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Buněčné a molekulární charakteristiky hemolymfy u raků
KIFAYATULLAH, NA
The cellular and molecular components of the hemolymph are the major arm of the innate immune system in decapod crustaceans. In-depth knowledge of the hemolymph components, including hemocytes and hemolymph proteins, can enhance our understanding of innate immunity in crustaceans. We utilized transmission electron microscopy and quantitative proteomics to study the cellular and molecular aspects of coagulation and phagocytosis in the hemolymph. Chapter 2 reviews the cellular and molecular parameters of the innate immune system and the effects of environmental stressors and their abiotic and biotic stress mechanisms in decapod crustaceans. The innate immune system of decapod crustaceans heavily relies on hemocytes in the circulating hemolymph. Generally, three types of hemocytes are accepted based on their morphology, however, there is still a lack of consensus among researchers regarding the classification of hemocyte types. The key innate immune functions such as coagulation and phagocytosis are still poorly understood and require further investigation especially at a molecular level. Environmental stressors can adversely affect the immune responses of decapod crustaceans, increasing their susceptibility to diseases. However, the abiotic stress mechanism is poorly understood due to the lack of available literature and needs further investigation. In Chapter 3, transmission electron microscopy was used to investigate the ultrastructural behavior of hemocytes during coagulation and phagocytosis in the early stages of leg amputation injury in marbled crayfish Procambarus virginalis. The granular hemocytes were the first to be activated, and the morphology of cytoplasmic granules changed from electron-dense to electron-lucent forms in an expanding manner. The transformed granules containing amorphous electron-lucent materials merged and discharged their contents into the extracellular space for coagulation. We observed that the leftover nucleus from degranulated hemocytes participates in the process of coagulation, which could be confused with hyalinocytes in some previous studies. In addition, leg amputation caused massive muscle degeneration, followed by a significant influx of phagocytic hemocytes that removed a substantial amount of muscle fibers and organelles, such as mitochondria, generated from disintegrating and decaying muscle. Furthermore, we found that phagocytic hemocytes contained varying numbers of granules in their cytoplasm and, for the first time, discovered that these cells incorporate necrotic bodies resulting from degenerated muscles into their organelles, such as cytoplasmic granules and nucleus. The granular hemocytes were found to be the main cells that carry out phagocytic activity in the injury site. This study provides a comprehensive description of all the stages of morphological changes in hemocytes during coagulation and phagocytosis after injury in crayfish for the first time. In Chapter 4, proteomic analysis of non-clotted and clotted samples indicated that quantities of most proteins remained unchanged during the coagulation process, suggesting that necessary proteins for coagulation are pre-synthesized and stored before clot formation. Due to their open circulatory system, decapod crustaceans possess robust clotting mechanisms. Upon injury, pre-synthesized clotting factors are released, resulting in clot formation. Therefore, only a few proteins, such as C-type lectin domain-containing proteins, Laminin A chain, and Tropomyosin, were down-regulated during clotting, suggesting their possible roles in the structural integrity of cells. Their downregulation could facilitate degranulation, a crucial step for clot formation.
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Micronuclei and their connection with intracellular innate immunity and viral infection
Knoblochová, Kateřina ; Bruštíková, Kateřina (advisor) ; Španielová, Hana (referee)
Micronuclei are tiny structures that contain nuclear DNA and a membrane derived from the nucleus. They emerge in cells that have been exposed to severe stress factors, such as viral infections, radiation, or genotoxic substances. While micronuclei have long been used as markers of genotoxic stress, the mechanism of their formation and internal processes are not yet fully understood. DNA enclosed inside micronuclei is restructured in an atypical manner, which may induce mutations and accelerate oncogenic transformation of the cell. Due to these processes micronuclei can also act as reservoirs of immunostimulatory nucleic acids, which may potentially be detected by molecular sensors. Therefore, studying micronuclei is significant in relation to the activation of signaling pathways that are part of the innate intracellular immunity. This work summarizes the current knowledge about micronuclei and their connection to innate intracellular immunity and viral infection. Keywords: micronuclei, innate immunity, molecular sensors, chromotripsis
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Polyomavirus minichromosomes: interactions with components of innate imunity
Satratzemis, Christos ; Forstová, Jitka (advisor) ; Trejbalová, Kateřina (referee)
The genome of polyomaviruses is a circular dsDNA (double-stranded DNA) which is associated with cellular histones within virions and during the entire viral replication cycle. Given the structural similarity to eukaryotic chromatin, the complex of polyomaviral DNA with histones is called minichromosome. The chromatin state of minichromosomes influences viral transcription and replication which could be exploited by the host innate immune response. One of the components of innate immunity, that affects viral chromatin, is the non-canonical histone H3.3, its chaperone DAXX- ATRX (death domain associated protein 6-alpha-thalassemia, mental retardation X-linked syndrome) and protein complexes called PML (promyelocytic leukemia protein). In order to trigger the innate immune response, foreign and/or stress molecules have to detected. During mouse polyomavirus (MPyV) infection, the innate immune response is initiated via the DNA sensor cGAS (cyclic GMP- AMP synthase). In this master's thesis, the distribution of histone H3.3, its chaperone DAXX-ATRX and the PML protein was analyzed during infection with MPyV. Using mass spectrometry, the histone was detected within viral chromatin. The data suggest that the chaperone complex and PML are involved in the regulation of H3.3 incorporation into the chromatin...
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Host-microbiota, pro-inflammatory immunity and physiological senescence in wild birds
Těšický, Martin
Triggered by microbial ligands, inflammation serves as a "double-edged sword" to fight infections on the one hand, but on the other hand causing tissue damage due to oxidative stress if it is dysregulated. For example, chronic inflammation can contribute to inflammaging, which is now widely regarded as one of the causes of ageing. In my interdisciplinary dissertation, my colleagues and I investigated three interrelated aspects of inflammation, using an evolutionary framework and various free-living birds as models: (1) ecological and evolutionary determinants of gut microbiota (GM) composition and diversity, a driver of wild bird immunity, (2) diversity in immune genes affecting inflammatory responses in wild birds and (3) inflammation-related physiological senescence in a free-living passerine bird, the great tit (Parus major). Firstly, using 16S rRNA gene metabarcoding, we revealed high intra- and interspecific variation in passerine gut microbiota (GM) dominated by the major phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Although in mammals GM depends strongly on host phylogeny and diet, in birds we found only moderate effects of phylogeny and very limited effects of host geography and ecology on GM composition. While microbiota diverged between the upper and lower...
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Host-microbiota, pro-inflammatory immunity and physiological senescence in wild birds
Těšický, Martin ; Vinkler, Michal (advisor) ; Tschirren, Barbara (referee) ; Štěpánek, Ondřej (referee)
Triggered by microbial ligands, inflammation serves as a "double-edged sword" to fight infections on the one hand, but on the other hand causing tissue damage due to oxidative stress if it is dysregulated. For example, chronic inflammation can contribute to inflammaging, which is now widely regarded as one of the causes of ageing. In my interdisciplinary dissertation, my colleagues and I investigated three interrelated aspects of inflammation, using an evolutionary framework and various free-living birds as models: (1) ecological and evolutionary determinants of gut microbiota (GM) composition and diversity, a driver of wild bird immunity, (2) diversity in immune genes affecting inflammatory responses in wild birds and (3) inflammation-related physiological senescence in a free-living passerine bird, the great tit (Parus major). Firstly, using 16S rRNA gene metabarcoding, we revealed high intra- and interspecific variation in passerine gut microbiota (GM) dominated by the major phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Although in mammals GM depends strongly on host phylogeny and diet, in birds we found only moderate effects of phylogeny and very limited effects of host geography and ecology on GM composition. While microbiota diverged between the upper and lower...
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Mechanisms used by SARS-CoV-2 for escape from innate host defense
Rybová, Lucie ; Němečková, Šárka (advisor) ; Pospíšek, Martin (referee)
SARS-CoV-2 of the Coronaviridae family causes the disease called Covid-19. It has a number of structural, non-structural and accessory proteins that interfere with the host's immune response and help the virus escape this response and survive in the host. The most important escape mechanism is suppression of host interferon function. Molecules causing inhibition of the host's innate immunity are encoded from the region of the virus genome ORF1a and ORF1ab. This work provides a summary of the characterization, morphology, genome, replication cycle, innate immunity mechanisms involved in host defense against infection, and escape mechanisms of SARS-CoV-2. Keywords: SARS-CoV-2, virus, morfology of SARS-CoV-2, SARS-CoV-2 genome, structural proteins, nonstructural proteins, innate immune system escape, IFN dysregulation, TLRs, SARS-CoV-2 variants, Covid-19
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Cellular factors restricting mouse polyomavirus infection in host cells: Studies of PML protein isoforms
Anderová, Karolína ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Promyelocytic leukaemia nuclear bodies (PML NBs) are multifunctional nuclear spherical structures formed by the PML protein shell and other interaction partners that have been described to be involved in many cellular processes and immune defences. In the antiviral immune response, PML NBs and their components act as direct restriction factors as well as in the regulation of the interferon response. On the other hand, viruses have developed antagonistic mechanisms to resist this inhibition. This work deals with the role of PML NBs in infection with model Murine polyomavirus (MPyV) and focuses on the study of PML protein isoforms. The first aim of the work was to analyse the formation of human (hPML) and mouse (mPML) NBs in a mouse embryonic fibroblast (MEF) model. Subsequently, the localization of hPML and mPML NBs during infection was determined. Close localization with viral replication centres was observed for both PML species. In the next step, the effect of infection or interferon α (IFNα) on mPML protein expression was tested. Infection and treatment with IFNα led to an increase in mPML expression at the level of both gene transcription and protein synthesis. At the same time, the data indicated the largest increase in transcription of the mPML3 isoform. The work also addressed the potential...
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Role of peripheral blood monocytes and innate immunity in diabetes
Zinková, Alžběta ; Daňková, Pavlína (advisor) ; Novota, Peter (referee)
Introduction: Diabetes mellitus is a polygenic disease and its development is influenced to some extent by environmental factors as well. Innate immunity triggers nonspecifically first defense reactions after penetration of the pathogen into the body, while overstimulation components of innate immunity may give rise to autoimmune diseases, including diabetes type 1. The components of innate immunity are, among others, Toll-like receptors (TLRs) belonging to a group of the structures recognizing preserved molecular structures characteristic of pathogens. Toll-like receptors are abundantly expressed by monocytes which produce prolactin (PRL) having an immunostimulatory function. To clarify the role of innate immunity in the pathogenesis of diabetes, we focused on the expression of mRNA and protein expression of TLR2 and TLR4. The expression of PRL was studied only at the level of mRNA. Monocytes were separated by flow cytometry into classical (CD14++) and nonclassical (CD14+). We monitored their percentages and the degree of expression of CD14 antigen on their surface.The operational objective of this dissertation was to optimize the stimulation of monocytes for the planned study of the function of non-pituitary prolactin in vitro and determine the appropriateness of the use of healthy donors' buffy...
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POSITIVE AND NEGATIVE HOST FACTORS OF AVIAN SARCOMA AND LEUKOSIS VIRUSES
Krchlíková, Veronika ; Elleder, Daniel (advisor) ; Růžek, Daniel (referee) ; Španielová, Hana (referee)
Identification and characterization of the host cell factors that either support or inhibit virus replication constitutes a major direction in virological research. In this work we focus on several such host factors in the context of avian cell. Chicken Tva, cell entry receptor for subgroups A and K of Avian sarcoma and leukosis virus (ASLV), was identified to be orthologous to human receptor for cellular uptake of cobalamin (Cbl). Here, we describe Cbl uptake in chicken cells and its dependency on Tva. Additionally, we characterize in vivo Tva knockout in chicken. Chicken Tvb receptor conferring susceptibility to subgroups B, D and E of ASLV was previously shown to participate in virus-induced cytopathic effects. In this work, we identify a natural ligand of Tvb and investigate its participation in apoptosis. RIG-I-like receptors (RLR) are a key family of cytosolic viral RNA sensors. The activation of these receptors leads to establishment of an antiviral state in the cell. In this study, we describe repeated evolutionary losses of RLR genes in birds: the loss of MDA5 in two avian orders and the loss of RIG-I in multiple species. Tetherin is an antiviral restriction factor blocking the release of newly formed viral particles. We identify tetherin orthologs in avian species and investigate...
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Inflammation and cancer in germ-free vs. conventionally reared animals
Čaja, Fabián ; Vannucci, Luca Ernesto (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Smrž, Daniel (referee)
Inflammation is considered as one of the main defence mechanisms of the immune system against threats that occur in the body. When present in its acute form, minimal or no detectable subsequent damage of original affected tissue exists. The more pathological form, chronic inflammation, is associated with permanent damage of the tissue and typically a hallmark of various diseases such as ulcerative colitis or colon carcinogenesis. These two pathologies are evolving in the unique colon microenvironment, where intensive interaction between the host cells and bacteria is present. The aim of our study was to investigate the immunological (ELISA, FACS, RT-PCR) and structural (histology, confocal microscopy) changes in the colon mucosa of Wistar-AVN rats induced by dextran sodium sulphate (DSS) to produce colon colitis and by azoxymethane (AOM) to produce colon carcinogenesis. Conventional (CV) and also germ-free (GF) reared animals were used to investigate the effects of the mucosal inflammation activated by the administered inducers as well as the role of colon microbiota - as promoters of a continuous immune activation - in the modulation of immunity and collagen scaffold remodelling. Our results showed that even in the early period after the induction, both inducers produced a smouldering...
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