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Monitoring the efficacy and safety of systemic treatment for atopic eczema
Tánczosová, Milena ; Gkalpakiotis, Spyridon (advisor) ; Rob, Filip (referee) ; Fikrle, Tomáš (referee)
Atopic dermatitis (AD), syn. atopic eczema, is one of the most common chronic inflammatory skin diseases of a non-infectious nature, which, although not life-threatening, has a significant negative impact on the patient's quality of life. The treatment of severe atopic dermatitis had long been very difficult due to the fact that the available treatment options were not suitable for long-term use and were associated with side effects and frequent laboratory examinations. The goal of therapy is to achieve long-term remission with minimal side effects and to improve patients' quality of life. Options of topical therapy are limited to corticosteroids and topical immunomodulators. Systemic therapy, including biological therapy, is inevitable in patients with severe atopic dermatitis. Based on intensive research and a better understanding of the etiopathogenesis of AD, we have at our disposal not only conventional immunosuppressive systemic therapy, but also newly targeted therapy with small molecules and also targeted biological therapy with dupilumab, which has shown a remarkable reduction in clinical severity with a good safety profile. The aim of this study was to describe the efficacy and safety of dupilumab treatment for patients with severe atopic dermatitis. Dupilumab is a fully human monoclonal...
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Panels of steroid receptor reporter cell lines for compound profiling and development of selective ligands for estrogen receptor alpha and beta
Sedlák, David ; Bartůněk, Petr (advisor) ; Anzenbacher, Pavel (referee) ; Kasal, Alexander (referee)
of the Ph.D. thesis Steroid hormone receptors represent a major target in the drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. The first part of this work describes a preparation of two panels of potent, selective and robust luciferase reporter cell lines on the unified cellular background in U2OS osteosarcoma cell line. This system consists of two panels of stable luciferase reporter cell lines for estrogen receptor α (ERα), estrogen receptor β (ERβ), androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and progesterone receptor (PR). The first panel of reporter cell lines relies on the expression of the chimeric steroid receptors created by the replacement of the N-terminal part of the steroid receptor molecule by Gal4 DNA binding domain (Gal4 DBD) binding to 9 copies of Gal4 upstream activation sequences (Gal4 UAS) in the promoter of the pGL4 luciferase reporter vector. In the second panel of reporter cell lines the activation of either synthetic promoter containing multiple hormone response elements or viral promoter derived from MMTV LTR is mediated by full- length exogenously expressed steroid receptors. We have extensively validated both panels using 28 well established ligands, carefully...
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Synthesis of photoactive derivatives of phthalocyanines and study of their biological activity
Rychvalská, Lucia ; Zimčík, Petr (advisor) ; Vícha, Robert (referee) ; Zitko, Jan (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Author: Mgr. Lucia Rychvalská Supervisor: prof. PharmDr. Petr Zimčík, PhD. Doctoral Thesis: Synthesis of photoactive derivatives of phthalocyanines and study of their biological activity Phthalocyanines (Pc) are macrocyclic compounds with conjugated system of double bonds with absorption maximum around 650 - 750 nm. They are characterized by high singlet oxygen production which makes them suitable candidates for photosensitizers in photodynamic therapy. However, they form inactive aggregates in water environment because of their planar hydrophobic aromatic core. There are many strategies to solve this problem, and this thesis is focused on fighting aggregation of Pc by their supramolecular interactions with cucurbiturils (CB). Subphthalocyanines (SubPc) are similar macrocyclic compounds composed of only three (instead of four) isoindol units with boron as a central atom, which has also axial substituent. Because of their cone-shape and axial substituent they are less aggregated in water but usually not very soluble, which could also be solved by interaction with CB. First part of this thesis was focused on synthesis of Pc with aminoadamantyl substituents on...
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Sperm morphology and molecular mechanism associated with sperm fertilizing ability in mice.
Šebková, Nataša ; Komrsková, Kateřina (advisor) ; Jonáková, Věra (referee) ; Linhart, Otomar (referee)
The process of sperm capacitation and acrosome reaction (AR) are highly dynamic processes essential for the fertilization, including cytoskeleton proteins in the sperm head. The study of the distribution of actin, spectrin and α-tubulin in the head of representative mammalian sperm indicates that before and after the AR there were changes in the distribution of the cytoskeleton structures in the sperm head of all representatives, mainly in the apical part of the acrosome, but also in the equatorial and postacrosome part of the sperm head. The particularly well-studied actin cytoskeleton plays an important role during the capacitation and before and after the AR. Environmental estrogens can interfere with the function of endogenous hormones in very low concentrations. They can interact with specific receptors affecting several signalling pathways leading to sperm capacitation and AR. The effect of 17β-estradiol, estrone, estriol and synthetic 17α-ethynylestradiol was evaluated by the ratio of tyrosine phosphorylation and the state of the acrosome during in vitro capacitation in the sperm head. This study has provided the evidence that estrogens significantly stimulate capacitation progress in a concentration-dependent manner. Estrogens decrease number of sperm after the induced AR too. The raising...
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Intravital diagnostics of neurodegenerative diseases
Baranová, Soňa ; Holada, Karel (advisor) ; Kaňovský, Petr (referee) ; Žáková, Dana (referee)
Prionopathies, also called transmissible spongiform encephalopathies (TSE) and synucleinopathies are neurodegenerative diseases that are associated with the accumulation of misfolded proteins (prion and α-synuclein) mostly in the central nervous system. To this day, early and definite diagnosis remains unavailable during the patient's lifetime, mainly due to the absence of reliable biomarker which makes clinical diagnosis more challenging. Therefore, the gold standard in diagnostics remains direct post-mortem evaluation of misfolded proteins within brain tissue by western blot and immunohistochemistry. In the recent years, seeding amplification assays (SAAs) like Real-Time Quacking-Induced Conversion (RT-QuIC) emerged for ultra-sensitive ante-mortem diagnosis of neurodegenerative diseases. SAAs exploit ability of pathological misfolded proteins present in patient's samples to change the conformation and initiate aggregation of native recombinant protein substrate by prion-like seeding mechanism. In the presented dissertation thesis, we exploited second-generation RT-QuIC assay (55řC, 700 rpm, cycles of 1 min double-orbital shaking and 1 min incubation) utilizing recombinant hamster shortened prion protein (rHAPrP90-231) to evaluate prion seeding activity in post-mortem TSE (n=38) and non-TSE (n=30)...
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Role of variable chains at the interface between subunits in forming ATP-binding pocket and function of P2X4 receptor
Tvrdoňová, Vendula ; Zemková, Hana (advisor) ; Novotný, Jiří (referee) ; Vlachová, Viktorie (referee)
7 ABSTRACT Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. The role of these domains in forming of ATP-binding pocket and receptor function was investigated by using alanine scanning mutagenesis of the R203- L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor and by examination of the responsiveness to ATP and orthosteric analog agonists 2- (methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4- benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'- triphosphate. ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. These experiments, together with homology modeling, indicate that residues of the first group located in the upper part of...
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Crystal structure of inorganic phosphates
Fejfarová, Karla ; Mička, Zdeněk (advisor) ; Žák, Zdirad (referee) ; Sýkora, Jan (referee)
½ž ËÙÑÑ ÖÝ Ó ×× ÖØ Ø ÓÒ ½ž½ž Ñ Ó ×× ÖØ Ø ÓÒ The aim of this thesis was to solve and describe the crystal struc- ture of selected inorganic phosphates. Nine different compounds have been studied, including the NASICON type phosphates Co0.5Ti2(PO4)3, Mn0.5Ti2(PO4)3, Na3.215Fe2(PO4)3, the iron(II) diphosphate Fe2P2O7 and hydrates of rare-earth hydrogen diphosphates LnHP2O7·3H2O (Ln = Y, Tb, Dy, Yb) and TbHP2O7·4H2O. While NASICON type phos- phates and rare-earth hydrogen diphosphates have a conventional three- dimensional crystal structure, the structure of α-Fe2P2O7 is incommen- surately modulated and has been described in (3+1)-dimensional super- space. ½ž¾ž ÇÚ ÖÚ Û Ó Ø ÓÒØ ÒØ Ó ×× ÖØ Ø ÓÒ Dissertation is divided in six parts. Part 1 introduce the crystal struc- ture analysis, the concept of aperiodic crystals and the superspace ap- proach. Part 2 describes course of X-ray diffraction experiment and used experimental equipment. The next three parts, Part 3, Part 4 and Part 5, in which the structures of NASICON-type phospates, iron(II) diphos- phate, and rare-earth hydrogen diphosphates are discussed, are the main parts of this work. Part 1. Introduction and Initiation Chapter 2, "Basic principles of crystal structure analysis" con- cerns by fundamentals of crystal structure analysis, i.e. behaviour of X-...
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Investigation of the protein interactions involved in the acylation of Escherichia coli α-hemolysin
Nemčeková, Lucia ; Osičková, Adriana (advisor) ; Šulc, Miroslav (referee)
Uropathogenic bacteria Escherichia coli are the primary cause of urinary tract infections. One of the virulence factors of these bacteria is α-hemolysin (HlyA), a protein belonging to the cytolytic RTX (Repeats in ToXin) toxins secreted by some gram-negative pathogenic bacteria. RTX toxins share several characteristic structural and functional domains and segments: (1) an N-terminal hydrophobic pore-forming domain, (2) an acylated segment where two lysine residues are modified by fatty acid chains, (3) a repetitive (RTX) domain binding calcium ions, and (4) a C-terminal secretion signal recognized by the type 1 secretion system. HlyA is synthesized as an inactive protoxin (proHlyA), which is activated by covalent acylation of the ε-amino groups of two conserved lysine residues, K564 and K690, by the co-expressed acyltransferase HlyC. Acyl-acyl carrier protein (acyl-ACP) serves as the acyl chain donor. However, the molecular mechanism by which the acyltransferase HlyC interacts with acyl-ACP and proHlyA is currently poorly understood. The aim of this bachelor thesis was to identify amino acid residues involved in the interaction between HlyC and ACP proteins. Based on an in silico interaction model of HlyC and ACP, positively charged residues in HlyC and negatively charged residues in ACP were...
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Presence and function of glycosidas in the reproductive tract of mammals
Slavík, Martin ; Postlerová, Pavla (advisor) ; Tuzhilkin, Roman (referee)
Glycosidases are enzymes that hydrolytically cleave polysaccharide structures and have fundamental importance above all for processing the glycocalyx on the surface of the sperm during the fertilization process, so that the sperm can finally successfully interact with the zona pellucida and fuse with the oocyte membrane. The bachelor thesis investigates the activities of several important glycosidases (β-galactosidases, α-galactosidases, α-mannosidases and neuraminidases) in mixed samples of reproductive fluids, specifically it is epididymal fluid, seminal plasma and oviductal fluid, originating from the domestic pig. Another sample in which the activity was measured of the aforementioned enzymes was ejaculated sperm. The activities of the given enzymes were determined using fluorescent substrates in slightly acidic and neutral pH. In addition to determining specific activity of individual enzymes, this bachelor's thesis was enriched with qualitative information regarding glycoproteins in male and female reproductive fluids and ejaculated sperm lysate. Glycoproteins are the dominant component of the sperm glycocalyx and many of them also represent the corresponding naturally occurring substrates for the above-mentioned glycosidases. Based on electrophoretic separation followed by staining, the...
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Hull-White model: forward-looking vs backward-looking approaches
Kárný, Jakub ; Flimmel, Daniela (advisor) ; Večeř, Jan (referee)
The subject of the thesis is the estimation of the parameters of the Hull-White model. The formula for the time-dependent parameter θ is derived. The constant parameters α and σ are first calibrated on the prices of the chosen interest rate derivatives in the forward-looking approach and secondly on the historical yield curves in the backward- looking approach. Calibrated Hull-White models are simulated and then the approaches used are compared in terms of available data. 1
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