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Expression of immunogenic cell death markers on lung cancer cells
Kobosilová, Linda ; Špíšek, Radek (advisor) ; Černý, Jan (referee)
Immunogenic cell death (ICD) is characterized by presence of specific molecules including surface exposed calreticulin (CRT) and the heat shock proteins HSP70 and HSP90. Release of ATP and high- mobility group box protein 1 (HMGB1) belongs to other typical characteristics. For induction of ICD in lung cancer cells high-hydrostatic pressure (HHP) was used. Treatment by HHP induces expression of immunogenic markers CRT, HSP70 and HSP90 on the cell surface. HHP also induces secretion of ATP to the extracellular milieu. Dendritic cells (DC) pulsed with HHP-treated tumor cells showed fenotypic maturation characterized by upregulation of maturation molecule CD83, costimulation molecules CD80 and CD86, chemokine receptor CCR7 and MHC class II molecule HLA-DR. Pulsed DCs have also higher rate of phagocytosis of HHP-treated tumor cells and they induce lower numbers of regulatory T cells compared to immature DCs. Moreover, activation of caspases (-8, -9, -3) and other proteins (phosphorylation of eIF2α) which are crucial in ER-stress mediated apoptotic pathway, was observed after HHP treatment. Using wide range of methods it was confirmed that HHP treatment is able to induce ICD in lung cancer cell lines, fenotypic and functional characteristics were described and the decreased induction of regulatory T-lymphocytes...
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Lectin receptor-ligand interaction important in experimental tumor therapy
Grobárová, Valéria ; Černý, Jan (advisor) ; Filipp, Dominik (referee) ; Krulová, Magdaléna (referee)
Lectin-saccharide interactions are involved in many biological processes essential for the survival and proper function of multicellular organisms. C-type lectin-like receptors, predominantly expressed by cells of the innate immune system, recognize saccharide structures on microbes and also aberrant glycosylation pattern of cancer cells. The NKR-P1 receptor family was among the first natural killer (NK) receptor families that were identified, however ligands for some of members remain still elusive. Recently, publications describing N-acetylglucosamine-terminated oligosaccharide structures as possible ligands for NKR-P1 receptor have been subjects for correction/retractions after investigation of the Ethical Committee of the Institute of Microbiology, ASCR, v. v. i. and Charles University in Prague. Re-evaluation of glycodendrimer effect, particularly effect of N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P), revealed mostly indirect role of NK cells on modulation of immune responses. Properly folded soluble recombinant rat NKR-P1A and mouse NKR-P1C lack binding activity to neoglycoproteins modified with GlcNAc-terminated structures. Moreover, new possible target cell populations (NKT cells and macrophages) for saccharide binding were identified.
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The community and protection of the environment from the legal point of view
Černý, Jan ; Žákovská, Karolina (advisor) ; Snopková, Tereza (referee)
The aim of this thesis is to analyse position and possibilities of a municipality in the Czech Republic in the field of environmental protection. The thesis is divided into six basic parts. The Introduction briefly illustrates the importance of municipalities and explains the system of this thesis. Chapter One deals with the environment, especially with its protection under the Czech constitutional law. Municipalities and their bodies are characterised in Chapter Two. The next chapter describes the town and country planning and examples of bylaws issued by a municipality within its separate power in order to protect the environment. Chapter Four introduces the legal instruments which can be used by a municipality in environmental protection within delegated power. In the Conclusion, the author draws attention on several problems connected with participation of municipalities in environmental protection, nevertheless he highlights that municipalities play an unquestionable role in this field.
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TAL Effectors: Tools for DNA Targeting
Jankele, Radek ; Svoboda, Petr (advisor) ; Černý, Jan (referee)
Two decades of research on interactions between Xanthomonas phytopathogenic bacteria and their hosts resulted in discovery of a novel Transcription Activator-Like Effector (TALE) protein family, which confers bacterial virulence in plants. TALEs bind selectively to plant promoters and activate expression of cognate genes enabling bacterial reproduction and dissemination. TALEs mediate recognition of specific promoter boxes in a simple and predictable manner. The TALE central repeat domain contains tandem repeats, which specifically contact single consecutive nucleotides in the target sequence via polymorphic amino acid residues. Repeats stack together in an unique right-handed superhelical assembly, which wraps around the DNA duplex. Validated TALE-DNA binding code shows, that two polymorphic amino acids NI, HD, NH, NG and NN in each repeat mediate recognition of A, C, G, T and A/G, respectively. The order of repeats determines recognized sequence in DNA sense strand. Custom TALE DNA-binding domains with desired specificities can be created within one week at low cost. Such designed domains fused to nuclease or activation domains are useful in research, biotechnology and gene-therapy for targeted gene editing and gene regulation. Notably, gene editing with custom-designed TALE nucleases (TALENs) allows for...
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The significance of autophagy and its communication with the apoptotic machinery for cellular survival or cell death
Pazour, Vítězslav ; Anděra, Ladislav (advisor) ; Černý, Jan (referee)
Autophagy is a cellular proces, taht allows degradation of a portion of cytoplasm, protein aggregates or entire organelles. Major function of autophagy is the maintainance of cellular homeostasis, the protection against stress and mobilization of internal resources. However, autophagy also has a role in imunity, development and differentiation. Autophagic signaling can interact with apoptotic machinery at several levels via regulatory proteins of both pathways or via mutual degradation or cleavage of the components of both pathways. Autophagy can communicate with both extrinsic and intrinsic pathways of apoptosis. Under certain circumstances can autophagy by itself also induce cell death. Autophagic cell death called also programmed cell death of type II is accompanied by massive vacuolization and lysosomal autodestruction of the affected cell. Autophagic cell death was documented during Drosophila development but also in mammalian cells. Autophagy also play importamt role in tumorogenesis, where it can either protect tumor cells against various stresses or it can contribute to their death. Further research of autophagic signaling and mechanisms of communication between autophagy and apoptosis may ont only extend our knowledge on these essential processes but can also contribute to cancer therapy. Powered...
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Impact of Toll-like receptor 4 polymorphism on pro-inflammatory responsiveness in great tit (Parus major)
Vinklerová, Jitka ; Bryja, Josef (advisor) ; Černý, Jan (referee)
Toll-like receptor 4 (TLR4) belongs among chief bacteria-sensing Pattern recognition receptors. Endotoxin (lipopolysaccharide, LPS) recognition by TLR4 triggers signalling leading to release of cytokines that direct leukocyte infiltration into the inflammatory site and cause swelling. Effector mechanisms that ensure pathogen elimination include phagocytosis and oxidative burst. It has been repeatedly reported that the polymorphism in TLR4 may affect host resistance to various diseases. TLR4 may be, therefore, an important molecule in host-parasite co-evolution. Herein, I focused on TLR4 amino acid substitution Q549R which is associated with ornamentation in great tits. In tits I describe immune responsiveness to LPS stimulation on morphological and molecular level and examine effects of the Q549R substitution on inflammation and general body condition. In LPS- treated individuals I found decrease in heterophil-lymphocyte ratio (H/L) that might be caused by attraction of the blood-borne cells into the inflamed tissue. This is in striking contrast with increase in H/L in PBS-treated animals resulting from the stress response. There was no effect of Q549R on general condition and haematological parameters but I revealed a significant effect of the interaction between host Q549R genotype and the type...
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Epigenetic regulation of HLA class II genes and its modification during the lifetime
Lamborová, Věra ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Černý, Jan (referee)
Background: The major histocompatibility complex (MHC) molecules play an important role in the immune response regulation and in the maintenance of the immune homeostasis. Regulation of their expression is therefore a key factor influencing the adaptive immune response. DNA methylation of gene regulatory regions is one of the mechanisms of gene expression control that affects the accessibility of DNA to transcription factors. Ageing is connected with changes in DNA methylation and increased predisposition to autoimmune diseases in older age could be associated with changes in MHC class II genes methylation. Aims: The aim of this diploma thesis is to analyze the methylation profile of DQA1 and DQB1 genes regulatory regions and to compare its differences between the generations and between individual alleles. The next aim is to compare DQA1 mRNA expression between the generations and between single alleles. Methods: DNA and RNA were isolated from blood of three age group donors. DNA was converted by the bisulfite treatment and regulatory regions of HLA class II genes were amplified and cloned into bacteria. Positive clones were sequenced and then analyzed. RNA was reverse transcribed and its expression level was determined by real-time PCR. Results: Statistically significant differences were found by...
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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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