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Inflammatory response induced by cardiac surgery with the use of cardio-pulmonary bypass; the impact of methylprednisolone
Koláčková, Martina ; Krejsek, Jan (advisor) ; Lonský, Vladimír (referee) ; Kročová, Zuzana (referee)
Most elderly people aged over 60 suffer from coronary artery disease (CAD), which can become very likely cause of death. Coronary artery bypass grafting (CABG) represents the standard way of treatment for CAD. CABG surgery is traditionally performed on an arrested heart with the use of cardiopulmonary bypass. The heart is usually accessed through a median sternotomy. Although this kind of surgery strongly stimulates both humoral and cell-mediated immunity, inflammatory response is self-terminating and only a small percentage of patients develop serious postoperative complications, such as organ failure, wound dehiscence, and sepsis. This work focuses on regulatory mechanisms controlling inflammatory response in cardiac surgical patients. It is a retrospective study that follows changes of humoral and cell-mediated immunity induced by cardiac surgery in two different groups of patients. Both groups were operated with the same surgical approach, when identical devices and types of equipment were used; the only difference was the content of 500 mg of methylprednisolone (MP) in priming solution of cardiopulmonary bypass. Blood samples were collected before surgery, after weaning from cardiopulmonary bypass, at the end of surgery, on the 1st , 3rd , and 7th postoperative day. Changes in number of...
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Molecular Mechanisms of the Interaction of Interacellular Pathogen Francisella Tularensis and Antigen Presenting Cells
Härtlová, Anetta ; Kročová, Zuzana (advisor) ; Černý, Jan (referee) ; Krejsek, Jan (referee)
Title of the PhD thesis: The molecular mechanisms of the interaction of Francisella tularensis and antigen presenting cells Abstract: Over the past few decades, reductionist approaches dominated in the study of biological systems and helped to understand many basic principles that underlie host- pathogen interaction. Nevertheless, recent advances in genomics, transcriptomics and proteomics have enabled to design new type of experiments and get thus novel information of all the components of biological systems, as well as to characterize interaction among them. Profiling of proteins, as main carriers of biological activity, is especially of great interest in order to elucidate the molecular mechanisms of infection in space and time. Nowadays, there is a need to develop new antibiotic and anti-viral drugs, extensively investigate the respective infectious agents in regard to pathogen-specific signaling processes or enzymes that do not occur in human cells. However, pathogens have evolved different strategies to avoid the host defense mechanisms. The fact that pathogens exploit many factors of the host cell signaling machinery for finding safe niche for their replication provides the basis for an alternative, host-directed strategy to prevent establishing of infection in a host. This PhD thesis applies...
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Changes in Expression of Membrane Molecules CD200R, CD95, CD95L, and Soluble CD200R Regulating inflammatory Responses in Patients Undergoing Cardiac Surgery
Holmannová, Drahomíra ; Krejsek, Jan (advisor) ; Brát, Radim (referee) ; Turánek, Jaroslav (referee)
Cardiac surgery is known to initiate a complex physiological response with the immune system activation (SIRS), neurohormonal response, metabolic changes, coagulopathies etc. SIRS is triggered by tissue injury, myocardial ischemia, reperfusion, use of anaesthesia, cardioplegia, extracorporeal circuit etc. Excessive immune system activation is associated with progression of SIRS, life-threatening multi-organ dysfunction (MOD), and increased morbidity/mortality in the postoperative period. The immune system response is regulated and terminated by both cellular and humoral regulatory and inhibitory mechanisms including changes in expression of in our study monitored molecules: CD200/CD200R, sCD200R and CD95/CD95L. Methods: The study included the measurement the expression of CD95, CD95L, CD200R, and sCD200R molecules in granulocyte and monocyte populations in blood samples of 30 patients who underwent heart surgery using CPB. Samples collected before surgery, after surgery, and in the postoperative period (1st , 3rd , 7th day) were analysed by flow cytometry and sCD200R by ELISA. Results: We discovered a significant increase in the percentage of granulocytes expressing inhibitory molecule CD200R (from 5% to 17.8%) instantly after surgery. It might be presumed that these cells are less susceptible to...
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Molecular mechanisms of Francisella tularensis pathogenesis
Fabriková, Daniela ; Beránek, Martin (advisor) ; Krejsek, Jan (referee)
Title of Ph.D. thesis: Molecular mechanisms of Francisella tularensis pathogenesis Key words: Francisella tularensis, pattern recognition receptors, inhibition, TRAF6 and TRAF3 complexes Annotation Francisella tularensis is a highly infectious intracellular pathogen and the causative agent of the disease called tularemia. An important aspect of Francisella tularensis virulence represents the capacity to subvert the host immune response by inhibiting or disrupting of the innate immune cell functions. The initial stage of infection is characterized by the massive bacterial replication without apparent inflammatory response, which is crucial for the development of effective host defense against invading pathogen. The aim of this Ph.D. thesis was to describe the early pattern recognition receptors (PRR) signaling response to Francisella tularensis subsp. holarctica LVS (F. tularensis LVS) in primary bone marrow- derived macrophages. The obtained data show the capacity of F. tularensis LVS to simultaneously activate and suppress Toll-like receptors, RIG-I-like receptors, and cytosolic DNA sensors signaling pathways. F. tularensis LVS modulates these PRR pathways by the suppression of K63-linked polyubiquitination events and by the inhibition of the assembly of TRAF6 and TRAF3 signaling complexes. The use of the...
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Identification of new virulence factors in intracellular pathogen Francisella tularensis
Daňková, Věra ; Szotáková, Barbora (advisor) ; Krejsek, Jan (referee) ; Prokešová, Ludmila (referee)
ABSTRACT Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Věra Daňková Supervisor: Doc. Ing. Barbora Szotáková, Ph.D. Title of Doctoral Thesis: IDENTIFICATION OF NEW VIRULENCE FACTORS IN INTRACELULLAR PATHOGEN FRANCISELLA TULARENSIS This dissertation thesis is focused on the role of bacterial tetratricopeptide repeat-like (TPR-like) proteins in the pathogenesis of infection. As a model organism we have chosen highly virulent intracellular bacterium Francisella tularensis (F. tularensis), whose mechanism of pathogenesis is not completely described. In the first part of dissertation thesis we took advantage of bioinformatic methods and identified three genes (FTS_0201, FTS_1680, and FTS_0778) with predicted TPR-like domains. Mutants defective in protein expression were prepared by TargeTron insertion mutagenesis. Prepared mutant strains were used for studying the role of selected proteins in pathogenicity and immunogenicity of F. tularensis subsp. holarctica strain employing in vivo and in vitro models and further for studying the involvement of these proteins in stress tolerance. Our results showed that the FTS_1680 protein is required for intracellular replication and full virulence of bacterium. We also described impaired ability...
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Tau protein, a biomarker of Alzheimer's disease: in vitro phosphorylation and tau-reactive antibodies characterization
Hromádková, Lenka ; Bílková, Zuzana (advisor) ; Fialová, Lenka (referee) ; Krejsek, Jan (referee)
Tau protein, a microtubule-associated protein localized in axonal projections of neurons, is a key molecule in the pathology of Alzheimer's disease (AD), the most common cause of dementia in the elderly population. Tau belongs to the group of natively unfolded proteins without globular structure and is prone to numerous posttranslational modifications (PTMs). Under pathological conditions, abnormal PTMs and misfolding of tau protein occurs and leads to oligomerization and aggregation into paired helical filaments forming neurofibrillary tangles, the histopathological hallmark of AD. Currently available drugs applied in AD treatment can only slow the disease progression and those, which halt the AD-specific neurodegenerative processes, are still missing. Very promising and evolving therapeutic approach is immunotherapy, and even immunomodulation by administration of intravenous immunoglobulin (IVIG) products, a reservoir of natural antibodies from the plasma of healthy donors, has been already tested. The discovery of naturally occurring antibodies directed to tau (nTau-Abs) in body fluids of both AD and healthy subjects and their presence in IVIG begin the investigation of their therapeutic potential. Considering a wide range of possible modifications of tau and of various tau species (oligomers,...
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Changes in Expression of Membrane Molecules CD200R, CD95, CD95L, and Soluble CD200R Regulating inflammatory Responses in Patients Undergoing Cardiac Surgery
Holmannová, Drahomíra ; Krejsek, Jan (advisor) ; Brát, Radim (referee) ; Turánek, Jaroslav (referee)
Cardiac surgery is known to initiate a complex physiological response with the immune system activation (SIRS), neurohormonal response, metabolic changes, coagulopathies etc. SIRS is triggered by tissue injury, myocardial ischemia, reperfusion, use of anaesthesia, cardioplegia, extracorporeal circuit etc. Excessive immune system activation is associated with progression of SIRS, life-threatening multi-organ dysfunction (MOD), and increased morbidity/mortality in the postoperative period. The immune system response is regulated and terminated by both cellular and humoral regulatory and inhibitory mechanisms including changes in expression of in our study monitored molecules: CD200/CD200R, sCD200R and CD95/CD95L. Methods: The study included the measurement the expression of CD95, CD95L, CD200R, and sCD200R molecules in granulocyte and monocyte populations in blood samples of 30 patients who underwent heart surgery using CPB. Samples collected before surgery, after surgery, and in the postoperative period (1st , 3rd , 7th day) were analysed by flow cytometry and sCD200R by ELISA. Results: We discovered a significant increase in the percentage of granulocytes expressing inhibitory molecule CD200R (from 5% to 17.8%) instantly after surgery. It might be presumed that these cells are less susceptible to...
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Proteome analysis of secerned proteins of Francisella tularensis
Konečná, Klára ; Hernychová, Lenka (advisor) ; Krejsek, Jan (referee) ; Bílková, Zuzana (referee)
Title of dissertation thesis: Proteome analysis of secerned proteins of Francisella tularensis The facultative intracellular bacterium Francisella tularensis is the causal agent of the infectious disease called tularaemia. Despite the available wide range of new knowledge focused on bacterium Francisella, till now, mechanisms of tularaemia disease pathogenesis were not completely clarified. The contents of our work was based upon analysis and identification of culture filtrate proteins of bacterium F. tularensis of three strains (LVS, FSC00, SchuS4). Among identified proteins, there were seeked protein candidates for secretion and proteins, which could help with explanation of molecular mechanisms of disease pathogenesis caused by F. tularensis. The best protein candidate for secretion is enzyme acid phosphatase with proven important role in bacterium F. tularensis escape from phagosome. The attention was also focused on the new described mechanism of bacterial secretion, mediated by membrane vesicles. By the help of transmission electron microscopy was demonstrated, that F. tularensis of the strains LVS and FSC200 secretes membrane vesicles into extracellular milieu. Key words: Francisella tularensis, secretion systems, cultivation filtrate proteins, secreted proteins, outer membrane vesicles
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Inflammatory response induced by cardiac surgery with the use of cardio-pulmonary bypass; the impact of methylprednisolone
Koláčková, Martina ; Krejsek, Jan (advisor) ; Lonský, Vladimír (referee) ; Kročová, Zuzana (referee)
Most elderly people aged over 60 suffer from coronary artery disease (CAD), which can become very likely cause of death. Coronary artery bypass grafting (CABG) represents the standard way of treatment for CAD. CABG surgery is traditionally performed on an arrested heart with the use of cardiopulmonary bypass. The heart is usually accessed through a median sternotomy. Although this kind of surgery strongly stimulates both humoral and cell-mediated immunity, inflammatory response is self-terminating and only a small percentage of patients develop serious postoperative complications, such as organ failure, wound dehiscence, and sepsis. This work focuses on regulatory mechanisms controlling inflammatory response in cardiac surgical patients. It is a retrospective study that follows changes of humoral and cell-mediated immunity induced by cardiac surgery in two different groups of patients. Both groups were operated with the same surgical approach, when identical devices and types of equipment were used; the only difference was the content of 500 mg of methylprednisolone (MP) in priming solution of cardiopulmonary bypass. Blood samples were collected before surgery, after weaning from cardiopulmonary bypass, at the end of surgery, on the 1st , 3rd , and 7th postoperative day. Changes in number of...
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