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Study of the prophylactic effect of glycoclusters on a model of bacterial adherence
Kurucová, Michaela ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF), an autosomal recessive genetic disorder, arises from mutations in the CFTR gene encoding the CFTR protein, which primarily functions as a chloride channel in the body. The malfunction of chloride ion transport leads to multiple organ dysfunctions, with the most significant impact on the respiratory system. A hallmark of CF is the increased adhesion of bacteria, especially Pseudomonas aeruginosa (PA), to the lung epithelium, resulting from the formation of dense mucus and glycosylation modification in the lungs. The pathogenicity of PA is significantly contributed to by its virulent factors, specifically PA-IL and PA-IIL lectins, which facilitate adhesion to host cells by binding to surface receptors containing D-galactose (PA-IL) or L-fucose (PA-IIL). The main objective of this study was to demonstrate the ability of synthetically prepared multivalent inhibitors based on fucose and galactose to inhibit PA adhesion to lung epithelial cells. Trivalent glycoclusters designed to target the PA lectins were studied ex vivo using an adhesion test employing immortalized epithelial cells - the CuFi-1 (from a CF patient) and NuLi-1 (from a healthy individual) cell lines. A control PA strain, denoted as PAK (ST 1763), was utilized to monitor bacterial adhesion. For visualization and...
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Preparation of growth factor TGF-β3 with coiled-coil tag
Otépka, Tomáš ; Vaněk, Ondřej (advisor) ; Kubíčková, Božena (referee)
Growth factors represent a group of significant substances in the metabolism of organisms. They are signaling molecules that control cell activity at the endocrine, paracrine, or autocrine levels. They act as key mediators binding to cell receptors, triggering a cascade of reactions leading to the regulation of genetic transcription in the cell nucleus and stimulation of cellular response. Growth factors influence various physiological functions such as cell proliferation, cell differentiation, and tissue healing. The utilization of growth factors is evident, for example, in regenerative medicine. For a similar purpose, research has been initiated to prepare the growth factor TGF-β3 with the possibility of attaching it to a polymeric carrier using a coiled-coil tag. This work focuses on the recombinant production of TGF-β3 - its analog with a latency-associated peptide (LAP) - and the application of techniques applicable to the intention of this work, specifically, attaching the protein to a polymeric carrier based on amino acids. Given the structural complexity with which growth factors are physiologically released from cells, the preparation of growth factors with a coiled-coil tag in vitro represents an unexplored challenge in the field of recombinant protein expression. In our HEK293T cell line...
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Chicken antibodies against the pathogenic yeast Candida albicans
Konečná, Lucia ; Heidingsfeld, Olga (advisor) ; Kubíčková, Božena (referee)
5 Abstract Candida albicans belongs to the most widespread pathogenic yeasts. As an opportunistic pathogen, it mainly colonises the mucous membranes of the host and causes infection, especially in individuals with a compromised immune system. The most common infection caused by C. albicans is vaginal mycosis, which at least 3 out of 4 women will experience during their lifetime. Antifungal drugs are used to treat this condition, but the yeasts are becoming less susceptible. For this reason, there is an effort to develop new treatment options for (not only) infections caused by C. albicans. Although vaginal mycosis is not a severe condition in most cases, it is a widespread infection that occurs repeatedly in many women, sometimes several times a year. Since vaginal mycosis reduces the quality of life in many women, we decided to focus on this disease in this paper. This project used hen antibodies, which have a number of advantages over mammalian antibodies. In oviparous animals, antibodies are not only found in the blood serum, as seen in mammals but are also secreted into their eggs. This makes the isolation of hen antibodies non-invasive and ethical. Moreover, the methods of isolating antibodies from eggs are also simpler than isolation from blood serum. Ultimately, hen antibodies are also more...
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Phytoremediation of pharmaceuticals
Forróvá, Barbora ; Bělonožníková, Kateřina (advisor) ; Kubíčková, Božena (referee)
Anxiolytics, sedatives, hypnotics and antidepressants are some of the most prescribed pharmaceuticals today. As their consumption increases, there is a growing risk of them leaking into surface water and soil, where they can be accumulated, interfere with key biological processes in animal cells or contaminate the food chain. With this is related an increasing need to find ways to ensure the removal of these substances from contaminated sites. There is a wide range of methods for decontamination, however not all of them are affordable and sufficiently environmentally friendly. Phytoremediation is one of the most environmentally and economically friendly ways used for removing pharmaceuticals and other xenobiotics from the environment. In this work, the effects of the drugs diazepam, rohypnol and trazodone on the antioxidant system of maize (Zea mays, L., cultivar DKC 3969) plants grown under sterile conditions hydroponically in vitro were studied. The effects these drugs had on the plants were monitored through the activity of important antioxidant enzymes, peroxidases and glutathione-S-transferases, as well as through non-enzymatic markers of antioxidant capacity and the content of substances indicative of oxidative stress, malondialdehyde and H2O2. A significant increase in phenolic content was...
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Study of glycocluster prophylaxis on bacterium adherence model
Áčová, Andrea ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF), as autosomal-recessive inherited disease caused by mutation in CFTR gene, induces an ion channel disorder which triggers big amount of CF patients' health problems. The gravest are complications associated with the respiratory system. Accumulation of thick mucus and altered glycosylation in lung lead to increased adherence of bacterial cells, mainly Pseudomonas aeruginosa (PA) and Burkholderia cepacia complex, to lung epithelial cells of CF patients. The presented thesis, focusing on one of the virulence factors of PA, the PA-IIL lectin, studies the adherence of the PA control strain (PAK; ST 1763) under the influence of anti-PA-IIL chicken IgY antibodies and multivalent glycoconjugates based on fucose, to lung epithelial cells collected from healthy person (NuLi-1) and CF patient (CuFi-1). Before the adherence testing, the presence of PA-IIL in PAK culture was checked by "Western Blotting" followed by immunodetection. For quantitative evaluation of the adherence testing, both lung epithelial and bacterial cells were first fluorescently labeled with PKH dyes. The results of spectrofluorimetric measurement after the adherence testing were also compared to microscopic observation of PAK adherence. Different fractions of chicken anti-PA-IIL antibodies from egg yolks showed...
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Production of IL-2 fusion antibodies and determination of their biological activity
Frantová, Eliška ; Vaněk, Ondřej (advisor) ; Kubíčková, Božena (referee)
Interleukin 2 is a small cytokine with many important immune functions. It is used mainly as a T cell growth factor, but it also acts on other immune cells, especially NK and NK-T cells. IL- 2 at higher doses induces the differentiation and proliferation of the cell population of effector and memory T cells, which are characterized by cytotoxicity and are able to effectively defend the organism against pathogens and / or tumor cells. In contrast, low-dose IL-2 stimulates the Treg population, which suppresses immune responses and helps prevent autoimmune diseases. However, in cancer therapy, stimulation of this cell population is undesirable. Because free IL- 2 is toxic to the body at high doses, strategies have previously been proposed to potentiate the biological effect of IL-2. One of the most promising appears to be the single-stranded recombinant fusion construct, where IL-2 is covalently linked via an oligopeptide linker to an anti-IL-2 monoclonal antibody (mAb). Based on the findings of the studies of IL-2 / anti-IL-2 mAb immunocomplexes, this immunocytokine (IC) could provide significant therapeutic benefits in vivo, as compared to free IL-2, especially very robust strengthening of biological activity, selective stimulation of specific cell populations according to the selected antibody and...
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Antioxidant properties of tobacco plants grown in vitro with different nitrogen sources
Vlčková, Denisa ; Ryšlavá, Helena (advisor) ; Kubíčková, Božena (referee)
The established concept of plant nutrition with NO3 − and NH4 + ions, as the only absorbable forms of nitrogen, has been expanded in recent years to find that some plants are also able to use amino acids and peptides as a nitrogen source. In this work, the content of proteins, phenolic compounds and flavonoids, antioxidant capacity and activity of antioxidant enzymes in leaf extracts of six groups of tobacco plants grown in vitro were observed. The nutrition of these plants varied in the form and concentration of nitrogenous compounds. The experimental groups included: control plants grown on complete Murashige-Skoog agar rich in NH4 + and NO3 − ions (MS), plants grown in the presence of NO3 − ions, with reduced nitrogen concentration compared to MS (AD), plants grown in the presence of casein hydrolysate as the sole nitrogen source (H), plants grown in presence of both NO3 − ions and casein hydrolysate (AD+H) and AD and H plants grown in the presence of polyethylene glycol simulating drought stress (AD+PEG and H+PEG). Of all groups, plants H showed the highest content of phenolic compounds and flavonoids and the highest values of antioxidant capacity and antioxidant enzyme activity (catalase, glutathione reductase, superoxide dismutase, total peroxidases, ascorbate peroxidase, glutathione-S-...
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Current approaches to cystic fibrosis therapy
Chmelíková, Barbora ; Kubíčková, Božena (advisor) ; Marková, Vendula (referee)
This bachelor thesis is focused on possible herapeutic methods for cystic fibrosis with main focus on modulators. The individual modulators are described in terms of their function, their differences and application possibilities. The thesis are also discuss other methods of treatment of cystic fibrosis, both for the respiratory tract and gastrointestinal tract. The thesis itself will be divided into three complementary parts. The first theoretical part defines the basic concepts of terms related to cystic fibrosis, the second follow-up practical part is focused on the comparison of the various methods of treatment of cystic fibrosis and the third part in the form of detailed analysis of the individual modulators. As for the methodology of the work, a method of literature search using secondary verified sources will be used. Keywords: cysticfibrosis, gene therapy, therapy, modulator, potentiator, correctors
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Biochemical aspect of investigation of the natural-origin binders and varnishes in the paint layer of old paintings
Pecháčková, Soňa ; Hudeček, Jiří (advisor) ; Kubíčková, Božena (referee)
The subject of this work are choosen bioanalytical methods, used for the identification of organic binders and varnishes in coloured layer of ancient paintings. In the past, the plant and animal substances were used as a binders and varnishes. To those materials belong proteins, oils, polysacharide gums and resins. Succesful analysis in the history of using binders and varnishes demands a good knowledge of their chemical qualities and reactivity during the process of aging. Nowadays are prefered the methods and technics, which don?t destruct the sample and which are non-invasive towards the whole artwork. Recently, the imunofluorescence technics have been started to use as the alternative methods of classical and easier microchemical tests. The spectroscopic methods are widely used in material investigation of ancient artworks. The main is infrared spectometry (FTIR) and Raman spectroscopy. The methods of nuclear magnetic resonance are significant mainly for studying changes of connective and isolating material during the process of aging. To indentify mainly protein binders, the technic of mass spectrometry (MALDI-TOF) is widely used. Due to the progress of technic the paper and thin-layer chromatography were replaced by very effective high performance liquid chromatography (HPLC), gas chromatography (GC)...
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