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Analysis of the intracellular localization of nucleophosmin: effect of C-terminal mutations
Kráčmarová, Markéta ; Brodská, Barbora (advisor) ; Čáp, Michal (referee)
C-terminal mutations of the phosphoprotein nucleophosmin (NPM) are the most frequent genetic aberration detected in adult acute myeloid leukemia (AML). I focused on characterization of type A, B and E of AML-related C-terminal mutations. The plasmids bearing fluorescently labeled wild type or mutated NPM have been constructed to characterize mutation-induced changes in the localization of NPM. Mammalian cell lines HEK293T, HeLa and NIH 3T3 were used for production of the chimeric proteins. The intracellular localization of the mutated forms of NPM was analyzed by immunofluorescence staining and fluorescence microscopy of the living cells. The localization of the mutNPM type A and B was almost identical and predominantly cytoplasmic, while mutNPM type E was detected in nucleolus and cytoplasm simultaneously. However localization of the mutated forms was greatly influenced by the used cell line. It has been demonstrated that the exogenous NPM interacts with the endogenous NPM and that they mutually affect their intracellular localization due to heterooligomer formation. Detailed analysis of the relationship between the C-terminal mutations and the localization of the mutated NPM improves understanding of specific mutation effect on the formation and progression of AML and also specifies its prognostic...
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The Design of siRNAs and the Influence of Modifications on Their Stability and Efficiency
Kuldanová, Kateřina ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
Design of small interfering RNA (siRNA) is a basic step to successful RNA interference (RNAi). However, it is not trivial to design a suitable siRNA duplex at all, because there are some problematic areas like stability of siRNA duplexes in the presence of nucleases, efficiency of target mRNA elimination, sequence-specific elimination of non-target mRNAs, cytotoxicity and immunogenicity for example, which can influence the aliveness of siRNA duplexes and consequently also their utility in medicine and other fields. The understanding of the principles of siRNA duplexes function during RNAi is a necessary step to solution of the obstacles on the way to efficient drugs. The mean of optimisation of described troublesome properties is an alteration of siRNA duplexes design by use of different modifications. Phosphodiester backbone, bases and also ribose can be modified. Some modifications beneficial for siRNA duplexes and consequently RNA interference are known in every one of these groups. This work presents existing pieces of knowledge about some of the most known modifications, just like phosphorothioate substitution, 2′-deoxy-2′-fluoronucleotides, 2′-O-methyl ribonucleotides and LNA nucleotides are, as well as about a higher number of less known modifications like boranophosphate substitution,...
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Transgenic technologies based on transposons
Dobiášovská, Ivana ; Kozmik, Zbyněk (advisor) ; Čáp, Michal (referee)
Genenetic engineering is one of the leading technologies in biological research. Transgenesis, one of the most important genetic engineering technologies, enables to study genetic aspects of organismal systems and thus helps us to better understand to the functional characteristics of genomes. Transposons are naturally occuring mobile genetic elements, which can be used to artificially integrate transgenes into host cell genomes. Catalysis of this essential step during transgenesis makes from transposons an useful genetic tool.The aim of this work is to present eukaryotic DNA transposons that transpose in a cut-and-paste-fashion, together with particular mechanisms affecting their function, that can be used as gene delivery system.
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Analysis of essentiality of glmM gene coding for phosphoglucosamine mutase of Streptococcus pneumoniae.
Krupička, Jiří ; Branny, Pavel (advisor) ; Čáp, Michal (referee)
Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...
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Impact of the regulatory T cells detection and differences in expession of tumor antigens in ovarian cancer
Kloudová, Kamila ; Sojka, Luděk (advisor) ; Čáp, Michal (referee)
Regulatory T cells (Treg) play a key role in maintaining the immune tolerance. They suppress development of autoimmune diseases and contribute to maintaining the homeostasis of the immune system. Expansion and excessive ability of regulatory T cells to suppress the immune response is increasingly observed also at many types of cancer. Due to the active inhibition of the antitumor immune response Treg contribute to tumor progression. Specific phenotype based detection and analysis of Treg functional properties may contribute to the successful monitoring of Treg accounts and to the effective cancer immunotherapy itself. Tumor cells express high amounts of so-called tumor antigens, which may play a key role in the antitumor immune response. Expression level of the tumor antigens gives the evidence about relevancy of each antigen in the specific immune response and efficiency of cancer immunotherapy. These data are obviously important to be obtained from the tumor cell lines as well as primary tumor cells. In the first part of the thesis I was focusing on the quantitative analysis of regulatory T cells in tumor tissue and peripheral blood of patients with ovarian cancer. For this purpose I used the newly introduced methyl-sensitive quantitative PCR (MS-qPCR) method and compare the data with the widely...
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Differentiation in Saccharomyces cerevisiae colonies
Čáp, Michal
Yeast colonies are, alongside to bacterial biofilms, multicellular communities formed by unicellular microorganisms. These specific communities differ in many ways from populations cultivated in planktonic cultivations. Gradients of nutrients, metabolic by- products and other factors are formed and preserved within these structures, which provides a basis for cellular differentiation. Current literature concerning these issues with emphasis on yeast colonies and biofilms is summarised in the Introduction of this work. Section Results then describes my contribution to the knowledge on the differentiation of the colonies of Saccharomyces cerevisiae as a model system for studying microbial multicellular structures. Previously described horizontal differentiation, i.e. differentiation between colony centre and margin, is further characterised with respect to ammonia signalling and stress resistance. The importance of genes conferring the cell`s oxidative stress resistance in colony differentiation was studied and it was concluded that not stress resistance but rather metabolic and other changes promoted by ammonia signal are important for colony differentiation and survival. A new type of colony differentiation - the horizontal, i.e. differentiation between upper and lower part of the colony, is...
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Translational control in immune response.
Hlaváček, Adam ; Valášek, Leoš (advisor) ; Čáp, Michal (referee)
Immune reaction often requires a prompt modification of gene expression that in turn alters cellular physiology. There are an increasing number of articles supporting a critical role of translational control in this aspect of cellular biology. The aim of this work is to present some of cellular and molecular mechanisms that connect translational control and immune reaction in immune and somatic cells and can be possibly misused by some viruses. Perhaps not surprisingly, many immunologically relevant translational control mechanisms are similar to those acting during the stress response. Over the years it has been documented that the T cells, dendrocytes, Natural killer cells and macrophages utilize translational control for their immunological activation following stimulation. Combination of general and gene-specific translational control mechanisms enables fast changes in proteome and physiology that are characteristic for immune cell activation. The overall impact of translational control on immune response is further illustrated by the fact that it acts upon each stage of life of immune cells - from their activation, through survival, to a programmed cell death. Even in some non-immune cells the translational control plays an important role with respect to immunity, as these cells are known to have an...
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Properties and function of middle T antigen of the murine polyomavirus
Fabiánová, Anna ; Forstová, Jitka (advisor) ; Čáp, Michal (referee)
Polyomaviruses are small DNA viruses, which are able to induce a broad variety of tumors. The main oncoprotein of the mouse polyomavirus (MPyV) is middle T antigen (MT antigen) which is able to transform cells. MT antigen has not an enzymatic activity of its own. It is able to activate signal transduction of host cells through its interactions with certain cellular proteins. These proteins include protein phosphatase 2A (PP2A), Src kinase, phosphatidylinositol 3 kinase (PI3K), Shc protein, 14-3-3 protein and phospholipase Cγ1 (PLCγ1). This work is focused on interaction between MT antigen and cellular proteins and on the impact of this interaction on cell transformation. Since MT antigen is a potent oncogene, the work also deals with the character of transformed cells and tumor development in mouse mammary epithelium. Keywords: polyomaviruses, MT antigen, PP2A, PI3K, PLCγ1, Shc protein, 14-3-3 protein
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Differentiation in Saccharomyces cerevisiae colonies
Čáp, Michal ; Palková, Zdena (advisor) ; Pichová, Iva (referee) ; Demnerová, Kateřina (referee)
Yeast colonies are, alongside to bacterial biofilms, multicellular communities formed by unicellular microorganisms. These specific communities differ in many ways from populations cultivated in planktonic cultivations. Gradients of nutrients, metabolic by- products and other factors are formed and preserved within these structures, which provides a basis for cellular differentiation. Current literature concerning these issues with emphasis on yeast colonies and biofilms is summarised in the Introduction of this work. Section Results then describes my contribution to the knowledge on the differentiation of the colonies of Saccharomyces cerevisiae as a model system for studying microbial multicellular structures. Previously described horizontal differentiation, i.e. differentiation between colony centre and margin, is further characterised with respect to ammonia signalling and stress resistance. The importance of genes conferring the cell`s oxidative stress resistance in colony differentiation was studied and it was concluded that not stress resistance but rather metabolic and other changes promoted by ammonia signal are important for colony differentiation and survival. A new type of colony differentiation - the horizontal, i.e. differentiation between upper and lower part of the colony, is...
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Incidence and molecular typing of Clostridium difficile strains in the Czech republic
Malinová, Anna ; Jirásková, Alena (advisor) ; Čáp, Michal (referee)
Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Clostridium difficile-associated disease (CDAD) is of gaining importance now due to its increasing incidence and severity. However, little is known about the C. difficile infections in the Czech Republic. The aim of the study was to characterize C. difficile strains recently isolated (2008 to 2011) from patients hospitalized with gastrointestinal disease in four Prague health care institutions using molecular typing methods; PCR toxinotyping, PCR ribotyping and MLVA (multilocus variable number tandem repeat analysis). Among 273 C. difficile strains, we identified 8 toxinotypes (0, III, IV, V, VI, VIII, IX a XXIII) and 63 ribotypes, of which ribotypes 596 (23,4 % patient), 017 (13,9 %) and 176 (7 %) were the most frequent. According to PCR ribotyping, the situation in the Czech Republic is the most similar to the situation in Poland. Within ribotypes 017, 017/1 and 017/2 and ribotypes 596 and 596/1, 5 and 4 distinct clusters were identified by MLVA, none of which was institution-specific. Additionally, pathogenic C.difficile were isolated from piglet faeces (63,3 %) in a single piglet farm, evaluating the role of C. difficile as an emerging animal pathogen. All piglet isolates belonged to the toxinotype 0 and the ribotype...
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