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Proteins mimicking epitopes of broadly neutralizing HIV-1 antibodies
Zosinčuková, Tereza ; Malý, Petr (advisor) ; Osička, Radim (referee)
HIV-1 is a dangerous retrovirus which represents one of the world's leading health problems. HIV-1 infection is incurable and without proper treatment by antiretroviral therapy it leads to death within several years. Despite intensive research, no HIV vaccine is currently available. This thesis presents a new and unique approach which has not been used for vaccine development yet. The promising strategy is based on small binding proteins that can elicit broadly neutralizing HIV-1 antibodies by mimicking their epitopes. The aim of this project was to select and characterize small binding proteins that can successfully mimic the surface of viral envelope glycoproteins that is recognized by the broadly neutralizing HIV-1 antibodies PGT121 and PGT126. Proteins were selected from a highly complex combinatorial protein library derived from a new type of scaffold called Myomedin. Firstly, the extent of the protein library was narrowed down using the ribosome display. Then the direct sandwich ELISA screening was applied to select scaffold variants that interact with the target antibodies. In total over 200 variants were tested and several promising candidates were found. These Myomedin variants were purified, biochemically and biophysically characterised and the best ones were used to immunize mice....
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Preparation and Characterization of Protein Binders Mimicking Epitopes of HIV-1 Neutralizing Antibodies
Šulc, Josef ; Malý, Petr (advisor) ; Novotný, Marian (referee)
For three decades, the ongoing HIV pandemic has taken the lives of tens of millions of people. Still, more tens of millions are fighting this incurable disease today. Current failures in combating this global problem are caused mainly by the virus's extreme ability of mutation, its very effective molecular shield which repels the immune system's attacks, and its immense variability. A breakthrough, achieved relatively recently, is the discovery of the so-called broadly neutralizing antibodies against HIV-1, which carry a very efficient and broad neutralizing response. So far, it's not known how to elucidate the production of these antibodies in the infected hosts to quell or altogether eliminate the virus. This work deals with experimental results, which led to both in vivo and in vitro proof-of-concept of the so-called protein mimetics, the ability to imitate viral surface epitopes, and therefore stimulate an efficient immune response carried by targeted broadly neutralizing antibodies. This effect is mediated by recombinant binding proteins, based on the Myomedin scaffold. This work describes the selection and characterization of these binding proteins mimicking the epitopes of one of the most effective broadly neutralizing antibodies, 10E8. It shows that the binding affinities of selected...
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Femtosecond absorption spectroscopy for study of stimulated emission
Streshkova, Neli ; Malý, Petr (advisor) ; Zukerstein, Martin (referee)
The aim of the thesis is the building and optimization of an experimental setup for me- asuring the transient transmission and optical gain by the femtosecond pump and probe technique. The setup combines the technology of parametric generation of pump pulses with tunable wavelength and the generation of femtosecond continuum as a spectrally wide probe. In the thesis, conditions of supercontinuum generation in bulk fused silica are extensively studied from a practical viewpoint. The performance of the setup was tested on two types of samples: the solution of rhodamine 6G in methanol and the semiconduc- tor nanocrystalline sample - CdSSe-doped glass. Time resolved spectra of the transient transmission of both samples were measured and the spectral dependence of the optical gain in rhodmine 6G was obtained. 1
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The role of ORMDL proteins in mast cell signaling
Paulenda, Tomáš ; Dráber, Petr (advisor) ; Heneberg, Petr (referee) ; Malý, Petr (referee)
1. Abstract (EN) This thesis is collection of work focused mainly on the understanding of mast cell activation and its regulation by Orm1-like (ORMDL) proteins. ORMDL family is a group of endoplasmic reticulum (ER) membrane resident proteins that are highly conserved amongst mammalian species. ORMDL proteins can be found in diverse range of organisms from plants through fungi to animals. ORMDL proteins were first discovered in yeasts and the interest in these proteins skyrocketed after the discovery that ORMDL3 is associated with childhood onset asthma in genome wide association studies. Following research connected ORMDL3 also with allergic inflammation and inflammatory bowel disease. Since mast cells are mainly known for their role in allergy and allergen induced inflammation, we decided to investigate the role of ORMDL proteins in regulation of mast cell activation and signaling. In our first study we focused on the role of ORMDL3 in mast cell activation via the high affinity IgE receptor 1 (FcεRI). We prepared bone marrow-derived mast cells with decreased (ORMDL3-KD) or increased (ORMDL3-OE) ORMDL3 expression. We showed that ORMDL3 is a negative regulator of mast cell activation events like degranulation, cytokine release and migration, without any effect on calcium mobilization. ORMDL3 was previously...
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Novel protein binders targeting marker of epithelial cells
Huličiak, Maroš ; Malý, Petr (advisor) ; Anděra, Ladislav (referee)
Fast and precise quantification of circulating tumour cells (CTC) in lung adenocarcinoma is a pivotal step in acceleration of diagnosis, selection of early therapy and estimation of treatment prognosis. Development of a new type of microfluidic device based on detection and quantification of epithelial- and mesenchymal-type CTC by high-affinity and cell-type specific protein binders anchored to a microfluidic chip surface represents a highly innovative approach. In this work, we used EpCAM membrane glycoprotein as a target for generation of epithelial cell- specific protein binders by a directed evolution of proteins selected from highly complex combinatorial libraries derived from albumin-binding domain scaffold (ABD) or human muscle protein domain-derived "Myomedin" scaffold. Collections of EpCAM-binding candidates from the both used libraries were generated and particular binding variants were further characterized by DNA sequencing, biochemically and by functional cell-surface binding assays. The best candidates might serve as robust anchor proteins of a microfludic chip. Key words: epithelial cell, EpCAM, protein binder, ribosome display, combinatorial library, protein scaffold
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Effects of a foam roller during stretching
Pětíková, Nikola ; Šteffl, Michal (advisor) ; Malý, Petr (referee)
Title: Effects of a foam roller during stretching Aims: The main aim of the thesis was to compare effects between the foam roller and stretching exercises. Methods: 10 female students aged 23-24 from UK FTVS participated in this study. All the participants were active athletes. The study was realized as a cross-over trial. There was used tensiomyography using the TMG 100 to measure all the participants. The musculus vastus lateralis were tested in both sides. A generalized linear model was used to evaluate the results. Results: The foam rolling tended to better activate muscle before training and statistically significantly accelerated post-load recovery processes compared to normal stretching. Conclusions: The foam roller could be used effectively after sports performance as a tool to accelerate regeneration, thereby enhancing sports performance. Keywords: warm-up, stretching, TMG, tensiomyography
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Novel protein binders targeting marker of epithelial cells
Huličiak, Maroš ; Malý, Petr (advisor) ; Anděra, Ladislav (referee)
Fast and precise quantification of circulating tumour cells (CTC) in lung adenocarcinoma is a pivotal step in acceleration of diagnosis, selection of early therapy and estimation of treatment prognosis. Development of a new type of microfluidic device based on detection and quantification of epithelial- and mesenchymal-type CTC by high-affinity and cell-type specific protein binders anchored to a microfluidic chip surface represents a highly innovative approach. In this work, we used EpCAM membrane glycoprotein as a target for generation of epithelial cell-specific protein binders by a directed evolution of proteins selected from highly complex combinatorial libraries derived from albumin-binding domain scaffold (ABD) or human muscle protein domain-derived "Myomedin" scaffold. Collections of EpCAM-binding candidates from the both used libraries were generated and particular binding variants were further characterized by DNA sequencing, biochemically and by functional cell-surface binding assays. The best candidates might serve as robust anchor proteins of a microfludic chip. Key words: epithelial cell, EpCAM, protein binder, ribosome display, combinatorial library, protein scaffold
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Novel binding proteins derived from small protein domains targeting diagnostically important molecules
Vaňková, Lucie ; Malý, Petr (advisor) ; Brynda, Jiří (referee) ; Trefil, Pavel (referee)
The rapid development of the gene engineering techniques, especially methods for in vitro directed evolution and combinatorial mutagenesis, has triggered the generation of new binding agents to almost any antigen of interest as an alternative to broadly used antibodies. These so-called non-Ig scaffolds are often derived from proteins with useful biophysical properties. While the therapeutic market is still dominated by monoclonal antibodies, the easy option of desired customization of non-Ig binders by conventional methods of gene engineering predestine them largely for the use in the diagnostic area. The ABD scaffold, derived from a three-helix bundle of albumin-binding domain of streptococcal protein G, represents one of the small non-Ig scaffolds. In our laboratory, we have established a highly complex combinatorial library developed on the ABD scaffold. This ABD scaffold-derived library was used to generate unique binders of human prostate cancer (PCa) biomarkers PSP94, KLK2, KLK11 for the more precise diagnosis of PCa. The second part of the thesis describes the generation of ABD-derived binders selectively recognizing different phenotypes of circulating tumor cells as a binding component of the cell capture zone of microfluidic chip for lung adenocarcinoma diagnosis. Beside this already...
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Compensation for injury to the natural rights of man
Malý, Petr ; Thöndel, Alexandr (advisor) ; Frinta, Ondřej (referee)
This diploma thesis deals with the issue of harm to natural human rights and its compensation. The thesis focuses on the current regulation of this institute contained in The Civil Code No. 89/2012 Coll. Furthermore, in this work, the current regulation is compared with the previous regulation, namely Civil Code No. 40/1964 Coll. The main aim of this work is to provide a basic and comprehensive insight into the issue of harm to natural human rights, focusing on the description of the changes brought about by the adoption of the new Civil Code. Another aim of the thesis is to outline the consequences of these changes. At the beginning the diploma thesis focuses on all the articles of the first part of the Civil Code, which deal with the regulation of natural rights. In the next part of the thesis it provides a general insight into the issue of liability for the damage caused to natural rights. This general basis is necessary for the next part of the work, which deals with claims for compensation of immaterial harm and decision on its amount according to the new Civil Code. The most extensive third part of the thesis therefore deals with specific remedies, namely compensation in the event of injury and the death and indemnification of secondary victims. At the end, the diploma thesis mentions another...
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Selection approaches in directed evolution of binding proteins
Huličiak, Maroš ; Malý, Petr (advisor) ; Hlouchová, Klára (referee)
Artificial binding proteins derived from small protein domains attract attention as a promising alternative to monoclonal antibodies and can be used in many kinds of applications. They are useful in diagnosis of human diseases, seem to be a clue for more efficient vaccine development preventing from global diseases such as AIDS, can exhibit a therapeutic potential or improve purification techniques. For the selection of protein variants with desired properties such as high specificity and binding affinity, more than 10 different selection techniques have been developed. So called display techniques such as phage display, yeast display, retroviral display or baculovirus display are based on protein expression from different vectors. Contrary that, ribosome display, mRNA display and CIS display are cell-free systems based on in vitro translation. Development of different selection approaches allows production of post- translationally glycosylated, phosphorylated and acetylated proteins, increased yield of the produced binders and improved their binding properties. The submitted work provides an overview of current selection techniques, compare their parametres regarding to combinatorial libraries, describes their advantageous properties and limitations, and focus on a practical utilization of...
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