National Repository of Grey Literature 129 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Preparation, synthesis and activity of new inhibitors of cystein proteases from Porphyromonas gingivalis
Gistrová, Tereza ; Konvalinka, Jan (advisor) ; Rýček, Lukáš (referee)
This bachelor thesis deals with modification of known inhibitor KYT-36 specific for the Porphyromonas gingivalis protease called gingipain (Kgp).1 Proposed modification with an oligoethylenglycole linker in suitable position will enable the use of the derivative in the iBodies concept.2 These polyfunctional macromolecules based on a polymer backbone are capable of replacing antibodies in biochemical experiments in the study of enzymes. The main aim of the thesis is to propose and perform an effective synthetic strategy. This thesis includes an analysis of a crystal structure of enzyme Kgp with the inhibitor KYT-36 (PDB ID: 6I9A).3 The analysis enabled a choice of an appropriate modification site. A suitable functional group was added to the selected fragment allowing attachment of the linker. The linker's design was based on requirements for the intended use within the iBodies concept. One of them being a suitable functional group at the end of the chain ensuring an effortless conjugation with polymer backbone. Based on a retrosynthetic analysis, a synthetic strategy of the inhibitor core was proposed. This strategy builds upon the Passerini reaction. Key words: Porphyromonas gingivalis, gingipain, inhibitor, KYT-36, iBodies, Passerini
Synthesis of peptides as potential ligands of GCPII protein as identified by phage-display method
Trajhan, Filip ; Konvalinka, Jan (advisor) ; Žáková, Lenka (referee)
Glutamate carboxypeptidase II (GCPII, PSMA-prostate-specific membrane antigen) is a human transmembrane metalloprotease highly expressed in prostate cancer, which makes it a suitable target for diagnostics or targeted cancer therapy. Therefore, specific and tight-binding ligands of GCPII are needed. One way of finding potential low molecular weight peptide ligands is the screening of combinatorial libraries. One of the methods of high-throughput screening is phage display, which uses genetically modified bacteriophages for the study of interaction between peptides and target protein. A library of potential peptide ligands was prepared based on the results of phage display targeted against various forms of GCPII. Parallel peptide synthesiser SPENSER was used for the synthesis and subsequent cyclization of linear peptides. The prepared bicyclic model peptide was characterised and his binding affinity for studied enzyme was confirmed. The possibilities of enhancing the cyclisation efficiency and the use of optimized cyclisation step for preparation of other peptide libraries are discussed. Key words Glutamate carboxypeptidase II, phage display, peptide synthesis, bicyclic peptides, combinatorial chemistry
Development of instrumentation and high-throughput screening methods for peptide ligand discovery and validation
Kryštůfek, Robin ; Konvalinka, Jan (advisor) ; Jiráček, Jiří (referee)
Peptides are used as synthetically available and easily derivatizable scaffold upon which it is possible to develop ligands targeting broad spectrum of biological targets. A time-tested approach to peptide binder identification is the preparation and screening of combinatorial libraries. Bypassing of this complicated procedure is possible by using biological systems for presentation, identification and selection of peptides based on the principle of in vitro evolution - i.e. display techniques. There are two complementary automated solutions for peptide binder identification described in this work. First is the SPENSER parallel peptide synthesizer, developed as a part of this diploma project, which can be used for peptide ligand discovery and optimization as well as validation of ligands identified using display techniques. Several libraries consisting of a total of 1 052 peptides have been prepared and then used to describe its potential applications. A sample of 154 preparations, representing 14.6 % analytical coverage of the prepared libraries, showed an average purity of 67 ± 19 % according to LC-MS. The libraries presented illustrate that SPENSER is a suitable tool for the parallel synthesis of linear and disulfide-cyclized peptides with limited variability, or libraries consisting of short...
Design, preparation and characterisation of polymer conjugates for protein recognition using peptide ligands
Kryštůfek, Robin ; Konvalinka, Jan (advisor) ; Hořejší, Václav (referee)
Antibodies stand as a cornerstone of many laboratory assays and are used as indispensable tools in biomedicine. iBodies polymer conjugates have been proposed as a possible alternative to antibodies in several traditionally immunological methods. iBodies are based on methacrylamide copolymers decorated with affinity anchors, reporters and targeting ligands which mediate conjugate specificity. The necessity to design ligands and their linkage is a significant obstacle to the generalization of this technology. One way of overcoming this obstacle is the development of secondary antibody mimetics which will use antibodies to obtain specificity for target identification and/or visualization. Based on the literature, several cyclic peptide Fc-binding ligands were designed with linker moieties for attachment to the polymer conjugate. Free and conjugated ligands were compared based on their interaction constants with human IgG. Conjugation with polymer carrier in all cases led to an increase in affinity. Human isotype specificity is dependent on the cyclization method, with single disulfide bridge cyclization exhibiting selectivity for IgG1-4 and IgM, and N→C cyclization leading to abolition of IgM binding. The polymer conjugates have been compared in terms of sensitivity and selectivity using dot blot with...
Mechanical properties of materials of knee joint endoprostheses and of mechanically significant tissues in a knee joint
Dvořák, Matouš ; Konvalinka, Jan (referee) ; Burša, Jiří (advisor)
This bachelor thesis deals with the issue of total knee endoprosthesis and describes its division in terms of its construction and attachment style. It summarizes the mechanical properties of human tissues occurring in the knee joint and simultaneously characterizes the most used materials for the production of implants. The thesis contains a calculation focusing on determining the tension around the area of contact of the tibia with the stem of the tibial component. The calculation is performed analytically and subsequently verified by the finite element method in ANSYS software.
Identification of small compounds disrupting protein-protein interaction in influenza A polymerase.
Hejdánek, Jakub ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...
Interaction of a surface marker of immune cells with low-molecular weight ligands and their polymer conjugates
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...
BIOPHYSICAL AND FUNCTIONAL CHARACTERIZATION OF DDI1-LIKE ASPARTIC PROTEASES INVOLVED IN REPLICATION STRESS RESPONSE
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Čermák, Lukáš (referee)
Accurate, timely replication of a DNA molecule is a pivotal moment in the life cycle of every living organism. Any temporal or spatial defect putting the fine-tuned replication machinery off balance causes the so-called replication stress. As the replication machinery consists mainly of enzymes and other proteins, it is not surprising that many of the obstacles most severely blocking the replication machinery progress are of protein origin. Therefore, specialized proteases responsible for relieving replication stress matured during evolution. However, neither the full repertoire of proteolytic enzymes and their particular substrates taking place in countering the DNA replication stress nor detailed molecular mechanisms involved remain unknown. This thesis describes how conserved putative aspartic proteases of the Ddi1-like family engage in countering DNA replication stress via a proteolysis dependent mechanism. We structurally and biophysically characterized yeast and human members of the Ddi1-like family, explored their interactions with ubiquitin and polyubiquitin chains, and identified hypersensitivity to DNA replication inhibitor hydroxyurea in a yeast strain double deleted for DDI1 gene together with a DNA dependent metalloprotease WSS1. Detailed analysis of the DDI1 role in hydroxyurea...
Analysis of impact of mechanical properties of components of pathological arterial wall on its state of stress
Bartoňová, Petra ; Konvalinka, Jan (referee) ; Burša, Jiří (advisor)
The thesis deals with measurements of the atherosclerothic carotid plaques mechanical properties with wiew towards the improvements of their constitutive models and then towards the impact of application of those constitutive models in fibrous cap. The introductory part provides a brief summary of medical knowledge needed to understand atheroma formation and the risks involved. Next, there's a survey of plaque mechanical testing methods and the obtained results on tissue properties. The previously used constitutive models of atherosclerothic tissues are listed as well. Main body of the thesis describes the preparation of testing samples from obtained atheroma, methodology and course of experiments execution and subsequent processing of the experimental data to evaluate the behavior of the atheromatous tissue individual parts. Then the discussion and evaluation of our results and findings is given. Along with the evaluated mechanical properties, experimental data fitted to models are presented with respect to their predictive abilities. The final part is dedvoted to the finite element analysis of an idealized 2D model with emphasis on the effect of necrotic core and fibrous tissue properties on von Mises equivalent stress in the fibrous cap.
Relationship of force and deformation characteristics to the energy of deformed system
Kupka, Oldřich ; Konvalinka, Jan (referee) ; Horníková, Jana (advisor)
The bachelor´s thesis deals with the description of energy in deformed systems and their use to determine the force acting on the system and displacements caused by forces. In the first part of the thesis there are described the basic types of stresses and how it is possible to determine their strain energy and displacements. After that, strain and potential energy including their complementary components are described. Next part is explanation of the principle of virtual work, Betti's theorem and Maxwell's reciprocity theorem. In the second part of thesis, the first and the second Castigliano´s theorems are derived. Castigliano´s theorems are applied to a statically determinate and indeterminate problems.

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