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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.
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Regulation of the B7-H1 (CD274) inhibitory molecule expression on tumour and immune cells.
Hrušková, Veronika ; Reiniš, Milan (advisor) ; Krulová, Magdaléna (referee)
The co-stimulatory/inhibitory molecules of family B7 that are expressed on the surface of antigen-presenting and other cells, including tumour cells, play an important role in immune responses regulations. Owing to binding on the T-lymphocyte receptors, they regulate their positive and also negative responses. An important co-stimulatory member of B7 family is B7-H1 molecule. This molecule is presented in many different tissues, not only on haematopoietic cells. However, it is also expressed on tumour cells. Its two binding receptors are in the cytoplasmic membrane of T-cells. The first receptor is unknown and the second receptor is PD-1 (CD279). The linkage between B7-H1 - PD-1 regulates negatively proliferation, differentiation and cytokine secretion of T-cells. This negative regulation is crucial for the constitution of peripheral immune tolerance and also for escape of tumour cells from T-lymphocyte based anti-tumour responses. Although the mechanisms underlying the B7-H1 cell-surface expression have been intensively studied, and they are not fully understood, especially in tumour cells. IFNγ and TNFα regulate B7-H1 expression on transcription level as well as on post- transcription level. Binding sites for transcription factors IRF-1 and NF-κB are located in B7- H1 promoter region. The IRF-1...
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Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography
Vaněk, Ondřej ; Bezouška, Karel (advisor) ; Hrabal, Richard (referee) ; Bařinka, Cyril (referee)
Department of Biochemistry, Faculty of Science, Charles University in Prague 2010 Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography Abstract of Ph.D. thesis Ondřej Vaněk Supervisor: Prof. RNDr. Karel Bezouška, DSc. Natural killer cells (NK cells) were found out for their ability to spontaneously kill certain allogeneic tumour cell lines, without any previous sensitization. NK cells are part of non- adaptive immune response with very short reaction time against pathogens such as viruses, intracellular bacteria, parasites, and they are responsible for elimination of certain tumour cells and thus they are able to fight against malignancy and formation of metastasis. Activity of NK cells is regulated by the balance between activation and inhibitory signals mediated by the NK cell surface receptors. From the structural point of view, the majority of NK cell surface receptors could be classified as the C-type lectin or immunoglobulin-like receptors. One of many C-type lectin subgroups are type II lymphocyte receptors that are expressed on the NK cell surface. This study had two main aims. The first one was to find suitable expression and purification systems for selected C-type lectin receptors of NK cells and the other one was to perform their...
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Functional studies of selected members of the Arabidopsis formin family
Oulehlová, Denisa ; Cvrčková, Fatima (advisor) ; Binarová, Pavla (referee) ; Černý, Jan (referee)
Formins are multidomain proteins containing a conserved formin-homology 2 (FH2) domain, which catalyzes de novo nucleation of actin filaments. In yeast and animal cells, both mechanisms and regulation of formin function have been extensively studied, yet much less is known about action of plant formins, which considerably differ from yeast and animal ones in the domain composition. In higher plants, formins are classified into two groups, Class I and Class II, and so far, experimental data are available only for the first group members. Here I present results of experimental study of several members of the large formin family in Arabidopsis, including the characterization of a Class II formin AtFH16. Arabidopsis genome contains 21 formin-encoding genes, and though they greatly differ in their expression levels and pattern, all of them are transcriptionally active. We selected 17 homozygous T-DNA insertional mutants in 14 formin genes. Under standard cultivation conditions, no obvious phenotypic discrepancies between wild type and mutant plants were found. To impair two dominant pollen formins, an atfh3atfh5 double-mutant was prepared and even in this case, both microspore development and pollen tube growth remained unaffected. Consistently, polarized growth of tobacco pollen tubes was not altered...
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In-vitro analysis of amoeboid-mesenchymal transition of A375m2 melanoma cells
Kasalová, Lenka ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee)
The invasion of cancer cells is an important aspect of cancer progression. Single tumor cells exhibit at least two types of invasion in 3D environment, mesenchymal and amoeboid invasion. Tumor cells can switch between these two modes of movement depending on cellular status and surrounding environment. Amoeboid-mesenchymal transition (AMT) is less explored then mesenchymal-amoeboid transition (MAT). We performed a proteomic analysis of amoeboid-mesenchymal transition of human melanoma cell line A375M2. We have induced amoeboid-mesenchymal transition by treatment with a ROCK inhibitor Y27632 in 3D matrigel matrices and in 2D environment. Induction of the amoeboid-mesenchymal transition has changed a level of expression of 92 proteins and a level of phosphorylation of 15 proteins. Expression of only 17 proteins and phosphorylation of 8 proteins was identically changed in both of these environments. We found that PKCα regulates amoeboid migration and that treatment of cells with a PKCα inhibitor Gö6976 induces amoeboid-mesenchymal transition. Analysis of the proteomics data have further shown that induction of AMT by the ROCK inhibitor Y27632 leads to activation of antiapoptotic signals and activation of signaling pathways involved in regulation of actin cytoskeleton especially regulation of focal...
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Photoreceptors properties in strictly subterranean African mole-rats
Krejčová, Tereza ; Němec, Pavel (advisor) ; Kott, Ondřej (referee)
4 Photoreceptor properties and distribution in strictly subterranean African mole-rats (Rodentia, Bathyergidae) The visual system of subterranean rodents is assumed to be regressed in response to their lightless ecotope. According to the prevailing doctrine, negative or non-selective processes have driven evolutionary regression of the visual system in many unrelated groups of rodents that has adopted strictly subterranean mode of life. Among rodents this has been exemplified by detailed studies of the blind mole-rat Spalax ehrenbergi. However, recent studies involving a larger spectrum of subterranean rodent species demonstrated unexpected diversity of retinal properties and visual system designs among subterranean rodent species and thus challenged the widely held view that the visual system has undergone extensive convergent evolution in subterranean mammals. This paper examines the presence, the distribution, and photoreceptor properties in the African mole-rats (Bathyergidae, Rodentia). It describes rodopsin, S- and L-opsin expression patterns, photoreceptor densities and rod/cones proportions in five species of bathyergid mole-rats, namely Bathyergus suillus, Georychus capensis, Fukomys damarensis, Cryptomys natalensis and Heliophobius argenteocinereus. Spectral cone types and rods were assessed...
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A tool for generating code supporting reading of text files
Černý, Ondřej ; Týnovský, Miroslav (referee) ; Kolovratník, David (advisor)
In the present work we deal with the concept and implementation of tool AutoComp for generating programs for processing structured text files. The tool accepts description of the text whith making use of regular expressions and state machines. The output of the tool is a code in a programming language which gives interface for integrating code to a final program. It is possible to define points for calling specified functions and returning controll to the caller program in the input description. The use of generated code is to tokenize text or parse elementary gramatics. The tool provides two target programming languages and few character encodings. The concept was focused on extensibility to more languages and character encodings.
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Comparison of Immune System of Newborns and Adults
Dusilová, Adéla ; Zajícová, Alena (referee) ; Hrdý, Jiří (advisor)
In general, it is possible to characterize neonatal immune system (IS) as immature in comparison to adult IS. From a clinical point of view, newborns show an increased susceptibility to infections. Breastfeeding can contribute to the descent incidence of illnesses, because it supplies the intestinal mucosal system with antibodies of the mother`s origin, important nutrients and other immunoregulatory components. Breast milk compensates decreased newborn's capacity to produce immunoglobulins- especially IgA, that concentration reaches adult levels in two years, but even later (to the pubescent period). Other classes of antibodies are found in cord blood only sporadically except IgG, which is transferred transplacentary. Reduced ability of B lymphocytes to produce antibodies is caused by insufficient expression of surface costimulatory signals of Th2 cells. T lymphocytes are not able to react properly to low doses of stimulators (polyclonal activators - phytoid lectins: ConA or PHA), which bind to T cell receptors in complex with CD3 and proliferate in a response to anti-CD3 monoclonal antibodies. Most of the cord blood T lymphocytes display "naive" phenotype CD45RA. During intrauterine development, neonatal IS is in contact with mother IS and because a pro-inflammatory Th1 response could lead to...
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Contribution of ten ectodomain cysteine residues to function of ATP-gated P2X4 receptor
Tvrdoňová, Vendula ; Teisinger, Jan (referee) ; Zemková, Hana (advisor)
Extracellular adenosine-5'-triphosphate (ATP), released from damaged cells or coreleased as a cotransmitter from synaptic vesicles, acts on its plasma membrane receptors termed purinergic. Purinergic P2X receptors are ATP-gated cation channels. To date seven P2X isoforms designated P2X1-7 have been cloned that are organized as trimeric homomers or heteromers. All P2X subunits share a similar structure consisting of a large extracellular loop, two transmembrane domains and intracellular N- and C- termini. An additional structural feature is conserved aminoacids, these include ten conserved cysteine residues in the extracellular loop. All ectodomain cysteines form disulfide bonds which are organized in two areas: three disulfide bridges are localized in the N-termini half and two in the C-termini half at P2X receptor. ATP binding pocket is apparently localized between two neighbouring subunits. The aim of this Diploma Thesis was to examine the relevance of ectodomain cysteine residue and/or disulfide bonds for the expression, function and ATP binding properties of the P2X receptor. All ten, one by one, ectodomain cysteines were substituted by alanines and ATP-induced currents was recorded in HEK293 cells expressing wild-type P2X4 receptor and its mutants. Low responsible or nonfunctional mutants...
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Hallucinogenic and other drugs as a cultural phenomenon
Vaňková, Jana ; Soukup, Václav (advisor) ; Matějů, Martin (referee) ; Křivohlavý, Jaro (referee)
The subject of the work is a culturological theoretical-empirical analysis of two mutually connected and related topics: l-Hallucinogenic and other drugs, their consumption-abuse and addiction 2- Prevention of abuse of hallucinogenic and other drugs - forms of prevention and content. The subject of the dissertation is the problems associated with hallucinogenic and other drugs and their interpretation in the context of factors "hallucinogenic drugs, the individual and environment-society". The dissertation will be a culturological analysis including professional contributions on the problems concerning a subject which spans multiple fields. The goal of the dissertation is to give culturological contribution both for multiple fields discusion and practical prevention Acceptance of findings and observations from other fields is therefore essential, as is finding central points and common ground for co-operation with other desired fields. The work has two parts - a theoretical part and a practical one. The theoretical section will include an analysis of three basic levels in the consumption of hallucinogenic and other drugs. We can call the first level Consumption of hallucinogenic and other drugs as a specific human behaviour - a specific human expression. The work maps the attitude to hallucinogenic and...
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