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Recombinant expression, purification and characterisation of the surface immunoreceptor CD47 (integrin associated protein)
Kuchyňová, Sarah ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
The human immunoreceptor CD47, also designated <Integrin Associated Protein= is a novel therapeutic target for cancer treatment. When bound to one of its ligands, a protein found on the surface of immune cells, SIRP, the phagocytosis of the CD47 expressing cell is inhibited. This interaction is used by malignant cells to escape detection by the immune system, thus making CD47 a promising candidate molecule for cancer immunotherapy. The literary overview focuses on the characterisation of CD47 in terms of its structure, localization, and function. Additionally, this sections thoroughly delves into the aforementioned interaction with SIRP as well as existing mechanisms for the targeting of the CD47- SIRP axis. The following experimental section centers around the preparation of the plazmid pTT28_hCD47. The process by which the gene for the extracellular portion of human CD47 was inserted into the linearized pTT28 vector is noted and its transfection into a mammalian expression system to produce the protein. Moreover, the section divulges into the purification of the recombinant protein by affinity purification using Ni-NTA agarose as well as gel permeation chromatography, resulting in the isolation of CD47 proteins differing in glycosylation. Their functionality was validated via SPR, in a study...
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Anti-CD20 nanobody production and its conjugation to a polymeric carrier
Cibiková, Tereza ; Vaněk, Ondřej (advisor) ; Čermáková, Michaela (referee)
Cancerous diseases causedby malignant transformationof B-lymphocytes are characterized by increased expression of surface marker 20 (CD20, cluster of differentiation 20). Traditionally, the treatment of this disease targets CD20, most often with monoclonal antibodies (e.g. rituximab). However, due to the side effects of these therapeutics, alternative approaches to therapeutic intervention through CD20 are being explored. The development of protein drugs conjugated to a polymeric carrier, which would not have the side effects of monoclonal antibodies and would have a sufficient duration of action in the body, seems promising. The aim of this work was to prepare and characterize a macromolecular protein-polymer conjugate whose interaction with CD20 would induce apoptosis of malignantly transformed B-lymphocytes.
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Structural biochemistry of protein NKp80, an activation receptor of human lymphocytes
Pazderová, Kristýna ; Vaněk, Ondřej (advisor) ; Konvalinka, Jan (referee)
Natural Killer (NK) cells can spontaneously destroy target cells, such as infected, damaged, or malignant cells. NK cell cytotoxicity is mediated by activating receptors on their surface, such as NKp80 (Natural Killer protein 80 kDa). The human receptor NKp80 stimulates cytotoxicity through its interaction with its ligand AICL (Activation Induced C-type Lectin), which is constitutively expressed by all myeloid cells. In pathological conditions, such as cancerous or damaged cells, AICL is often upregulated, resulting in the lysis of these cells by NK cells expressing NKp80. This interaction is thus a promising immunotherapeutic target for treating myeloid leukaemia. However, the structures of both proteins have remained elusive. Therefore, we have focused on the successful production of the extracellular domain of these proteins. To improve the production yield of NKp80, we introduced a series of mutations in the stalk region to study their effect on production, stability, and homodimer formation. The introduced mutations significantly increased the production yield allowing for a large-scale production with subsequent crystallisation of the protein. The crystallisation resulted in the elucidation of the hitherto unknown structure of NKp80 homodimer at a resolution of 2.9 Å. However, the large-scale...
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Specificita interakcí protein-protein a jejich modulace
Pham, Phuong Ngoc ; Schneider, Bohdan (advisor) ; Damborský, Jiří (referee) ; Vaněk, Ondřej (referee)
(EN) Protein-protein interactions (PPI) have essential roles in life processes, and abnormal PPI are associated with many human diseases. Given their importance, PPI have received increasing attention and became drug targets. However, the design of specific PPI and their modulation is challenging. Cytokine-receptor interactions are especially important in the regulation of the immune system. Interleukin-10 (IL-10) over-production results in excessive immunosuppressive effects, tumor growth and infection. The interaction between interferon gamma receptor 2 (IFN- γR2) and interferon gamma (IFN-γ) leads to activation of downstream signaling pathways but the mechanism of such interaction is elusive. Interleukin-24 (IL-24) is another cytokine that signals through receptors sharing the interleukin-20 receptor two (IL-20R2) subunit and has important roles in autoimmunity and cancer. The aims of this Ph.D. thesis are to study PPI from several aspects emphasizing their specificity. The first goal is to develop a novel protein scaffold and subsequently evolve it into a high-affinity binder specific for human IL-10. The second goal is to understand the structural basis for receptor specificity of human IFN-γ. The third goal is to modulate the binding affinity between human IL- 24 and its receptor IL-20R2 by...
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Optical speed sensor of surface
Vaněk, Ondřej ; Hadaš, Zdeněk (referee) ; Sedlák, Petr (advisor)
The aim of this work is development of an optical sensor for surface-velocity measurement based on ordinary optical mouse sensor. The proposed sensor should have find its usage in industrial applications for low-cost contactless measurement of conveyor belts or semi-finished products, thus some other gauges could check quality of products. Theoretical part of this thesis includes introduction to optical sensors and their usage. Rest of the work describes practical part consisting of programming of communication between sensor and PC in MATLAB and Arduino environments, development of simple device based on Arduino hardware, the set-up of experimental workplace and providing speed tests.
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Sanitation installation and gas installation in multifunctional building
Vaněk, Ondřej ; Bárta,, Ladislav (referee) ; Vrána, Jakub (advisor)
Bachelor Thesis ,,Technical and gas installations inmulti-building‘‘ is processed in the form of project documentationplumbing and gas installations for building construction in accordance with applicable regulations. Two-storey building is designed, besement. The building is used on the first floor of the municipal office and on the second floor of meeting andbanqueting facilities. Project part focuses on the design and solvingplumbing and gas installations. Part of the thesis are the total property assessmentsand literature review focused on the issue of hot water.
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Sanitation installation and gas installation in residential building
Vaněk, Ondřej ; Vaščáková, Alena (referee) ; Vrána, Jakub (advisor)
The thesis deals with sanitary engineering and gas installation in an apartment building with a healthcare facility. The theoretical part is focused on the installation systems, which are addressed to the suitability of the system in the given project, and the advantages and disadvantages of these systems. Computational and project part addresses the sewer, water mains and gas in the specified object. Designed building is without a basement and eight storeys. On the first floor is located technical facilities around the building and three residential units. All other floors are located on each floor of four residential units. This thesis is done on the basis of current Czech and European regulations.
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Comparative study of body temperature measurement using various technologies
Vaněk, Ondřej ; Sekora, Jiří (referee) ; Provazník, Ivo (advisor)
The aim of this work is to compare the body temperature measurement with different contact thermometers. The theoretical part includes studying the topic, familiarization with the situation at the Faculty Hospital Brno, Bohunice and suggestion of method for comparison. The practical part includes thermometers measurement in the laboratory to verify the accuracy, clinical measurement on patients in different parts of the body and under various conditions and statistical evaluation of results.
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Activating antitumor immune response using bispecific fusion proteins
Chytrá, Gabriela ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
Natural killer (NK) cells are lymphocytes of the innate immune system that recognize and eliminate transformed and potentially harmful cells in a mechanism termed immunosurveillance. Malignant cells strive to escape immunosurveillance, and if successful, oncological disease develops. To restore immune recognition, immunotherapy utilizing NK cell-directed therapeutic fusion proteins can be employed. Therapeutic fusion proteins target tumour markers expressed on the surface of malignant cells and, at the same time, stimulate immune response through binding to NK cell activating receptors, for example receptor NKG2D or NKp30. A relevant example of a tumour marker is the HER2 receptor, which is often overexpressed in several types of cancer, most notably breast carcinoma. This thesis describes the preparation of several bispecific fusion proteins with potential use in immunotherapy. Bispecific fusion proteins consist of an NK cell activating ligand (ligand MICA or B7-H6) and nanobody targeting selected tumour marker (receptor HER2), which are connected by flexible glycine-serine linker. The constructs of fusion proteins were prepared in two configurations - with nanobody located on the N-terminus and the ligand on the C-terminus and vice versa. In addition, bispecific fusion proteins introducing...
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Effect of selected compounds on NMPylation of nsp9 SARS-CoV-2 catalyzed by NiRAN domain of nsp12 SARS-CoV-2
Fedák, Michal ; Šilhán, Jan (advisor) ; Vaněk, Ondřej (referee)
SARS-CoV-2 pandemic that begun in the end of 2019 caused over 770 milion confirmed cases of infection and almost 7 milion deaths worliwide. Despite pandemic being suppressed thanks to vaccines and restriction measures, the research of new medication targeting this virus continues. One of the possible targets of SARS-CoV-2 is nonstructural protein 9 (nsp9), which is essential for virus repliation. Just recently it was revealed that this protein can covalently bind nukleoside monophosphate (NMPylation) or RNA chain of various length (RNAylation). These reactions are catalyzed by nidovirus RNA-dependent RNA polymerase (RdRp) asociated nucleotidyltransferase (NiRAN) domain of nonstuctural protein 12 (nsp12). RNAylation of nsp9 showed to be crucial step in synthesis of RNA cap. The function of NMPylation was not jet revealed, but it is believed that this modification of nsp9 could play a role in priming of RNA synthesis. Some studies have already investigated inhibition of NMPylation. In this diploma thesis recombinant nsp9 was prepared and used in NMPylation reactions. Negative effect of DMSO on nsp9 NMPylation was observed. Three compounds (saquinavir, darunavir and conivaptan) selected based on the results of in silico studies did not show ability to inhibit NMPylation of nsp9. Next, it was confirmed...
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