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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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Congenital disorders of glycosylation and their impact on cellular and energetic metabolism
Rychtárová, Lucie ; Hansíková, Hana (advisor) ; Hodek, Petr (referee) ; Pecinová, Alena (referee)
Inherited disorders of glycosylation (CDG) are a large group of more than 160 types of metabolic disorders caused by genetic defects that lead to impaired glycan biosynthesis and modification. The lipid dolichol plays an essential role in glycan biosynthesis. Glycans play a key role in the function and structure of proteins and lipids and their deficiency leads to severe clinical symptoms. CDG usually manifests in childhood as a multisystem disorder. Families thus face a serious health problem due to the progressive and highly variable nature of the disease, the unfavorable prognosis and, with few exceptions, the unavailability of treatment. Currently, we still do not have a sufficient range of methods to recognize rare types of CDG and our knowledge of the pathophysiology of CDG is still limited. The first aim of this work was to optimize the method of determination of dolichol isoforms and to study them in physiology and pathology. The second aim of the work was to investigate the bioenergetic status and overall metabolism in the most common type of CDG - deficiency of phosphomannomutase 2 (PMM2-CDG), and in CDG caused by a defect in dolichol biosynthesis. The distribution of urinary dolichol isoforms in the population was characterized using an optimized method. The dolichol isoforms profile...
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Preparation and characterization of chicken antibodies against pathogenic yeast Candida albicans
Vodecký, Matúš ; Hodek, Petr (advisor) ; Dračínská, Helena (referee)
The increase of infections caused by yeast pathogens is an alarming global public health problem. People with compromised immune systems, such as after infection with human immunodeficiency virus or diabetes mellitus, are most at risk. Diagnosis is often inadequate, and treatment relies on a limited selection of antifungal drugs, to which resistance is also increasing. These aspects represent a major problem, as second-choice drugs, which may have toxic side effects (e.g., nephrotoxicity in the case of amphotericin B), must then be applied to resistant pathogens. Given the severity of yeast infections, scientists are trying to find new ways of protection against these pathogens. One option is passive immunotherapy. In this bachelor thesis, antibodies from hen's eggs were chosen for this purpose, as they have the potential for wide therapeutic use. The preparation of hen's egg yolk IgY antibodies is both ethically and economically acceptable. Moreover, egg yolk antibodies show relatively high stability. Polyclonal antibodies were isolated from eggs of hens immunized with either cell wall or whole C. albicans cells in a medium simulating the vaginal environment (VSF). Antibody reactivity was tested by ELISA. The cell walls of C. albicans in VSF medium or YPD medium were used as antigen. The results...
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Interaction of bacterial lectins with human lung epithelium
Vyhnalová, Kateřina ; Hodek, Petr (advisor) ; Bořek Dohalská, Lucie (referee)
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene encoding the same named chloride channel, which is present on the apical membrane of epithelial cells. As a consequence of these mutations, the transepithelial transport of ions and the regulation of other ion channels are impaired. Cystic fibrosis affects primarily respiratory system, where, as a result of the dysfunction of the CFTR protein, is an accumulation of excessively viscous mucus and altered glycosylation of saccharide structures on the surface of the lung epithelium. This pathological condition predisposes CF patients to bacterial lung infections, the leading cause of death in CF patients. The most common pathogen colonizing the respiratory system of CF patients is Pseudomonas aeruginosa (PA). For adhesion to host tissue, PA uses several virulence factors, including lectin PA-IIL, which exhibits an unusually high affinity for L-fucose. In this work the influence of selected monosaccharides and multivalent fucose-based inhibitors (glycoclusters) on PA-IIL binding to human lung epithelium was investigated. To assess the effect of these agens ex vivo, paraffin-embedded formalin-fixed human lung tissues (FFPE tissues) from a healthy individual and...
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New insulin-like growth factor 1 (IGF-1) analogs for receptor studies and therapeutic applications
Lin, Jingjing ; Jiráček, Jiří (advisor) ; Hodek, Petr (referee) ; Lapčík, Oldřich (referee)
The insulin/IGF system is an evolutionary conserved network that includes three closely related peptide hormones (insulin, IGF-1 and IGF-2), three homologous tyrosine kinase receptors (IR-A, IR-B and IGF-1R), a distinct IGF-2 receptor (IGF-2R), and six IGF-binding proteins. While insulin signals mainly via IR, playing a key role in glucose homeostasis, IGFs mainly signal via IGF-1R to mediate normal human growth. A tight control of ligands and receptors expression is crucial for the proper functioning of the organism. Aberrant signaling by these receptors is manifested in many pathological conditions, including cancer, growth disorders, neurodegenerative diseases and diabetes. Hence, the insulin/IGF axis represents a promising therapeutic target. In view of the multiple unsuccessful clinical anticancer trials performed either with tyrosine kinase inhibitors or antibodies targeted against IGF-1R, the development of novel selective and receptor-specific insulin and IGF analogs with minimized side effects would be of interest. Our study began with the recombinant preparation of IGF-1 dimers in which IGF-1 monomers are interlinked between their C- and N-termini with Ser-Gly linkers (length of 8, 15 or 25 residues). The goal was to investigate whether the binding of two covalently linked IGF-1 molecules...
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Terpene discovery combining in silico and molecular biology approaches
Smrčková, Helena ; Pluskal, Tomáš (advisor) ; Hodek, Petr (referee)
Terpenoids are the largest class of natural products with remarkable chemical and structural diversity, making them a significant source of compounds for drug discovery. Terpenoids are used as food flavours, therapeutics, cosmetics, hormones or fuel. The starting scaffolds for terpenoid biosynthesis are condensed and, in some cases, cyclic carbohydrates synthesized from linear isoprenyl pyrophosphate precursors by terpene synthases. The enzyme family of terpene synthases is extensive and possesses well-characterized motifs and function-determining domains that can be used to search for homologous proteins with unknown catalytical function that might produce new compounds. Thanks to a bioinformatic pipeline developed in the Pluskal Lab in the Institute of Organic Chemistry and Biochemistry CAS, candidate terpene synthases with the potential to produce new terpene scaffolds were mined in silico from large protein sequence repositories. I expressed nine selected proteins originating from plants, bacteria, and fungi in the engineered budding yeast strains JWY501 and ZX178-08 that overproduce isoprenyl pyrophosphates. I then analysed the resulting yeast cell culture extracts using gas chromatography- and liquid chromatography-mass spectrometry. Five out of nine proteins exhibited terpene synthase...
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Effect of selected perfumes on aromatase activity
Drejslarová, Iva ; Hodek, Petr (advisor) ; Smrček, Stanislav (referee)
Cytochromes P450 are enzymes involved in many physiological processes in the body, besides the metabolism of xenobiotics also in the biosynthesis and catabolism of endogenous substances such as hormones and steroids. Cytochromes P450 that are responsible for the transformation of endogenous compounds can be affected by xenobiotics, which interfere with the secretion, metabolism, transport or elimination of endogenous hormones by various mechanisms. These substances are also referred to as endocrine disruptors and include, for example, pesticides, insecticides, certain pharmaceuticals, or compounds contained in cosmetic products. One of their mechanisms of action is to interfere with steroid hormone biosynthesis through interactions with steroidogenic enzymes. One such enzyme is cytochrome P450 19, aromatase, which catalyses the conversion of testosterone to ß-estradiol in the final step of steroid hormone biosynthesis. Modulation of the activity of this enzyme by endocrine disruptors results in an imbalance of estrogen levels in the body, which can lead to impaired reproduction, osteoporosis, atherosclerosis, dementia, and the development of certain types of cancer. In this context, the effect of commercially available perfumes on the conversion of testosterone to ß-estradiol, which is catalyzed by...
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Effect of natural substances on transcriptional activity of nuclear and steroid receptors and expression of cytochrome P450 (CYP1A)
Bednaříková, Barbora ; Hodek, Petr (referee)
This work is based on two attached first authors' publications dealing with the effect of natural substances of plant origin on transcriptional activity of nuclear and steroid receptors and their action on cytochrome P450 expression, CYP1A subfamily. The studied substances are anthocyanidins and stilbenes, belonging to a broad group of phenolic compounds. The aim of the first study was to evaluate the effect of the most common anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, and peonidin) on the transcriptional activity of nuclear and steroid receptors. The activities of nuclear receptors: vitamin D receptor (VDR), retinoid X receptor (RXR), retinoic acid receptor (RAR), pregnane X receptor (PXR), thyroid receptor (TR), and steroid receptors: progesterone receptor (PR), estrogen receptor (ER), androgen receptor (AR), and glucocorticoid receptor (GR) were assessed using either stable or transiently transfected luciferase gene reporter cell lines. The cytotoxicity assays and gene reporter assay were performed after the 24h treatment of cells with increasing range of concentrations (10 nM to 50 µM) of selected anthocyanidins. The results of experiments indicate that none of the studied anthocyanidins in all tested concentrations caused remarkable changes of transcriptional activity of...
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Selected nutritional supplements as modulators of biotransformation enzymes
Bača, Ondrej ; Hodek, Petr (advisor) ; Ryšlavá, Helena (referee)
Flavonoids are secondary metabolisms of plants, that have wide range of biological activities. Between the most known activities are antioxidant activities, antibacterial activities, antivirus activities and chemoprotective activities. They are consumed as a part of plants to varying degrees by the population. They are consumed either in their plant forms, or as a nutritional supplement, such as pills. As nutritional supplement they don't fall under strict regulations and their effect or active quantity isn't always examined properly. As a foreign substance to the body that is xenobiotics, they can fundamentally influence so called biotransformation enzymes, which apart from other handle metabolism of xenobiotics. One of those enzymes is N-acetyltransferase. This biotransformative enzyme, which among others metabolises lot of pharmaceuticals, shows number of polymorphisms, where the occurrence of some of those relates to different illnesses such as breast cancer or bladder cancer, diabetes, or Parkinson. The modulation activity of those enzyme can either hurt the organism or help it. In this paper we will focus on inhibitive effect of flavonoids myricetin and dihydromyricetin to N-acetyltransferase especially the mechanism of this inhibition. Inhibition was measured in vitro for both steps of the...
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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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