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Influence of freezing and thawing process on cryopreserved cells nuclei and surfaces. Functions and physico-chemical properties of cryoprotectants.
Golan, Martin ; Kratochvílová, Irena (advisor) ; Raška, Milan (referee) ; Schneider, Bohdan (referee)
1 Abstract: Cryopreservation of cells is a complex process with many useful applications in basic biological research, medicine and agriculture. In this work we deepened the current understanding of the cryopreservation process both at physical and biological level. Results include characteristics of selected cryoprotectants (primarily DMSO, trehalose, antifreeze protein ApAFP752) in liquid phase, during phase transition and in solid phase, as well as their impact on cryopreserved cells states. Specifically, the level of cell viability, state of cell membrane and condition of cell nucleus (nuclear membrane, chromatin condensation, DNA strand breaks) are monitored over several time points after thawing. It is shown that S-phase cells (NHDF and MCF7 lines) suffer massive collapse of replication forks during cryopreservation which makes them much less suitable for cryopreservation than cells in other phases of the cell cycle. Several methods (most importantly Atomic Force Microscopy, Confocal Fluorescence Microscopy and Flow Cytometry) were used to examine the post-thaw state of cryopreserved cells. The acquired insights into cryodamage of cells can lead to optimization of current cryopreservation protocols and to more thorough evaluation of efficacy of future novel cryoprotectants.
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Mesenchymal stem cells and their regenerative and immunomodulatory potential
Brychtová, Michaela ; Lysák, Daniel (advisor) ; Horák, Vratislav (referee) ; Raška, Milan (referee)
Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...
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Influence of freezing and thawing process on cryopreserved cells nuclei and surfaces. Functions and physico-chemical properties of cryoprotectants.
Golan, Martin ; Kratochvílová, Irena (advisor) ; Raška, Milan (referee) ; Schneider, Bohdan (referee)
1 Abstract: Cryopreservation of cells is a complex process with many useful applications in basic biological research, medicine and agriculture. In this work we deepened the current understanding of the cryopreservation process both at physical and biological level. Results include characteristics of selected cryoprotectants (primarily DMSO, trehalose, antifreeze protein ApAFP752) in liquid phase, during phase transition and in solid phase, as well as their impact on cryopreserved cells states. Specifically, the level of cell viability, state of cell membrane and condition of cell nucleus (nuclear membrane, chromatin condensation, DNA strand breaks) are monitored over several time points after thawing. It is shown that S-phase cells (NHDF and MCF7 lines) suffer massive collapse of replication forks during cryopreservation which makes them much less suitable for cryopreservation than cells in other phases of the cell cycle. Several methods (most importantly Atomic Force Microscopy, Confocal Fluorescence Microscopy and Flow Cytometry) were used to examine the post-thaw state of cryopreserved cells. The acquired insights into cryodamage of cells can lead to optimization of current cryopreservation protocols and to more thorough evaluation of efficacy of future novel cryoprotectants.
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Mesenchymal stem cells and their regenerative and immunomodulatory potential
Brychtová, Michaela ; Lysák, Daniel (advisor) ; Horák, Vratislav (referee) ; Raška, Milan (referee)
Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...
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Elements of Immune Fitness
AbuAttieh, Mouhammed O. ; Krejsek, Jan (advisor) ; Raška, Milan (referee) ; Kročová, Zuzana (referee)
1 Charles University in Prague Faculty of Medicine in Hradec Kralove Elements of Immune Fitness Mouhammed O. Abuattieh Abstract of the thesis Doctoral study programme Medical Immunology Hradec Kralove 2013 2 Dissertation thesis was written during combined doctoral study (PhD) study programme Medical Immunology at the Department of Immunology and Allergy, Faculty of Medicine in Hradec Králové, Charles University in Prague and Transplantation Biology Programme, Department of Surgery, Mayo Clinic in Rochester, MN, USA. Author: Mouhammed O. Abuattieh M.D Department of Ophthalmology, Washington University Saint Louis MO, USA. Supervisor: Prof. Jan Krejsek, Ph.D. Chairperson, Department of Immunology and Allergy, Faculty of Medicine in Hradec Králové, Charles University in Prague, Faculty Hospital Hradec Králové, Czech Republic Consultant-Supervisor: Assoc. Prof. Marilia Cascalho M.D., Ph.D. Transplantation Biology Program, University of Michigan, Ann Arbor MI, USA Opponents: Assoc. Prof. Milan Raška, MD., Ph.D. University Palacky, Faculty of Medicine, Institute of Immunology, Olomouc, Czech Republic Assoc. Prof. Zuzana Kročová, Ph.D. University of Defence, Faculty of Military Medicine, Institute of Molecular Pathology Hradec Králové, Czech Republic This thesis will be defended at September 2013 at Department of...
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Francisella Tularensis infection of macorphage-like cell line J774.2 - changes of surface and intracellular molecules
Novosad, Jakub ; Krčmová, Irena (advisor) ; Raška, Milan (referee) ; Kročová, Zuzana (referee)
Summary: Background: Francisella tularensis (F.t.) is a facultative intracellular bacteria, enrolled at the list of Centre for Disease Control (CDC) as a high risk bioterrorism agent, category A. There is a long-term effort to understand to the immunopathogenesis of F.t. infection. The aim of our study was focused on phenotype analysis (CD54, CD16/32 and CD86) and nitric oxide (NO) production analysis of murine macrophage-like cell line J774.2 durig F.t. live vaccine strain (LVS) in vitro infection. J774.2 cells were either untreated or stimulated either before or after F.t. infection by interferon gamma (IFNγ), lipopolysaccharide (LPS) separately or in its combination. Method: We followed up the expression of cell surface markers and NO production 3, 6, 9, 12 and 24 hours after initiation of infection with or without stimulation using flow cytometry and Griess method respectively. The expression was followed as either absolute value of mean fluorescence index (MFI) or as relative change of MFI (∆MFI). Murine macrophage-like cells (J774.2) were incubated in cultivation flasks (2x106 cells/10ml of medium Dulbecco's MEM with Glutamax-1 with 10% BSA). The cells were activated with 10 or 50 ng of LPS / 1ml of medium or with 100 or 1 000 I.U. of IFNγ / 1ml of medium separately or in combination (10 ng/ml and 100...
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