National Repository of Grey Literature 78 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Preparation of fusion ligands and evaluation of their binding to NK cell receptors
Nepokojová, Tereza ; Vaněk, Ondřej (advisor) ; Grantz Šašková, Klára (referee)
Natural killer cells (NK cells) are an important part of innate immunity. On their surface they express a complex group of receptors that use different signalling motifs to activate or inhibit NK cell cytotoxic activity. NK cells are capable to kill aberrant cells (namely, viral, infected, and tumour cells) by using special cytotoxic mechanisms to trigger apoptosis. The activating receptors recognize tumour or stress-induced ligands, e.g., NKG2D receptor recognizes the MICA ligand and NKp30 recognizes the B7-H6 ligand. Therefore for human immune system it is only natural that cancer cells are destroyed by NK cells. The current therapeutic goals in the treatment of cancer are primarily focused on strengthening the body's own natural ability to fight with cancer and one possible way is stimulation of NK cells to win this deadly fight. In addition to NK cells, antibodies are also widely used for the treatment of cancer, as well as other immune-related disorders. Most of them are monoclonal antibodies, but antibody fragments are getting attention and are being tested more and more in recent years. This work describes the preparation of three bifunctional fusion proteins: B7-H6-L-aHER2, MICA-L-aHER2, and aHER2-L-MICA, which contain immunoligands for the activating receptors of NK cell and VHH fragment...
Preparation of receptor AICL in fusion with Fc fragment of human IgG
Runová, Alžbeta ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer cells (NK cells) are one of the basic elements of innate immunity. They play a key role in immune response against virus-infected, cancerous or otherwise stressed cells. NK cells express surface activating and inhibitory receptors. Activating receptors trigger cytotoxic mechanisms that lead to the target cell's apoptosis. Inhibitory receptors provide cellular tolerance. The balance between these receptor signals determines the resultant NK cell response to the target cell. C-type lectin-like receptors include the activating receptor NKp80 and its ligand AICL. AICL is a myeloid-specific activating receptor expressed on tumor cells. The NKp80:AICL complex that assists in the cytolysis of malignant myeloid cells is being studied in the context of cancer immunotherapy. This bachelor thesis describes the preparation of vectors containing genes encoding AICL expression constructs, and the subsequent production of proteins in the human embryonic renal cell line (HEK293S GnTI- ). The expression constructs contain the extracellular domain of AICL, TEV protease site and Fc fragment. Two different constructs were prepared - one containing the native AICL sequence and the other carrying C87S mutation.
Recombinant expression of protein NKp46 and its ligand Epa1
Houserová, Jana ; Vaněk, Ondřej (advisor) ; Kubíčková, Božena (referee)
Natural killer cells (NK cells) are cells of innate immunity that play an essential role in the immune response of an organism. In contact with infected, stressed or tumour cells, the NK cells can trigger cytotoxic mechanisms. The initiation of mechanisms depends on the presence of activating or inhibitory ligands on the surface of the cells. On the surface of the NK cells, there are activating and inhibiting receptors that upon binding their respective ligands send a signal to the NK cell. One of the activating mechanisms is the decrease of expression in MHC gp I molecules on the surface of the infected cells. This molecule is a ligand of the inhibiting receptors. One of the activating receptors of NK cells is NKp46. This receptor belongs to the natural cytotoxicity receptor (NCR) family. NKp46 has many ligands, one of them being the adhesin Epa1 of yeast Candida glabrata. This thesis aims at preparing plasmids and producing extracellular domains of the NKp46 receptor and its ligand adhesin Epa1.The interaction of NKp46 and its ligand Epa1 remains to be the subject of future research. Plasmids containing NKp46 and Epa1 genes were successfully prepared and verified by DNA sequencing. The NKp46 protein was produced in eukaryotic expression system of HEK293S GnTI- cell line. A stably transfected...
The influence of S159A mutation on the oligomeric state of human NK cell receptor NKR-P1A
Hausleitner, Filip ; Vaněk, Ondřej (advisor) ; Kožíšek, Milan (referee)
Natural killer cells (NK cells) are lymphocytes that possess cytotoxic activity against tumour or virally infected cells independent of preceding antigen sensitisation. To kill such cells, they utilise their activating and inhibitory surface receptors that interact with target cell surface molecules. The immune response carried by NK cells depends on the balance of both activating and inhibitory signals. Human NK cell surface receptor NKR-P1A belongs to the structural family of C-type lectin-like receptors. This receptor interacts with its ligand LLT1, which belongs to the same protein family, with low affinity and high specificity. The NKR-P1A:LLT1 complex formed between NK cell and its target cell inhibits NK cell cytotoxicity, and hence is a part of the regulation of immune response. This thesis studied the effect of S159A mutation on the stoichiometric state of soluble human NKR-P1A ectodomain in solution. Therefore, a mutant form of NKR-P1A G90-S225 S159A ectodomain was successfully produced in stably transfected human embryonic kidney cells 293 (HEK293S GnTI" ). This construct was purified by affinity and size-exclusion chromatography, and analysed by SDS-PAGE and analytical ultracentrifugation. Our results show that the preclusion of N-linked glycosylation in the position 157 promotes the...
Preparation of the expression vectors for the production of fusion protein NS2-NS3 of Dengue and Zika viruses
Novotný, Pavel ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
in English Zika and Dengue viruses, which are part of Flaviridae family, are responsible for fever diseases. Infection can be asymptomatic or with symptoms of light virosis, however, it could be also life threatening. In the case of Dengue, it could progress to haemorrhagic fever. Infection by Zika is also dangerous for pregnant women because of enhanced risk of microcephaly in new-borns. These viruses are transferred by mosquitoes from Aedes family and are part of arbovirus group. Vaccination is a good tool for control of these diseases, but development of virostatics are also in progress. Inhibitors of viral proteases are important clinically approved virostatics against HIV and HCV. It is not clear, if Zika of Dengue proteases can become drug targets, however these proteases are also interesting for basic research. Flavivirus protease is located in viral polyprotein synthesized by host cells as a part of NS3 domain. This enzyme needs activation domain NS2B; mechanism of this interaction is not fully understood. On top of the protease activity, NS3 domain includes helicase, NTPase and RNA 5'-triphosphatase activity. It is not known why these enzymes coexist as one protein domain. To address these unanswered questions, it is important to prepare expression systems for kinetic, structure and...
Targeted modifications of the protein-protein interactions: Ternary complex of interferon-γ as a model system
Zahradník, Jiří ; Schneider, Bohdan (advisor) ; Obšilová, Veronika (referee) ; Vaněk, Ondřej (referee)
A key prerequisite for a deeper understanding of biological processes at molecular level is a detailed description of the three-dimensional structure of interaction partners and their complexes. We adopted the IFN-γ complex as our model system. Even though IFN-γ is one of the key modulators of the immunity response, which has been studied intensively for more than 60 years, the structure of the accessory receptor chain and the understanding of the IFN-γ complex is still lacking. In this work we firstly discussed the binary system between IFN-γ and its high affinity receptor R1 which is structurally known. Using a new innovative methodology we focused on the modulation of the affinity between IFN-γ and its receptor R1. Our approach was based on the modulation of protein - protein stability by mutating cavities in the proteins' structure and increasing the affinity about seven-fold. Secondly, we crystallized and solved the structure of the IFN-γ receptor 2, the accessory receptor molecule. Our analysis of variable residues on the surface of the structures of type II family receptors, to which receptor 2 belongs, revealed the putative binding site for IFN-γ. In the third part of our work, we crystallized IFN-γ from olive flounder Paralichthys olivaceus and solved its structure at 2.3 Å resolution (PDB...
Neuroprotective effects of food intake regulating peptides in vitro and in vivo
Kasperová, Barbora Judita ; Maletínská, Lenka (advisor) ; Vaněk, Ondřej (referee)
Nowadays, Alzheimer's disease (AD) is one of the most serious health problems among elder. To this day, pathogenesis of AD is still unknown and therefore no effective treatment has been found. AD is characterized by neuropathological features, the formation of extracellular senile plaques of amyloid β and intracellular neurofibrillary tangles of Tau protein. Numerous experimental studies have confirmed that metabolic disorders such as type 2 diabetes mellitus or obesity contribute significantly to the development of cognitive impairment and therefore the development of AD. In this diploma thesis, the potential neuroprotective effect of peptides regulating food intake was investigated in in vitro and in vivo experiments. The potential neuroprotective effect of liraglutide, a glucagon-like peptide-1 analog used as a type 2 diabetes mellitus treatment, a prolactin-releasing peptide (PrRP) and its palmitoylated analog, palm11 -PrRP31, was investigated on SH-SY5Y cell line. The peptides were used as a pretreatment on SH-SY5Y cells in methylglyoxal-induced cytotoxicity. It has been proven that the peptides themselves are not toxic and do not significantly reduce the viability of SH-SY5Y cells. The tested peptides showed prophylactic effects against cytotoxicity and apoptosis induced by toxic...

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