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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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Study of the expression and toxicity of catechol derivates in MCF-7 cell line
Kleplová, Dominika ; Carazo Fernández, Alejandro (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Dominika Kleplová Supervisor: PharmDr. Alejandro Carazo, PhD. Title of the Diploma thesis: Study of the expression and toxicity of catechol derivatives in MCF-7 cell line Estrogens perform many important functions in the human body. They act by binding to estrogen receptors (ER) and thus regulate reproduction, the menstrual cycle, bone density, cholesterol metabolism or brain function. They also play an important role in the development and onset of breast cancer, where the amount of ER expressed is used as a very important biomarker in patients suffering from this disease. Despite ongoing research, breast cancer is considered to have the highest mortality rate. Catechols are organic compounds. In the human body, they can occur as metabolites in the degradation of benzene and estrogens or other endogenous compounds such as neurotransmitters and their precursors. Catechols are known to be involved in redox processes in the body, to exert antioxidant and toxic effects, to interfere with protein function and to cause DNA strand breaks. Their positive effect on breast cancer therapy is the subject of research, but it has not yet been fully explored. The subject of this thesis is to study the...
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CHARACTERIZATION OF GILTERITINIB-RESISTANT LEUKEMIC CELL LINE
Zlesáková, Lucie ; Čečková, Martina (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucie Zlesáková Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Consultant: Mgr. Simona Suchá, Ph.D. Title of diploma thesis: Characterization of gilteritinib-resistant leukemic cell line Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis for which therapy failure is often responsible. Development of drug resistance is among the most common causes of treatment failure. The exact mechanisms leading to the resistance are not known, especially for drugs recently introduced into the clinical practice such as gilteritinib. Therefore, the gilteritinib-resistant leukemic cell line (referred to as HL-60 g75) has been established in our lab and further characterized. The aim of this study was to evaluate the sensitivity of HL-60 g75 cells to gilteritinib and to clarify the stability of acquired resistance. We revealed that the resistance of HL-60 g75 cells is transient, since only after 4 weeks of gilteritinib-free cell culture, sensitivity of these cells was restored. While gilteritinib induced apoptosis similarly in both gilteritinib-sensitive (HL-60 wt) and -resistant cells, cell cycle analysis revealed lower responsiveness of HL-60 g75 to gilteritinib than HL-60 wt....
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Antiproliferative activity of novel dexrazoxane analogues and their effect on antitumor effectiveness of anthracyclines
Martinková, Pavla ; Jirkovská, Anna (advisor) ; Pohanka, Miroslav (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavla Martinková Supervisor: PharmDr. Anna Jirkovská, PhD. Title of diploma thesis: Antiproliferative activity of novel dexrazoxane analogues and their effect on antitumor effectiveness of anthracyclines Athracycline antibiotics (such as daunorubicin, doxorubicin or epirubicin) belong to the most common terapeutics of both solid tumors and hematological malignities. Unfortunately the serious and life-threatening adverse effect cardiotoxicity compromises their clinical usefulness. The only approved protection against anthracycline cardiotoxicity so far is dexrazoxane. Despite the outstanding cardioprotective ability, dexrazoxane use is very limited mainly due to its possible side effects. So we were directed towards synthesis of dexrazoxane analogues with better pharmacological properties. The aim of this diploma thesis was to assess the antiproliferative activity of novel analogues of both dexrazoxane (MK-15 and ES-5) and ADR-925 (JR-159 and KH- TA4) and their influence on the antiproliferative effectiveness of anthracyclines. Moreover, we aimed to study their chelating properties and their inhibition of the topoisomerase II in solution. We tested the antiproliferative activity of...
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Dynamics of de novo DNA methylation and its impact on transgene expression and CRISPR/Cas9 mutagenesis
Přibylová, Adéla ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Fajkus, Jiří (referee)
Genetic information must be protected, maintained and copied from cell to daughter cells, from generation to generation. In plants, most of the cells contain complete genetic information, and many of these cells can regenerate to a whole new plant. Such a feature leads to the need for precise control of which genes will be active and which not because in growth and differentiation, only the activity of specific genes for the individual cells, tissues, organs are required. One of the mechanisms controlling the gene activity is RNA interference (RNAi), which down- regulates or blocks the expression of specific genes at the transcriptional or post-transcriptional level. The crucial part of the RNAi is guiding the RNAi machinery to the target. It is mediated via sequence complementarity of the target with a small RNA (sRNA), which is diced from a double- stranded RNA (dsRNA) precursor. The molecular mechanism of dsRNA and sRNA formation and also the target origin predestinates the subsequent silencing pathway. In transcriptional gene silencing (TGS), the gene expression is regulated through chromatin epigenetic modifications. One of the epigenetic marks is cytosine methylation, which is established mainly by RNA-directed DNA-methylation (RdDM) pathway. Although the protein machinery was relatively...
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Characterization of newly developed fluorescence probes in cellular systems
Kadlecová, Julie ; Hubálek Kalbáčová, Marie (advisor) ; Hendrych, Tomáš (referee)
Nanoparticles (NP) are currently a progressive area of scientific research. The possibility of synthesizing them according to the required parameters opens up possibilities for their wide use also in biomedicine. One example is a nanoparticle that can detect cellular processes, such as pH. We already know that the pH of healthy and cancer cells differs by the opposite gradient on the intracellular and extracellular side of the membrane. In this context, this work deals with the study of fluorescent silicon nanoparticles (SiNP) tested on a human keratinocyte cell line from a healthy donor (HaCaT) and from skin cancer donor (A431). Once found that even the highest concentrations of SiNP used are not cytotoxic, they can be further studied by fluorescence, confocal and super-resolution microscopy. In order to assess the pH detection properties of these SiNPs, a method for measuring intracellular pH with a fluorescent raciometric probe SNARF-1 using fluorescence spectroscopy and flow cytometry was introduced. Since the pH values of the intracellular environment are closely related to cellular metabolism, the metabolism of A431 and HaCaT cells was characterized and compared. To do this, methods for measuring analog glucose consumption (2-NBDG) and another new method for measuring real-time metabolism...
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The effect of IgY on bacterial adhesion on epithelial cells ex vivo
Vašková, Lucie
0 Abstract Cystic fibrosis is an autosomal recessive disease caused by mutation in CFTR gene coding for a chloride channel in apical membrane of epithelial cells. This disorder leads to the change in ion transport causing the increase in mucus viscosity in airways as well as changes in glycosylation of saccharide structures on the cells. Because of that these cells are the target for bacterial adhesion. Chronic bacterial infections, which lead to gradual decline of lung function and damage of lung tissue, are the major cause of death of patients suffering with cystic fibrosis. Pseudomonas aeruginosa is the main pathogen causing chronic infections in cystic fibrosis patients. This bacterium produces a biofilm protecting them from host immune system and antibiotics. Once the colonization with PA occurs, it is difficult to get rid of this pathogen. The prophylactic treatment with orally administered hen antibodies against the PA virulence structures could be a prevention of chronic PA infections. In this work we tested the antibody against the bacterial lectin PA-IIL, which is suggested to be involved in the adhesion of the pathogen on epithelial cells. First, it was verified that the prepared antibody from egg yolks of a hen immunized with the bacterial lectin PA-IIL recognizes this antigen expressed...
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The effect of IgY on bacterial adhesion on epithelial cells ex vivo
Vašková, Lucie ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
0 Abstract Cystic fibrosis is an autosomal recessive disease caused by mutation in CFTR gene coding for a chloride channel in apical membrane of epithelial cells. This disorder leads to the change in ion transport causing the increase in mucus viscosity in airways as well as changes in glycosylation of saccharide structures on the cells. Because of that these cells are the target for bacterial adhesion. Chronic bacterial infections, which lead to gradual decline of lung function and damage of lung tissue, are the major cause of death of patients suffering with cystic fibrosis. Pseudomonas aeruginosa is the main pathogen causing chronic infections in cystic fibrosis patients. This bacterium produces a biofilm protecting them from host immune system and antibiotics. Once the colonization with PA occurs, it is difficult to get rid of this pathogen. The prophylactic treatment with orally administered hen antibodies against the PA virulence structures could be a prevention of chronic PA infections. In this work we tested the antibody against the bacterial lectin PA-IIL, which is suggested to be involved in the adhesion of the pathogen on epithelial cells. First, it was verified that the prepared antibody from egg yolks of a hen immunized with the bacterial lectin PA-IIL recognizes this antigen expressed...
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The effect of IgY on bacterial adhesion on epithelial cells ex vivo
Vašková, Lucie
0 Abstract Cystic fibrosis is an autosomal recessive disease caused by mutation in CFTR gene coding for a chloride channel in apical membrane of epithelial cells. This disorder leads to the change in ion transport causing the increase in mucus viscosity in airways as well as changes in glycosylation of saccharide structures on the cells. Because of that these cells are the target for bacterial adhesion. Chronic bacterial infections, which lead to gradual decline of lung function and damage of lung tissue, are the major cause of death of patients suffering with cystic fibrosis. Pseudomonas aeruginosa is the main pathogen causing chronic infections in cystic fibrosis patients. This bacterium produces a biofilm protecting them from host immune system and antibiotics. Once the colonization with PA occurs, it is difficult to get rid of this pathogen. The prophylactic treatment with orally administered hen antibodies against the PA virulence structures could be a prevention of chronic PA infections. In this work we tested the antibody against the bacterial lectin PA-IIL, which is suggested to be involved in the adhesion of the pathogen on epithelial cells. First, it was verified that the prepared antibody from egg yolks of a hen immunized with the bacterial lectin PA-IIL recognizes this antigen expressed...
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Antiproliferative activity of novel dexrazoxane analogues and their effect on antitumor effectiveness of anthracyclines
Martinková, Pavla ; Jirkovská, Anna (advisor) ; Pohanka, Miroslav (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavla Martinková Supervisor: PharmDr. Anna Jirkovská, PhD. Title of diploma thesis: Antiproliferative activity of novel dexrazoxane analogues and their effect on antitumor effectiveness of anthracyclines Athracycline antibiotics (such as daunorubicin, doxorubicin or epirubicin) belong to the most common terapeutics of both solid tumors and hematological malignities. Unfortunately the serious and life-threatening adverse effect cardiotoxicity compromises their clinical usefulness. The only approved protection against anthracycline cardiotoxicity so far is dexrazoxane. Despite the outstanding cardioprotective ability, dexrazoxane use is very limited mainly due to its possible side effects. So we were directed towards synthesis of dexrazoxane analogues with better pharmacological properties. The aim of this diploma thesis was to assess the antiproliferative activity of novel analogues of both dexrazoxane (MK-15 and ES-5) and ADR-925 (JR-159 and KH- TA4) and their influence on the antiproliferative effectiveness of anthracyclines. Moreover, we aimed to study their chelating properties and their inhibition of the topoisomerase II in solution. We tested the antiproliferative activity of...
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