|
|
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)
|
|
| |
|
|
Preparation of the constructs for analysis of expression of nuclear receptor nhr-97 by using transgenic techniques in the model system Caenorhabditis elegans
Boušová, Kristýna ; Stiborová, Marie (advisor) ; Hudeček, Jiří (referee)
The aim of this work was to prepare two constructs of the promoter of a gene coding for nuclear hormone receptor nhr-97 in C. elegans. Nuclear receptors belong to a large group of genes sharing homologous sequences in some vertebrate nuclear receptors. The first part of the work describes the structure of nuclear hormone receptors, their function and significance in the nematodes C. elegans. The model organism C. elegans, its anatomy, life cycle and genome were also described. The work also discusses the structure and use of green fluorescent protein (GFP), which serves to localize the expression of the nhr-97 gene in C. elegans. In the practical part of the work, the preparation of two constructs of the promoter is described. Isolation of genomic DNA of C. elegans, PCR amplification of the promoters and their subsequent cloning into vector pPD95.67 containing a gene coding for green fluorescent protein were performed. To verify the successful cloning of the promoter constructs, sequencing DNA was performed. Cloned promoters of nhr-97 will be used for microinjetions to C. elegans gonads and the expression of this gene regulated from particular promoters will be subsequently monitored using expression of green fluorescent protein in progeny.
|
|
|
Preparation of expression system of gamma-lactamase and expression testing
Magyerková, Monika ; Ingr, Marek (advisor) ; Šácha, Pavel (referee)
γ-lactamase is an enzyme clearing five-membered lactam cycles. Polyvinylpyrrolidone (PVP) is one of its potential substrates. Degradation of PVP by γ-lactamase is being studied due to its eventual use in waste-water purifying plants. The aim of the work was to prepare a synthetic gene from the bacterium Comamonas acidovorans and to clone it into the expression vector pET22b. PCA method was used for the synthesis of the γ-lactamase gene. 1725 bp long sequence of the γ-lactamase gene was split into two parts (synthons) which were synthesized individually. After the synthesis restriction cleavage and ligation to the vector pUC19 were performed. Competent cells E. coli, strain DH5α, were transformed by the obtained construct. After the sequence confirmation both synthons were cleaved by restriction endonucleases and connected by single-step ligation to the plasmid pET22b. Expression bacterial cells E. coli, strain BL21(DE3)RIL, were transformed by the recombinant plasmid containing the connected synthons and expression of the recombinant γ-lactamase was tested. Sequence of the clone producing a protein of the expected length was confirmed by sequencing analysis. The prepared plasmid will be used for the expression of recombinant γ- lactamase. (In English)
|
|
|
Performing a Relay Attack on Mifare Smart Cards
Činčala, Martin ; Henzl, Martin (referee) ; Malčík, Dominik (advisor)
This thesis deals with performing a relay attack on MIFARE smartcards while using off-the-shelf readers only. These readers are not designed for such attacks therefore implementation of the attack that would succeed against every smartcard was not possible. Since various attacks on smartcards have already been implemented, I have focused on the latest and still insufficiently explored card MIFARE Ultralight C. Relay attack has been implemented with simplified emulation of 4-bytes long UID and successfully tested on MIFARE Ultralight C. With the use of other readers it should be possible to perform attack also on other cards like MIFARE Plus, Desfire and SmartMX. For the purpose of cloning MIFARE Classic open-source tools are introduced.
|
|
|
Isolation of the antimicrobial peptide gene (defensin) from the hard tick \kur{Ixodes ricinus}, challenged by the pathogen infection
SKLADANÁ, Veronika
Antimicrobial peptides are major components of the innate immune response of epithelial cells. In hematophagous organisms, which acts as vectors of parasitic diseases, in particular Lyme borreliosis, the gut pathogen induce the expression of defensin, that provide the first barier of host defence. This raises the posibility that defensin may play a key role in the development of parasitic infection. The gene expressed in midgut was isolated from cDNA of the hard tick, Ixodes ricinus. The gene is coding the protein, that is produced and secreted during tick infection, and is known as defensin. Expression of the gene for defensin (224 bp) was induced by the pathogen. The gene was cloned into bacterial expression system.
|
|
|
The transcriptional regulation by the nuclear receptor NHR-25 in \kur{Caenorhabditis elegans}
MERGLOVÁ, Linda
NHR-25 is a one of few conserved nuclear receptors in C. elegans and its family is involved in many developmental processes not only in the worm but also in flies, fish, mice and humans. Yet, the cellular mechanism of the action of this gene family is poorly understood. In C. elegans, it is likely to function as a transcription factor but its direct target gene has not been identified to date. In this study, I utilized the defined NHR-25 binding (target) sequence and GFP as a marker to visualize a possibility that NHR-25 regulates transcription of other gene(s) in vivo. I have also tried the "candidate approach" of two genes utilizing existing transgenic worm strains carrying promoter::GFP fusion transgenes expressed in epithelial cells to see if those genes can be regulated by NHR-25. According to the results it seems, that there could be an interaction between one of these genes and nhr-25 and that NHR-25 can play some role in endocytosis.
|
|
|
Genetická selekce a klonování u metody GMDH-MIA
Jiřina, Marcel ; Jiřina jr., M.
The GMDH MIA algorithm is modified by the use of selection procedure from genetic algorithms and including cloning of the best neurons generated. The selection procedure finds parents for a new neuron among already existing neurons according to fitness and with some probability also from network inputs. The essence of cloning is slight modification of parameters of copies of the best neuron, i.e. neuron with the largest fitness. The genetically modified GMDH network with cloning (GMC-GMDH) can outperform other powerful methods. It is demonstrated on some tasks from Machine Learning Repository.
|
|
|
Molecular cloning, E.coli expression and purification of SCFV antibody fragments of diagnostic/therapeutic interest
Král, Vlastimil ; Fábry, Milan ; Hořejší, Magdalena ; Závada, Jan ; Sedláček, Juraj
We describe molecular cloning, expression, purification and properties of two single chain antibody variable fragments (scFv) of potential diagnostic use, namely scFv M75 and scFv Tu-20. The former scFv is derived from a monoclonal antibody M75 specific for a cell surface protein MN/CA IX, strongly associated with many types of human carcinomas. The latter scFv is derived from a monoclonal antibody TU-20 specific for neuronal beta-III-tubulin.
|
|
| |
guest ::
