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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš ; Schneider, Bohdan (advisor) ; Pichová, Iva (referee) ; Kukačka, Zdeněk (referee)
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Tracking membrane permeabilization on single lipid vesicles - method development.
Gücklhorn, David ; Šachl, Radek (advisor) ; Heřman, Petr (referee)
Protein complexes are challenging systems to study, especially when these complexes form on lipid membranes only for a short period of time. This is also the case of fibroblast growth factor 2 (FGF2), a protein that has many physiological and pathological functions in the human organism. It plays major role in the development of cancer as it promotes cell survival and angiogenesis. It also serves as a basis for development of novel treatments of nerve injuries. Despite being heavily studied for many years, it remains unclear how the protein is translocated into the extracellular space where it performs its function. To study complex systems such as FGF2 that self-assembles on the membrane into membrane penetrating pores we decided to develop a simple and efficient fluorescent microscopy method. This method is called double leakage single GUV assay (DLSGA). It utilizes giant unilamellar vesicles (GUVs) mimicking native cellular membranes. In a single experiment, up to 300 individual GUVs are imaged for the content of a leakage dye that reports on the presence of FGF2 pores. During three measurements and under different conditions, detailed information about pore-opening dynamics is gained for each GUV. Results of these measurements are then used to divide GUVs into six groups based on formation and...
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Study of molecular mechanisms of the signaling proteins regulation
Kylarová, Salome ; Obšilová, Veronika (advisor) ; Mikšík, Ivan (referee) ; Novák, Petr (referee)
EN The aim of this study was to investigate the regulatory mechanisms of two important signaling proteinkinases and promising therapeutic targets, ASK1 and CaMKK2. ASK1 kinase is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-JNK kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress. The function of ASK1 is associated with the activation of apoptosis and thus plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration or cardiovascular diseases. The natural inhibitor of ASK1 is a ubiquitous oxidoreductase, thioredoxin, which is probably bound to N-terminus of ASK1, thus preventing a homophilic interaction and subsequent ASK1 activation. It has been suggested, that upon oxidative stress and oxidation of thioredoxin active site, thioredoxin dissociates from ASK1, but the structural basis of this interaction remains unclear. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of CaM kinase pathway that activates CaMKI, CaMKIV and AMPK involved in gene expression regulation or apoptosis activation. Function of this protein is often associated with neuropathology, carcinogenesis and obesity. CaM kinases are activated via binding Ca2+ sensor protein...
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Growth of graphene by thermal decomposition of silicon carbide for applications in biophysics
Hodoš, Matúš ; Kunc, Jan (advisor) ; Čechal, Jan (referee)
This bachelor thesis is focused on growth and characterization of graphene from SiC powder at different conditions. In the introduction graphene properties are briefly described. Afterwards three methods of graphene growth are discussed, with particular emphasis on thermal decomposition of SiC. Further, the characterization methods which were used by us are explained. In experimental part, three sets of graphene samples prepared by thermal decomposition of SiC will be described, each will differ in one of the factors: temperature, time and ambient atmosphere. Growth samples will be subsequently used in an experiment on yeast. The effect of graphene prepared at different conditions on membrane potential will be determined for possible application in biophysics.
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Study of structural features of single stranded DNA by biophysical techniques and crystallography
Svoboda, Jakub ; Schneider, Bohdan (advisor) ; Pavlíček, Jiří (referee)
DNA is the fundamental molecule in all domains of life, its role in heredity is well established. Although the famous double helical complementary form is indispensable for replication mechanism DNA can occupy wide range of conformations. In the past studies performed in the laboratory, DNA oligomers related to single stranded bacterial Repetitive Extragenic Palindromic (REP) showed spectral behavior suggesting complex equilibria including double helical, hairpin, and tetraplex conformations. The studies presented in this thesis extended the scope of analyzed sequences and employed circular dichroism spectroscopy and X-ray crystallography. We report spectral data and X-ray structures of three successfully crystalized oligonucleotides. All three structures acquire double helical architecture with two consecutive T- T mismatches in the center. To improve the convergence of the refinement process of the crystal structures we used novel dinucleotide conformational classes, NtC classes. The NtC class classification was also used to analyze geometries of selected non-canonical base pairs in all DNA crystal structures in the Protein Data Bank. We measured the fit between geometries of the dinucleotides involved in the non-canonical base pairing and the NtC classes and correlated this fit to the electron...
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Growth of graphene by thermal decomposition of silicon carbide for applications in biophysics
Hodoš, Matúš ; Kunc, Jan (advisor) ; Čechal, Jan (referee)
This bachelor thesis is focused on growth and characterization of graphene from SiC powder at different conditions. In the introduction graphene properties are briefly described. Afterwards three methods of graphene growth are discussed, with particular emphasis on thermal decomposition of SiC. Further, the characterization methods which were used by us are explained. In experimental part, three sets of graphene samples prepared by thermal decomposition of SiC will be described, each will differ in one of the factors: temperature, time and ambient atmosphere. Growth samples will be subsequently used in an experiment on yeast. The effect of graphene prepared at different conditions on membrane potential will be determined for possible application in biophysics.
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Study of molecular mechanisms of the signaling proteins regulation
Kylarová, Salome ; Obšilová, Veronika (advisor) ; Mikšík, Ivan (referee) ; Novák, Petr (referee)
EN The aim of this study was to investigate the regulatory mechanisms of two important signaling proteinkinases and promising therapeutic targets, ASK1 and CaMKK2. ASK1 kinase is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-JNK kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress. The function of ASK1 is associated with the activation of apoptosis and thus plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration or cardiovascular diseases. The natural inhibitor of ASK1 is a ubiquitous oxidoreductase, thioredoxin, which is probably bound to N-terminus of ASK1, thus preventing a homophilic interaction and subsequent ASK1 activation. It has been suggested, that upon oxidative stress and oxidation of thioredoxin active site, thioredoxin dissociates from ASK1, but the structural basis of this interaction remains unclear. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of CaM kinase pathway that activates CaMKI, CaMKIV and AMPK involved in gene expression regulation or apoptosis activation. Function of this protein is often associated with neuropathology, carcinogenesis and obesity. CaM kinases are activated via binding Ca2+ sensor protein...
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Spectroscopic study of interaction of antimicrobial peptides with model membranes
Tesař, Adam ; Kopecký, Vladimír (advisor) ; Kočišová, Eva (referee)
Antimicrobial peptides (AMP) have a great potential in medicine and pharmacy. Mechanism of their impact is an interaction with a cell membrane leading to the penetration of the membrane. The way of disruption of the cell membrane is not completely understand, therefore we focused on the interaction of AMP HAL-1, isolated from the venom of the bee Halictus sexctinctus, with a model membrane of 100 nm liposomes consisting of phosphatidylcholin and phosphatidylglycerol. Circular dichroism and infrared spectroscopy (FTIR) proved the change of the secondary structure from the random coil of free HAL-1 to -helix in an interaction with the membrane. The next step was preparation of the lipid bilayer on the surface of ATR prism, which will enable usage of the polarized FTIR spectroscopy to study the interaction of AMP with model membranes in future. Therefore, the ATR-FTIR spectroscopy and factor analyses were applied to study dynamics of drying of the liposomes and their subsequent hydration also with an addition of HAL-1. We focused on the stabilization of the system. Hydratation of the lipid bilayer by 2 µl sample showed stability for minutes, nevertheless after dilution the stabilization decay in minutes. The protective influence of the peptide on the lipid bilayer and slowing down of the drying out of the...
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