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The role of membrane microdomains and transmembrane adaptor proteins PRR7 and SCIMP in the regulation of immunoreceptor signaling
Hrdinka, Matouš ; Drbal, Karel (advisor) ; Černý, Jan (referee) ; Kalina, Tomáš (referee)
Dissertation summary The role of membrane microdomains and transmembrane adaptor proteins PRR7 and SCIMP in the regulation of immunoreceptor signaling Matouš Hrdinka How do the plasma membrane microdomains and transmembrane adaptor proteins (TRAPs) influence the outcome of immunoreceptor signaling? These have been the important questions of molecular immunology. In spite of the years of intensive research, these problems remain incompletely understood. The plasma membrane is a highly dynamic heterogeneous bilayer spontaneously organized into microdomains of various size, composition, and lifetime. The lipid rafts are one example of such microdomains and have been implicated in many biological processes, including immunoreceptor signaling. Because rafts are enriched in many signaling proteins, they are believed to function as platforms for signal initiation and propagation. The TRAPs are important organizers and regulators of immunoreceptor signaling. For example, LAT is indispensable in T cell receptor (TCR) signaling and T cell development, PAG for the regulation of Src family tyrosine kinases (SFKs), and NTAL is a multifunctional negative and positive regulator. The presence of these TRAPs in lipid rafts seems to be crucial for their functions, however, is still a matter of debate. Moreover, other so far...
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Studies on immunoreceptor signaling molecules
Ormsby, Tereza ; Hořejší, Václav (advisor) ; Černý, Jan (referee) ; Špíšek, Radek (referee)
A delicate balance in the number, specific type and function of leukocytes is required for proper functionality of the mammalian immune system. Innate immunity, which quickly recognizes pathogens, represents the first line of defense. Later, a more specific response is generated via adaptive immunity. Deregulation of the immune system is manifested by the inability to control infection, development of allergic, autoimmune disorders or even cancer, and ultimately can lead to death. To fulfill their functions, cells develop an intricate network of intra- as well as extra-cellular molecules organized into signaling cascades, which allows them to communicate between each other. Better understanding of the molecular mechanisms of signaling pathways in leukocytes is critical for design of efficient therapies. In this thesis, leukocyte signaling was studied in several aspects. First, the role of adhesion molecules in pathogenesis of cervical cancer and the regulation of their expression was investigated. The second publication describes a new transmembrane adaptor protein (TRAP), called prolin rich 7 (PRR7), as a potentially interesting regulator of signaling and apoptosis in activated T cells. The final publication characterized the role of the Btk kinase downstream of the triggering receptor expressed...
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Adenylate-cyclase toxin of Bordetella pertussis as a marker for the study of the complement receptor CD11b/CD18 endocytosis.
Chvojková, Věra ; Bumba, Ladislav (advisor) ; Černý, Jan (referee)
Bordetella pertussis is an important human pathogen that causes an infection disease called whooping cough. This gram-negative bacterium produces an adenylate cyclase toxin (CyaA) that recognizes an integrin receptor CD11b/CD18 present on the surface of myeloid phagocytes and delivers an adenylate cyclase (AC) domain into the cell cytosol. This thesis deals with the endocytic machinery of CyaA and its potential use as a specific marker for endocytosis of the CD11b/CD18 receptor molecule. Detoxified mutant of CyaA, CyaA-AC- , that has the capacity to promote calcium influx as well the potassium efflux, was shown to trigger activation of the integrin receptor CD11b/CD18 followed with endocytic uptake by clathrin-dependent pathway. On the other side, the inactive mutant CyaA-KP-AC- that is unable to provoke integrin activation was endocytosed by clathrin-independent pathway. These results suggest that the various endocytic pathways of the CD11b/CD18 are determined by different conformational states of the receptor molecule.
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Characterization of 32,33-didehydroroflamycoin - secondary metabolite from Streptomyces durmitorensis
Koukalová, Alena ; Černý, Jan (advisor) ; Konopásek, Ivo (referee)
Streptomycetes are soil filamentous Gram-positive bacteria that produce wide variety of pigments and biologically active substances including macrolides. Some of them are used as very efficient antibiotics and strong antifungal agents in medicine, others have became useful tools for staining biomembranes and detecting cholesterol via their internal fluorescence. Actinomycete Streptomyces durmitorensis (wild type strain MS405T ) is a bacteria isolated from Durmitor National Park in Montenegro soil samples. It produces secondary metabolite that has been identified as 32,33-didehydroroflamycoin (DDHR) closely related to the macrolides roflamycoin and generaly used filipin. DDHR exhibits cytototoxic activity against mammalian cells and yeast Saccharomyces cerevisiae strain EGY48. In addition it has interesting fluorescence properties allowing visualization of some membrane components. DDHR interacts with biomembranes, causes their disintegration leading to changes of the actin and tubulin cytoskeleton organization and in higher concentrations it causes cells necrosis. DDHR-sterol interaction in cell membranes decreases fluorescence intensity of DDHR. The compound is able to fluorescently stain aberrant lysosomes and could be therefore potentially used in diagnostics of some lysosomal storage disease.
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Assessing biochemical properties of PDE8A1: Design of experimental system in living cells"
Galica, Tomáš ; Černý, Jan (advisor) ; Mašek, Tomáš (referee)
4 Abstract Phosphodiesterases (PDEs), enzymes that hydrolyze cyclic nucleotides, are important components of signal transduction pathways in eukaryotic cells. Second messenger 3'-5'- cyclic adenosine monophosphate (cAMP) is hydrolyzed by specific PDEs. By controlling concentration levels of cAMP in cell, PDEs preserve favorable environment for successful transmission of the cAMP signal. Moreover, PDEs are activated by protein kinase A (PKA) in response to elevated cAMP concentration, which is a feature crucial for signal termination. PDE8A1 is a high-affinity cAMP-specific IBMX insensitive phosphodiesterase, an enzyme important for cAMP signaling. However, mostly due to a lack of specific inhibitor, its role has not been assessed in detail. This thesis reports cloning of PDE8A1, identification of its posttranslational modifications and subcellular localization, as well as an alternative approach to address PDE biology by the use of cyclase toxin from Bordetella pertussis. Keywords: phosphodiesterase, cAMP, posttranslational modification, myristoylation, palmitoylation, adenylate cyclase toxin
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Defensins and autoimmunity: emerging alpha-defensin based model to study mechanisms underpinning autoimmune processes
Neuwirth, Aleš ; Filipp, Dominik (advisor) ; Černý, Jan (referee) ; Michálek, Jaroslav (referee)
The process of immune "self-nonself discrimination" is of utmost importance for the survival of all species as the biodestructive force of immune system can be directed towards the host as much as to pathogens. Thus, to shift this balance towards the latter, T cells bearing self- recognizing receptors are removed in the thymus (central tolerance) or their reactivity is harnessed through various additional mechanisms in periphery (peripheral tolerance). If the selfreactive T cells are not deleted and persist in the body, the regulation of self-tolerance can be breached, leading to the onset of autoimmunity. Presented thesis revolved around α-defensins, very effective bactericidal peptides that represent an important part of humoral innate immunity. There are two types of α-defensins: myeloid, expressed predominantly in neutrophils, and enteric, synthesized by intestinal Paneth cells. Data presented inhere are first to characterized the involvement of α-defensin- expressing cells in two types of autoimmune diseases, insulin-dependent diabetes mellitus (T1D) and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). The former relates to the identification of transcriptionally activated myeloid α-defensin- expressing eosinophils present in the thymus of diabetes prone rat. In...
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The role of innate immunity cells in the pathogenesis of celiac disease
Dáňová, Klára ; Palová Jelínková, Lenka (advisor) ; Černý, Jan (referee)
Celiac disease is an autoimmune disease which occurs in susceptible individuals after ingestion of food containing gluten. Gluten and its monomeric fraction gliadin induce inflammatory damage of the small intestine by activating the immune cells that react strongly to gluten peptides. Gluten peptides have the ability to activate cells of adaptive as well as innate immune system. This work is focused on the production of interleukin (IL)-1 in antigen presenting cells stimulated with peptic gliadin digest. We found that monocytes and peripheral blood mononuclear cells (PBMC) isolated from blood of celiac patients secrete significantly more IL-1α and IL-1β than cells of healthy donors after stimulation with gliadin digest. The gliadin-induced IL-1β expression is controlled by a signaling cascade that includes MAPK kinase family molecules and transcription factor NF-κB. Moreover, we found that the adaptor proteins MyD88 and TRIF as well as Toll-like receptor (TLR) 2 and 4 play a role in the signaling cascade underlying gliadin-induced IL-1β expression by using murine bone marrow derived dendritic cells (BMDC). The precursor form of IL-1β in gliadin- stimulated PBMC and murine BMDC is maturated by caspase-1. In celiac PBMC the gliadin- induced maturation and secretion of IL-1β depends on the potassium...
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Studying immune system using MHC II/ EGFP knock-in mouse
Zadražil, Zdeněk ; Černý, Jan (advisor) ; Tlaskalová - Hogenová, Helena (referee)
The immune system is essential for keeping the integrity of multicellular organisms. We were able to make a step forward in studying the complex immune reactions in mammals in vivo and/ or in situ using the major histocompatibility complex (MHC) class II/ enhanced green fluorescent protein (EGFP) knock-in mouse model. Due to the EGFP visualization of MHC II expressing cells we were able to observe antigen presenting cells, which are essential for the onset of immune responses, in their natural environment. Thus, we report some original features of the immune system. We have identified MHC II+ cell clusters with unknown, probably unique function, in the intestine. We have also described MHC II+ cell migration to the lactating mammary gland and tested few hypotheses about the role of this phenomenon for the development of the mammary gland, milk secretion or infant immune system establishment. Lastly, we observed residential macrophages in the cornea. The presence of APCs in the cornea is a very contradictory issue due to the fact that cornea is an immunologically privileged tissue and therefore harbors special immune features. key words: antigen presenting cells (APC), major histocompatibility complex class II (MHC II), enhanced green fluorescent protein (EGFP), immune system, knock-in mouse model
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Mechanisms of retromer - dependent protein recycling from endosomes
Horázná, Monika ; Macůrková, Marie (advisor) ; Černý, Jan (referee)
Most processes in nature are very effective concerning saving energy and minimizing waste. A good example of saving on cellular level is receptor recycling. Whether it concerns receptors for lysosomal enzymes or for proteins destined for secretion, after releasing their cargo protein the fate of the receptor would be sealed in lysosomes. Nevertheless, some transmembrane receptors contain a signal motif through which they are recognized by specific proteins or protein complexes and they escape the degradation in lysosomes. One such complex is the retromer. Its first discovered function was the recycling of receptors for lysosomal hydrolases in yeast. Later it was proved that it has a similar role in transport of many other proteins in other eukaryotes. The task for retromer is to sort the cargo proteins on the endosomal membrane and together with others auxiliary proteins create a transport vesicle which is then transported to the Golgi. This makes the cell able to recycle proteins that would otherwise be transported from endosomes to lysosomes for degradation.
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Mechanisms of MHCII signaling in B lymphocytes
Kotlabová, Klára ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
During the initiation of an antigen-specific immune response, peptide fragments originating from the antigen are presented in complex with MHC class II glycoproteins (MHCgpII) on the surface of the antigen presenting cells (APC). Antigen recognition by T lymphocyte is accompanied by the formation of the molecular structure at the interface with APC called immunological synapse (IS). During this contact, signal transduction is initiated at both, T lymphocyte and APC, sides of the IS. For a long time it was thought that the only function of MHCgpII is presentation of antigen. However, later it was found that stimulation of MHCgpII led to triggering of signals contributing to decision about the further fate of APC. MHCgpII do not have any signaling motifs in their cytoplasmatic domains, and so associated molecules are necessary for the transduction of the signals. This work focuses on B lymphocytes in which the associated molecules are Ig alfa/beta, MPYS, CD19 and CD20. After the stimulation of MHCgpII these proteins mediate signaling events including activation of several families of protein kinases, phospholipase C, mobilization of calcium and activation of transcriptional factors NFAT and AP-1. In B lymphocytes, activities of these pathways may result in proliferation and differentiation but also in the...
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