified as synergistic, additive, indifferent or antagonistic. Methods: To evaluate the interaction of antibacterial drugs in combination, four antibiotics were selected and employed to a pair-wise combinations. The main antibiotic was daptomycin, which was combined with tigecycline, linezolid and vancomycin. The effect of pair-wise combinations was monitored against the clinical isolate Enterococcus faecium and against the collection bacterial strain Enterococcus faecalis (ATCC 29212). Determination of MIC values was performed using the microdilution broth technique according to the EUCAST...
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Myofunctional therapeutic concept in patients with dysfunction of orofacial area
Harudová, Kateřina ; Zahrádka Köhlerová, Michaela (advisor) ; Kejhová, Eva (referee)
BACHELOR THESIS ABSTRACT Author: Kateřina Harudová Supervisor: Mgr. Michaela Zahrádka Köhlerová Title: Myofunctional therapy in patients with orofacial disorders Abstract: This bachelor thesis deals with the issue of myofunctional disorders in the orofacial area and the possibilities of therapy. The work is a case study character and consists of theoretical and practical parts. The theoretical part provides an overview of the current knowledge about myofunctional disorders and therapeutic approaches. It covers an anatomy and ontogenetic development of the orofacial area, physiology of speech development, etiology, symptomatology, diagnosis, and possibilities of prevention of myofunctional disorders. The theoretical part also includes an overview of selected therapeutic concepts and part about cooperation with pediatric patients. The practical part demonstrates the use of myofunctional therapeutic concept in the form of case studies of two pediatric patients. A partial goal of the work was create an educational brochure to facilitate self-therapy for parents and their children at home. After studying the literature and applying the concept, it was shown that myofunctional therapy improve muscle tone and motor skills of the lips and tongue, increase tongue muscles strenght, and optimizes resting position of...
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Fetal DNA circulating in maternal plasma and possibilities of its genetic analysis
Soukalová, Adéla ; Korabečná, Marie (advisor) ; Vodička, Radek (referee)
Cell free DNA is short, fragmented DNA found in plasma and serum, into which it is released by various mechanisms. It was first identified in body fluids of cancer patients and soon became a biomarker for non-invasive diagnostics. Soon after, the presence of fetal cfDNA in maternal plasma was detected and became the subject of clinical utility. Non-invasive prenatal diagnosis is a screening method that helps to determine the likelihood of birth defects early in pregnancy, specifically using fetal cfDNA. The final results are determined by invasive methods such as aminocentesis and chorionic villus sampling. Invasive methods of prenatal diagnosis carry a risk of fetal loss of less than 1 %. Despite this low risk, scientists are constantly striving to improve non-invasive methods that can detect aneuploidies such as Down's or Edwards syndrome, as well as determine the sex of the fetus, monogenic diseases and the Rh factor. Polymerase chain reaction (PCR) or next-generation sequencing methods are used for detection.
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The Theory of "Loss of Chance" in the Field of Medical Law
Petr, Ondřej ; Salač, Josef (advisor) ; Šolc, Martin (referee)
The Theory of "Loss of Chance" in the Field of Medical Law Abstract This master's thesis explores an alternative approach to establishing civil liability in medical-legal relationships. Due to the disparities described in the thesis, the positions of the parties in medical-legal disputes are not equal. Therefore, the theory of "loss of chance" was developed to address this issue by introducing a new approach to proving causal connection in court. The point of the theory is to transfer the procedural deficiencies of the aforementioned disputes into the substantive realm by creating a new legally protected asset, namely, the "chance." The thesis is divided into three parts. The first part addresses the issues of medical-legal relationships and the resulting legal disputes. It explains the fundamental principles underlying the theory of "loss of chance" and highlights the differences between the conventional approach to proving causal nexus and the theory itself. The second part is devoted to the historical and foreign development of the theory. With the help of key decisions of foreign case law, the concept of theory in these foreign jurisdictions is explained, which then influences the decision-making of Czech courts in cases related to theory. The last part of the thesis deals primarily with the main...
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Synthesis and evaluation of potential antimicrobial agents based on steroidal scaffold
Požárová, Pavla ; Krátký, Martin (advisor) ; Matouš, Petr (referee)
Antibiotic resistance represents a significant threat to humanity. To combat this trend, the development of new effective substances can be employed to potentially reverse its progression. Due to the broad range of biological activities exhibited by steroid structures, estrone was chosen as the starting material and was modified through various reactions. In total, fifteen new compounds were synthesized, which were tested for their antibacterial, antifungal, and antimycobacterial activities, and additionally for cholinesterases inhibition. The modification of estrone primarily involved two different approaches, resulting in the formation of hydrazones and esters. Twelve compounds were prepared through these steps. For the remaining three derivatives, changes in the starting materials or procedures were necessary. In the case of four compounds, it was necessary to synthesize one of the reactants. The yields ranged from 62% to 95% for hydrazones and from 8% to 54% for esters. The antibacterial activity was tested against four selected Gram-positive and Gram-negative strains. Antifungal activity was evaluated against four yeasts and filamentous fungi. Antimycobacterial activity was tested against five mycobacterial strains. All these methods utilized the microdilution broth method. The tested...
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An identification of selective antagonists of constitutive androstane receptor
Ondraszková, Karolína ; Smutný, Tomáš (advisor) ; Pourová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Karolína Ondraszková Supervisor: PharmDr. Tomáš Smutný, Ph.D. Title of diploma thesis: An identification of selective antagonists of constitutive androstane receptor The constitutive androstane receptor (CAR) is an important ligand-regulated xenosensor that regulates the transcription of important drug biotransforming enzymes (e.g. CYP2B6). The pregnane X receptor (PXR) belongs to the same family of nuclear receptors as the CAR. The structure and function of both receptors share many common features. However, PXR has a larger and more flexible ligand-binding domain. At present, a selective, highly potent and non- toxic inhibitor of CAR is not practically available. This work aims to determine the affinity of previously identified compounds in our group with antagonistic activity towards CAR. This is a replication study. For this purpose, we tested the compounds using gene reporter and two-hybrid assay. Of all the tested substances, we obtained desired results with substance 1A. Using the gene reporter assay method, we found that substance 1A can reduce basal CAR expression but also suppresses CAR activity induced by the model agonist CITCO. The high affinity of the substance for the...
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