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Production and purification of recombinant receptor Clrb
Prokopová, Tereza ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
The Natural Killer (NK) cells play a vital role in the nonspecific immunity. They are capable of efficient immunity reaction without any antigen specific receptors on their surface. NK cells recognize non-self molecules and they also recognize their molecules serving as health markers, and absence of these molecules such as MHC glycoproteins on the target cell surface. The NK cells are able to recognize viral infection or tumor transformation in the organism. If natural killer cell is in contact with a cell carrying an abnormally low MHC class I glycoproteins, it will create a signal which informs the cell is infected with a virus. NK cells trigger apoptosis of the target cell without prior stimulation, proliferation and differentiation. They also promote inflammatory responses by the production of chemokines and cytokines. The response is always the interplay of activating and inhibitory signals that the cell receives from its surroundings. The latest research shows that the targeted modulation of NK cells leads to less complications in bone marrow transplantation. They can be potentially used in immunotherapy, e.g. in the treatment of autoimmune diseases. Therefore, NK cells are a highly-studied group of cells. This thesis is focused on a production of Clrb ("C- type-lectin-related protein b")....
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Production of mouse NK cell receptor NKR-P1C and seeking of tis ligand
Pucholtová, Helena ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
Natural killer or NK cells are immunocytes that mediate innate immunity against pathogens and tumors without pre-exposition to the antigen. They are holding rapid antiviral defense during the initial phase of immune response, before starting the production of antibodies and the development of specific cytotoxic T -lymphocytes. On the surface of NK cells is expressed wide range of inhibition and activation receptors. Important family of those receptors are C - type lectin like from which the family of NKR - P1 ("natural killer cell receptor - protein 1") was discovered first. Diploma thesis deals with the preparation/study of mice NK cell activation receptor NKR- P1C and searching for its binding partner. The soluble form of the protein NKR-P1C was prepared by recombinant expression using the transient transfection of HEK293 cell line (human embryonic kidney 293) with wild type or homogenous glycosylation as IgG - Fc fusion protein, from which was it possible to obtain pure dimer of NKR P1C, after process of affinity purification, TEV protease cleavage and HPLC chromatography. The fusion protein was bound to protein A labeled with a fluorescent probe DyLight 488. Mice tissues and cell lines were labeled by this complex for purpose of seeking ligand.
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Cell signalling and molecular complexes of the TRH receptor
Drastichová, Zdeňka
The first part of this thesis is preoccupied with the identification of protein alterations in the membrane fraction of HEK293-E2M11 cells after prolonged TRH treatment. The isolated membrane fraction enriched in plasma membranes contained high amounts of Na+ ,K+ -ATPase, TRH receptor and G-proteins compared to the postnuclear supernatant. By using 2D electrophoresis and mass spectrometry, the levels of 42 proteins were identified to be altered in samples of PM-enriched fractions from TRH-treated (16 h; 10 μM) cells. Out of these proteins only ezrin and stomatin-like 2 are known to be localized in the plasma membrane. Five proteins (mitofilin, MTHSP75, prohibitin, stomatin like-2, peroxiredoxin III) whose levels were increased after the prolonged TRH treatment represent proteins localized in mitochondria. All of them are important for proper structure and function of mitochondria. The ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was markedly higher in cells treated with TRH compared to control cells. Hence, it can be concluded that prolonged TRH treatment may significantly affect mitochondrial membrane and function of mitochondria. The second part of this thesis deals with the identification of molecular protein complexes of TRH-R and/or Gq/11 protein. The presumed effects of TRH on the...
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Preparation of expression vectors for NKp65 and KACL, new members of human NK cell receptor family
Mikulová, Barbora ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer cells create an important part of innate immune system. Their importance lies in their ability to recognize and kill abnormal cells, especially tumour cells and virally infected ones, without previous activation. To recognize their targets, NK cells use a wide variety of surface receptors, both activating and inhibitory. If a ligand binds to an NK receptor, immune response is triggered. Examples of such ligand-receptor pairs are NKp80-AICL and NKRP1-LLT1 on human lymphocytes. Another ligand-receptor system of this kind is NKp65 and KACL, two recently discovered lectin receptors on human immunocytes. KACL is the last and most recently characterized member of CLEC2 receptor family in humans. Its expression is almost exclusively restricted to skin. NKp65, a close relative of NKp80, is a glycoprotein which stimulates NK92MI cell cytotoxicity upon KACL engagement. NKp65 has been shown to bind to KACL with a fairly high affinity by surface plasmon resonance measurement. This thesis aims at describing the cloning of expression vectors coding for NK cell receptors NKp65 and KACL, expression of these proteins in HEK293T cell line and their purification. Keywords: NKp65, KACL, NK cell, lectin, receptor, plasmid (in Czech)
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Cell signalling and molecular complexes of the TRH receptor
Drastichová, Zdeňka ; Novotný, Jiří (advisor) ; Hodný, Zdeněk (referee) ; Říčný, Jan (referee)
1 Summary The first part of this thesis is preoccupied with the identification of protein alterations in the membrane fraction of HEK293-E2M11 cells after prolonged TRH treatment. The isolated membrane fraction enriched in plasma membranes contained markedly increased the amount of Na,K-ATPase, TRH receptor and G-proteins compared to the postnuclear supernatant. By using 2D electrophoresis and mass spectrometry, the levels of 42 proteins were identified to be altered in samples of PM- enriched fractions from TRH-treated (16 h; 10 μM) cells. Out of these proteins only ezrin and stomatin-like 2 are known to be localized in the plasma membrane. Five proteins (mitofilin, MTHSP75, prohibitin, stomatin like-2, peroxiredoxin III) whose levels were increased after the prolonged TRH treatment represent proteins localized in mitochondria. All of them are important for proper structure and function of mitochondria. The ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was markedly higher in cells treated with TRH than in control untreated cells. Hence, it can be concluded that prolonged TRH treatment may significantly affect mitochondrial membrane and function of mitochondria. The second part of this thesis deals with the identification of molecular protein complexes of TRH-R and/or Gq/11 protein. The presumed...
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Opioid receptors and their signaling system in the myocardium
Ladislav, Marek ; Novotný, Jiří (advisor) ; Neckář, Jan (referee)
The main objective of this bachelor thesis is to systematically collect and sort information about opioid receptors and their signaling system in the myocardium. Heart activity is controlled mainly by adrenergic signaling, and this work therefore contains also some data concerning the characteristic and significance of other relevant receptors. For better understanding, general basic information about opioid system, especially about the receptors and their signaling, is also provided. Relatively little is known about opioid receptors in the myocardium even though these receptors may have an important role especially in various pathophysiological conditions. There can be several reasons for this. The possibility of further characterization of opioid receptors in the myocardium is rather difficult due to the relatively small number of these receptors in heart tissue. The situation is somewhat complicated also by some differences in the modulation of cardiac function among different species. The complete molecular mechanism by which opioid receptors act on the myocardium has not yet been fully uncovered. Especially in the case of humans this knowledge can be crucial, because these receptors and their ligands could be used for medical purposes.
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Role of DD- and DED-containing adaptor proteins in apoptotic signaling
Čaja, Fabián ; Janštová, Vanda (referee) ; Anděra, Ladislav (advisor)
Proteins containing a bundle of six anti-paralel α-helices in so-called "death domain" (DD) and similar structures (DED, CARD) represent important players in apoptotic signaling. To DD/DED/CARD domains-containing proteins belong pro- apoptotic membrane receptors from the TNFR superfamily, then adaptor proteins and enzymes as proteases or kinases. These pro-apoptotic "death receptors" interact with adaptor proteins and initiator caspases containing DDs or DEDs and activate apoptotic signaling cascade. DEDs and DDs are in addition found in many proteins participating in activation of caspases or other non-apoptotic signaling. Many experimental models document that defects in and deregulations of proteins containing DDs and DEDs can have severe if not lethal consequences for an organism. Abberations in these proteins in many cases could lead to cancerogenesis, immunodeficiencies or developmental defects.
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