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In vitro expansion and activation of natural killer cells for cell therapy.
BENEŠOVÁ, Monika
NK cells are part of the non-specific immune response and are one of the main components of antitumor immunity. They do not need antigen stimul for thein activation but recognize damaged ( transformed ) cells by characteristic decreased expression of MHC I molecules. These natural killers become subjekt of many clinical studies based on the use of anti-tumor activity of NK cells for both solid tumors and in hemato - oncological diseases. The aim of this study was to find optimal conditions for in vitro expansion and activation of NK cells. NK cells were isolated from mononuclear cell fraction by magnetic separation and cultured in two types of media SCGM and X - VIVO 10 with the addition of interleukin-2 respectively OKT3 antibody. The influence of the mononuclear cells on proliferation of NK cells was tested. After a 6- day culture , the cells were passaged and growth of NK cells was determined using hematological analyzer and flow cytometry. Expression of the activation markers CD25 and CD336 (NKp44) was observed. All experiments were conducted under conditions of good manufacturing practice. Slightly higher gain of NK cells was observed in SCGM media without a significant differences in the other additives. Much higher number of NK cells was observed in culture with supporting irradiated mononuclear cells. There were no differences in cultures with autologous or allogeneic cells. We found that NK cells with higher proliferative potential express increasingly CD25, unlike the cells with decreased proliferation which had increased expression of CD336 marker. The work led to the definition of the optimal culture conditions for NK cells and became the basis for the further development of the medicinal product from in vitro activated NK cells.
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Characterization of bacterial genes encoding DbtC-like extradiol dioxygenase with bioremediation potential for aromatic compounds in locality Hradčany
Šnajdrová, Renata ; Bořek Dohalská, Lucie (advisor) ; Nešvera, Jan (referee)
Aromatic pollutants pose a serious environmental problem. Petroleum and its derivates belong to the most abundant contaminants in the Czech Republic and their sanation is a priority objective for improving the life quality of the population. Bioremediation is a technology taking advantage of the natural capacity of soil and water microbial community to degrade environmental pollutants. Deeper understanding and detailed knowledge on specialized bacterial species, pathways and genes is required for selection, optimization and application of targeted bioremediation approach and for monitoring of its results. Recent analysis of a metagenomic library constructed from highly contaminated soil of the former military air-base Hradčany has identified a novel group of catabolic genes encoding extradiol dioxygenase similar to DbtC of Burkholderia sp. DBT1. The DbtC-like enzymes are among the three priority groups of extradiol dioxygenases with biodegradation relevance for the locality. The present study of soil bacterial isolates and metagenomic fosmid clones harboring the genes of interest gained evidence about the dbtC-like genes as a part of highly mobile gene cassette. Transposon insertion mutagenesis identified the genes joined with the expression of the extradiol dioxygenase activity. The dbtC-like genes were...
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Dual role of CD9 protein in mast cell activation
Machyna, Martin ; Dráber, Petr (advisor) ; Černý, Jan (referee)
Mast cells are well known effector cells in immune system. They have been implicated in such important processes as host defense against bacteria, toxins or parasites. However, in some cases they can develop improper reaction against harmless environmental antigens and thus causing allergies. It is therefore essential to understand signaling events that lead to activation of these cells in order to develop new treatment strategies. Newly prepared rat monoclonal antibody of IgG1 subtype raised against murine mast cells was characterized and found suitable for flow cytometry, immunoblotting and immunoprecipitation. Employing of optimized procedure for immunopurification in combination with mass spectrometry led to identification of its target cluster of differentiation (CD)9 protein. CD9 is a member of large protein family called tetraspanins. Functional studies showed that binding of this antibody to mast cells induced degranulation and early activation events such as increased tyrosine phosphorylation and enhanced levels of free cytoplasmic calcium. Interestingly, subsequent activation of these cells via antigen-mediated aggregation of the high-affinity IgE receptor (FcεRI) led to decreased degranulation, calcium response and tyrosine phosphorylation of several substrates. Importantly, anti-CD9 antibody did...
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Peptidases of Trematodes
Kašný, Martin ; Mikeš, Libor (advisor) ; Kopáček, Petr (referee) ; Haas, Wilfried (referee)
90 3. SUMMARY The text above refers about the majority of characterized trematode peptidases; the fundamental enzymes for trematode existence, which are integrated in many physiological processes like pathogenesis, tissue invasion/migration, nutrition, immune evasion and host-parasite interactions. In the history (until 1996), the peptidase catalytic activities in trematode extracts have been monitored. During 1980s and 1990s, the information of first cloned trematode peptidase genes were published and during last three decades cca 90 trematode peptidase sequences belonging to 19 peptidase families of 5 clans have been identified. The most studied trematode peptidases have been of Schistosoma mansoni origin: the serine peptidase - cercarial elastase (of cercariae), cysteine peptidases - cathepsins B, L, F, C plus the asparaginyl endopeptidase SmAE and the aspartic peptidase - cathepsin D (of adult worms and some other life stages). The recent computational cluster analysis revealed that the sequence S. mansoni elastase (the main cercarial penetration enzyme) is quite divergent from other serine peptidases of the S1 family. Cercarial elastase gene was proved in S. mansoni, S. haematobium and Schistosomatium douthitti, but not in the related S. japonicum. Mass spectrometry analysis confirmed cercarial...
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Transcriptional regulation of differentiation of embryonal carcinoma and malignant melanoma cells: The Role of Proteins p21 (WAF1) and MITF
Šestáková, Blanka ; Vachtenheim, Jiří (advisor) ; Černý, Radim (referee) ; Pavel, Stan (referee)
Summary. F9 cells (embryonal carcinoma) can be induced to differentiate with retinoic acid and dibutyryl-cAMP into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation. Clones of F9 cells stably expressing ectopic p21 revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter, implying that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. Whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. As differentiation of F9 cells into parietal endoderm-like cells...
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Šlechtění révy na ŠSV Ampelos
Pokorný, Stanislav
The aim of this thesis is the description of the vine and history, as well as the beginnings of cultivation of vines in South Moravia. The main part of this work deals with our eldest šlechtitleskou Station Wine in Znojmo - Ampelos and since 1895 when this station was then describes the main osbnosti that are associated with this station. Are mentioned varieties that have been bred here, but also varieties that have yet whether they will be entered in the State Variety Book. The last section briefly describes the other station activities such as slekce Sauvignonových clones or slekce yeast.
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Possibilities of physiotherapy methods of psoriatic arthritis
DROBNÁ, Martina
This bachelor thesis deals with the problems of psoriatic arthritis, which has chronic evolution among most cases and can lead to articular deformities in patients. This disease has an unclear pathogenesis conditioned by genetic, external and immunologic factors. It affects above all patients with type I psoriasis with emerging symptoms such as swellings with articular pains, morning stiffness and overall fatigue. PsA often shows positive antigen HLA B27, without presence of rheumatoid nodules and negative rheumatoid factor (Štolfa, 2012). PsA causes both physical handicap in patients and substantial disturbance of their social and psychical life. The thesis is divided into theoretical and practical part. The theoretical part briefly outlines the anatomy of skin as well as structure and function of joints. Onward it describes psoriasis, its forms and methods of treatment. The next chapter explains the term arthritis and presents RA as an example. It is followed by detailed description of PsA, definition of the disease, its causes and occurrence, clinical picture and some therapy possibilities. In the practical part, the method of qualitative research was used, with participation of three female patients suffering from PsA. Therapy was initiated by input kinesiologic analysis, including specific examinations such as goniometric examinations of joints, grasp examinations, muscle palpation examinations and others. Patient appointments were held regularly each week for a period of eight-week therapy. On each appointment, previously applied therapy was consulted, exercises were repeated, possibly uncertainties were clarified. Each therapy lasted about 45 minutes, during which hypertonic muscles along with trigger points were relaxed according to specific patient issues. In the last session, output kinesiologic analysis was done and patients were informed about the importance of adhering to regimen measures. The results of the research clearly show overall posture improvement in all patients. A very substantial indicator of therapy success is noticeable reduction of swellings and pain, and hand function improvement. The therapy also focused on adjustment of faulty breathing stereotype manifesting itself with upper type, thereby causing overexertion of cervical spine muscles and frequent headaches. Strong emphasis was also put on activation of the transverse arch, which was collapsed in all patients. This bachelor thesis can be used in clinical practice of physiotherapists or as an educational material for patients.
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Monitoring of selected coagulation parameters in pregnancy.
Nováková, Radka ; Křivčíková, Lucie (referee) ; Hronek, Miloslav (advisor)
In the group of 73 patients with recurrent spontaneous abortions, which they had a personal history of at least one thrombophilic disposition, either congenital acquired mutation of factor V Leiden in heterozygous form, or 20210 mutation in the gene of factor II in heterozygous form or mutation in the gene for MTHFR 677T or 1298C in heterozygous or homozygous form, or they had acquired thrombophilic disposition of antiphospholipid syndrome, or hyperhomocysteinemia, we studied the level-progress of blood coagulation parameters during the entire pregnancy. During this period, blood samples were taken from the patients on a regular basis. By some patients the parameters were examined even before pregnancy. Patients were getting nadroparin (Fraxiparine) in the 25th - 43rd week of pregnancy, alternatively enoxaparin (Clexane) who had showed allergic reaction. With the indirect detection method using a chromogenic substrate for the analyzer Sysmex CA-1500 (Sysmex-TOA) we evaluated functional activity of plasminogen, plasminogen activator inhibitor and α2-antiplazmin. With the direct non-competitive ELISA method, we found out a quantitative amount of antigen plasminogen tissue activator, plasminogen activator inhibitor and trombomodulin. We documented that the levels of trombomodulin and plasminogen...
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Chemokines of epithelial cells in bronchial asthma
Volfová, Dominika ; Stříž, Ilja (advisor) ; Holáň, Vladimír (referee)
Asthma is an allergic disease caused by adverse reactions to harmless antigens (allergens) and is characterized by the recruitment of eosinophils, Th2 lymphocytes, mast cells and neutrophils into the tissue site of inflammation, to the lungs. This accumulation of leukocytes is mediated by the generation of chemotactic cytokines (chemokines). Chemokines are low molecular weight proteins, functioning by binding to specific receptors on the cell surface. Binding of chemokines and their receptors is highly promiscuous and subsequent activation of effector cells is very heterogeneous, which can often complicate research in this area. However, chemokines and their receptors are important potential therapeutic targets in allergic diseases including asthma, mainly because of their central role in cell activation and inflammation. Chemokines are secreted by cells of the immune system and cells of various tissues of the body. Recently, attention turns to the role of epithelial cells in the pathogenesis of asthma. Bronchial epithelial cells stimulated by antigens produce cytokines and defense molecules used for the amplification of inflammatory processes and regulate the aktivity of effector cells. Impaired cytokine regulation may lead to the development of various lung diseases including asthma. This work...
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Geny kodující acetyhydroxy syntázu u Streptomyces cinnamonensis a jejich regulační oblast:Mutace v katalytické podjednotce zjią»ující necitlivost k inhibici konečného produktu
Kyselková, Martina ; Kopecký, Jan ; Šigutová, Lucie ; Pospíšil, Stanislav ; Felsberg, Jürgen ; Spížek, Jaroslav ; Janata, Jiří
Reaction of acetohydroxy acid synthase (AHAS) is the key step in branched-chain amino acids biosynthesis. AHAS is feed-back inhibited by valin that binds its regulatory subunit. In Streptomyces cinnamonensis, production of a secondary metabolite, polyether antibiotic monensin A, is associated with valin biosynthesis via 2-oxoisovalerate. Thus, mutants with higher AHAS levels or AHAS insensitive to valin show increased monensin A production. Gene ilvB coding for the AHAS catalytic subunit of the S. cinnamonensis parental strain and four mutants resistant to valin analogues was cloned and sequenced. Nucleotide sequence of ilvB from the strain BVR-18 differed from the wild type in a single substitution C574A resulting in an amino acid change E139A. In the ACB-NLR-2 strain, a triplet AGC in the position 805-807 was deleted resulting in the deletion of Q217. A homology model of the catalytic subunit, created by a virtue of the known three-dimensional structure of the yeast AHAS catalytic subunits dimer, revealed that the mutation found in the BVR-18 was located in a loop at the subunit-subunit interface in a close proximity of the TPP binding site in the active centre. The Q217 deletion in the ACB-NLR-2 occurred in a helix distant from the catalytic site, and seemed to shorten it. AHAS assays in a crude cell extract from the S. cinnamonensis BVR-18 and ACB-NLR-2 strains showed not only the insensitivity to valin but even higher activities of both enzymes in the presence of 10 mM valine. Catalytic and regulatory subunits from the parental and mutant strains were separately expressed in E. coli and combined in vitro to reconstitute the holoenzyme. Properties of such in vitro prepared enzymes were tested. The ACBR-2 and NLR-3 strains with increased AHAS levels have no mutation in the AHAS catalytic subunit. The ilvB regulatory region of those two strains and of the parental strain was cloned and sequenced using the method of chromosome walking. A putative leader peptide and a transcription termination sequence were identified within this region indicating that ilvB expression is controlled by attenuation. However, neither the ACBR-2, nor the NLR-3 have any mutation in the ilvB regulatory region suggesting that some other regulatory mechanism may participate in ilvB expression.
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