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Regulation of mast cell activation at the level of the high-affinity IgE receptor and STIM1
Bugajev, Viktor ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Hašek, Jiří (referee)
(EN) This thesis is focused on two important gate-keepers of mast cell signaling. The first is the complex of the high-affinity receptor for immunoglobulin E (IgE) (FcεRI) associated with Lck/Yes- related novel tyrosine kinase (Lyn), which is involved in acquired immune responses and the second is the stromal interaction molecule (STIM)1, which senses calcium levels in endoplasmic reticulum (ER) and upon depletion of ER Ca2+ stores participates in opening of the plasma membrane Ca2+ release- activated Ca2+ (CRAC) channels. Although the structure of FcεRI is known for many years and numerous molecules associated with the receptor have been described, the exact molecular mechanism of initiation and termination of the FcεRI signaling is elusive. Therefore, we evaluated the current knowledge on the molecular mechanisms of FcεRI phosphorylation with emphasis on the newly described model according to which cross-talk between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) sets the threshold for FcεRI tyrosine phosphorylation (PTK-PTP interplay model). Furthermore, we extended the knowledge about topography of active phosphatases which are prone to oxidation within the clusters of transmembrane adaptor proteins non-T cell activation linker (NTAL) and linker for activation of T...
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Myosin 1c isoforms and their functions in the cell nucleus and in the cytoplasm
Venit, Tomáš ; Hozák, Pavel (advisor) ; Černý, Jan (referee) ; Hašek, Jiří (referee)
Nuclear myosin 1 (NM1) was the first myosin described in the cell nucleus. From its discovery, it has been found to function in processes of Pol I and Pol II transcription, chromatin remodeling, and chromosomal movements. However, direct mechanisms of how NM1 works in the cell nucleus were still missing. We therefore decided to prepare NM1 knock-out mice to answer questions about phyiological functioning of this protein. Myo1c is an isoform of NM1 protein, previously described in the cytoplasm. The only difference between these isoforms is 16 amino-acids at the N-terminus of NM1, which were thought to be the nuclear localization signal. However, we discovered that the nuclear localization signal is located in the neck domain of myosin, and therefore it is able to direct both isoforms to the nucleus. Moreover, we found that the ratio between both proteins is nearly the same in the nucleus and deletion of NM1 does not cause compensatory overexpression of Myo1c. NM1 KO mice are fully viable with minor changes in bone mineral density and red blood cells size. We found that the function of NM1 in processes such as Pol I transcription can be fully covered by Myo1c protein, suggesting redundancy and interchangeability of these two isoforms in the cell nucleus. We also found that PIP2, a phosphoinositol...
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Myosin-PIP2 interaction in the cell nucleus
Yildirim, Sükriye ; Hozák, Pavel (advisor) ; Černý, Jan (referee) ; Forstová, Jitka (referee)
Even though nuclear myosin 1 (NM1) and myosin 1C (Myo1C) are the products of the same gene, NM1 has additional 16 amino acids at the N-terminus due to alternative start of transcription. Studies claim that NM1 and Myo1C are nuclear and cytoplasmic proteins, respectively. Therefore, researchers thought that NM1 translocates into nucleus via nuclear localization signal (NLS) in its N-terminal extension. However, here we show that NLS is placed within second IQ domain where calmodulin (CaM) binds in a calcium- dependent manner. Since both NM1 and Myo1C have identical neck domains where NLS resides, we have confirmed that both myosin isoforms localize to nucleus. Based on findings indicating Myo1C binding to phosphatidylinositol 4,5-bisphosphate (PIP2) via its tail domain, we tested if NM1 and Myo1C can interact with PIP2 in the nucleus. We show that both isoforms can bind to PIP2 via their tail domains, and interactions with PIP2 can recruit other nuclear proteins into this lipo-protein complex. PIP2 makes complex with a subset of Pol I transcription and processing machinery proteins and modulate their functions in the nucleolus. Moreover, PIP2 depletion results in a dramatic loss of Pol I transcription activity. NM1 and actin were already shown to promote Pol I transcription. Here, we show that...
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Elucidating the interactions of interleukin-1alpha with components of the eukaryotic transcription machinery
Zámostná, Blanka ; Pospíšek, Martin (advisor) ; Černý, Jan (referee) ; Mělková, Zora (referee)
4 ABSTRACT Interleukin-1α (IL-1α) is a pleiotropic cytokine and a key mediator of host immune response. It is synthesised as a 31-kDa precursor, that is cleaved by the cysteine protease calpain into the 17-kDa mature IL-1α and the 16-kDa N- terminal peptide of IL-1α (IL-1αNTP). Although IL-1α can be secreted, act on target cells through the surface receptor IL-1RI and trigger the signal transduction pathway, increasing evidence points toward the involvement of IL-1α in certain nuclear processes. IL-1αNTP is highly conserved among higher eukaryotes and contains a nuclear localisation sequence; indeed, both the precursor and IL-1αNTP are found in the cell nucleus. Previously, a genetic interaction of IL-1α with nuclear histone acetyltransferase (HAT) complexes has been reported from mammalian cells and, interestingly, also from the heterologous yeast model. This thesis extends the research of the nuclear function of IL-1α and demonstrates that IL-1α physically associates with the HAT/Core module of yeast SAGA and ADA HAT complexes. Results of the HAT subunit gene knock-out experiments followed by a set of co-immunoprecipitations also suggest a novel model of the yeast SAGA complex assembly, in which ADA appears to represent only a partly functional HAT complex. In its natural milieu of mammalian cells, IL-1α...
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Processing of different substrates by mammalian Dicer
Faltýnková, Jana ; Svoboda, Petr (advisor) ; Černý, Jan (referee)
Small RNA pathways represent sequence specific mechanisms regulating gene expression or mediating antiviral defence in eukaryotes. The common feature of these pathways are ~20-30-nucleotide small RNAs, which function as sequence specific guides. Small RNA pathways differ from each other in their roles, biogenesis of small RNAs and mechanism of regulation their targets in different organisms. In mammals, there are three recognized small RNA pathways: RNA interference (RNAi), microRNA (miRNA) and PIWI interacting RNA (piRNA) pathways. Biogenesis of small RNAs of RNAi and miRNA pathways is dependent on the Dicer protein, which generates small interfering RNAs (siRNAs) and miRNAs from long double stranded RNAs (dsRNAs) and small hairpins, respectively. This bachelor thesis provides an insight into structure and function of mammalian Dicer, particularly into differences in Dicer processing of pre-miRNA and siRNA precursors.
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Tracing the fate of cell populations from regressive tooth primordia during ontogenesis
Řadová, Marie ; Hovořáková, Mária (advisor) ; Černý, Jan (referee)
(v anglickém jazyce) Development of tooth primordia in mice is an important model for study of odontogenesis. Several dental rudiments develop during the mouse embryogenesis. These structures develop in functional teeth in their phylogenetically older relatives. Similarly, we can initiate growth of teeth from these germs in some mutant mice. In my diploma thesis we have focused on the importance of rudimentary structures with odontogenic potential in postnatal individuals. As a model of development, we have chosen a cell population originating from rudimentary primordia MS (mesial segment) that develops in diastema of the lower jaw during the embryonic day 12.5. Using the inducible Cre-lox technology we have marked the cells which are part of the signal domain of primordia at this time. As a marker of these cells we have used gene Shh. We have found out that these cells persist prenataly and also postnatally. Further we have isolated this cell area and we have tested it using a variety of methods. We have shown that in the cells of postnatal individual are expressed markers of stem cells (Sox2, Bmi1, Gli1) and also genes for major enamel matrix structural proteins: ameloblastin and amelogenin. The same stem cell markers are also expressed in vitro culture of the isolated cells. This cell population...
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Autophagy in the immune system
Vávra, Dan ; Černý, Jan (advisor) ; Janštová, Vanda (referee)
Autophagy is an essential, homeostatic process - survival mechanism that protects cells by various ways: cells break down their own components to recycle nutrients, remodel and dispose unwanted cytoplasmic constituents. Autophagy is involved in the degradation of long-lived proteins and entire organelles, but paradoxically, considering important prosurvival functions, autophagy may be deleterious. It plays an important role during development, tumor suppression, immunity and is required for the adaptation to environmental stresses such as starvation. Recent studies indicate, that autophagy is a central player in the immunological control of bacterial, parasitic and viral infections. The process of autophagy may degrade intracellulal pathogens. This work describes the mechanism of autophagy and highlights the role of autophagy in innate and adaptive imunity, summarizes some advances in understanding the functions of autophagy and its possible roles in the causation and prevention of human deseases.
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Interactome of IL-1α N-terminal domain
Dolečková, Denisa ; Pospíšek, Martin (advisor) ; Černý, Jan (referee)
Interactome of IL-1α N-terminal domain Cytokines are highly effective mediators produced by various cell types within and outside of the immune system with the aim to influence the orientation, intensity, and duration of the immune response and inflammatory process. Their biological effects mediated through binding the high-affinity membrane receptors and triggering the signal transduction pathway are usually well defined. However, as it is more and more frequently observed, in addition to the exocrine function, some cytokines may show intracrine effects. For this type of cytokines, the term "dual function cytokines" has been adopted. One of these cytokines is Interleukin-1α, in which the recent research has concentrated on determining its intracellular functions. The intracellular function of interleukin-1α has not been clearly defined so far. However, apart from the absence of the conventional hydrophobic sequence, its existence is supported by the fact that the N-terminal peptide included in its precursor is highly conserved and contains nuclear localization signal. The aim of this work is to define the conditions of localization of the interleukin-1α N- terminal domain in different cellular compartments and to study proteins potentially interacting with it using fluorescent microscopy. Key words:...
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Cord blood T regulatory cells and their association with development of type 1 diabetes
Norková, Jindra ; Štechová, Kateřina (advisor) ; Černý, Jan (referee)
Type 1 Diabetes (T1D) is organ-specific autoimmune disease which causes pancreatic beta cells to be irreversibly destroyed. The only possible treatment represents life-lasting insulin administration. The real trigger of destructive insulitis isn't known. T1D is a multi- factorial disease involving both external and internal factors in the disease pathogenesis. The presence of autoreactive T lymphocytes in pancreas is necessary for development of diabetes. T regulatory cells have protective function in the destructive insulitis. The aim of this diploma thesis was to study cord blood T regulatory cells and their connection to type 1 diabetes development. We tried to find the difference among T regulatory cells in mononuclear cord blood cells (CBMC) in different study groups. Samples were collected from mothers suffering from T1D, gestational diabetes. Healthy controls were tested as well. Sixty-eight samples of cord blood were included in the study among the years 2009 - 2011. Samples were divided into 3 groups (CBMC from children born to T1D mothers, mothers with gestational diabetes and healthy mothers without T1D). CBMC were ana- lysed by flow cytometry. T regulatory cells (defined as CD4+CD25+) were isolated by magnetic separation (MACS). The functional capacity of these cells was studied as well by...
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The study on physiological importance of enteric alpha-defensin expression in the thymus
Dobeš, Jan ; Filipp, Dominik (advisor) ; Černý, Jan (referee)
Enteric α-defensins belong to a superfamily of antimicrobial peptides representing the humoral branch of innate immunity. They are produced by intestinal Paneth cells and play an irreplaceable role in the homeostasis of small intestine by modulation of its bacterial composition and the protection of intestinal stem cells residing in the intestinal crypts. Unexpectedly, the tissue-specific gene expression screening of enteric alpha-defensins on a panel of rat tissues revealed their presence in the thymus. The characterization of their cellular source pointed to the CD45- MHCII+ AIRE+ medullary thymic epithelial cells (mTECs). The subsequent analysis further confirmed that, analogous to the rat model, mouse enteric α- defensins (cryptdins) are in the thymus expressed exclusively in the mTECs. It is now well established that autoimunne regulator (AIRE)-dependent transcription of peripheral tissue restricted antigens (TRAs) and their presentation by mTECs leads to a deletion of self- reactive thymocytes. This process of negative section represents the central mechanism of immunological tolerance. Predictably, mutations and malfunctioning of AIRE lead to aberrations in the negative selection, occurrence of self-reactive T-cells and subsequent production of autoantibodies against TRAs. Consistent with...
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