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Stress resistance of bacterial pure cultures and mixed cultures
Chorvátová, Michaela ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
The purpose of this bachelor thesis was to investigate a stress resistance of bacterial monocultures and defined mixed culture. The theoretical part contains a literary review, which deals with the general stress resistance of bacteria and the dynamics of their growth. Furthermore, polyhydroxyalkanoates are introduced, as well as their involvement into stress response of bacteria. In this review was also elaborated the flow cytometry, significant modern method, which was important part of the experiment. In the experimental part, the resistance to osmotic and temperature stress was studied. For this purpose, monocultures of the PHA accumulating strain Cupriavidus necator H16 and the mutant strain Cupriavidus necator H16/PHB-4 unable to accumulate PHA were used. The defined mixed culture was prepared by cocultivating these two strains. Overall, the accumulation and utilization of PHA, namely poly (3-hydroxybutyrate), increased the resistance of bacteria. The most notable was the increase in the case of osmotic stress, when salt concentrations did not significantly reduce the viability of Cupriavidus necator H16 against a mutant strain which viability decreased with growing intensity of stress. On the other hand, experimental temperatures did not have a significant effect on cell viability. The highest growth of bacteria was recorded, in most cases, at their temperature´s optimum of 30 °C.
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Biological role of PHA cycle in bacterium Rhodospirillum rubrum
Šarköziová, Patrícia ; Obruča, Stanislav (referee) ; Slaninová, Eva (advisor)
The diploma thesis deals with the study of polyhydroxyalkanoates (PHAs) production and their protective effects during exposure of the bacterial strain Rhodospirillum rubrum to various stress factors. The theoretical part of this work is focused on the characterization of PHAs and used bacterial strain. In the first part of the experimental work carbon substrates for biomass growth and PHA production in different time intervals (24, 48, 72 and 96 hours) were investigated. Acetate, fructose, hexanoate and malate served as a carbon source. The second part of this work was focused on the selection of a suitable fluorescent probe for the bacterium Rhodospirillum rubrum (propidium iodide, SYTOXTM, fluorescein diacetate), which was used to determine the viability of bacterial cells using flow cytometry. This method was used to determine the viability of bacterial cells after their exposure to various stress factors (thermal stress, freezing and thawing, ethanol stress, osmotic stress, peroxide stress, pH stress and UV stress). Finally, the growth curve of bacteria Rhodospirillum rubrum was determined by measurement of the optical density.
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Biological role of PHA cycle in bacterium Rhodospirillum rubrum
Šarköziová, Patrícia ; Obruča, Stanislav (referee) ; Slaninová, Eva (advisor)
The diploma thesis deals with the study of polyhydroxyalkanoates (PHAs) production and their protective effects during exposure of the bacterial strain Rhodospirillum rubrum to various stress factors. The theoretical part of this work is focused on the characterization of PHAs and used bacterial strain. In the first part of the experimental work carbon substrates for biomass growth and PHA production in different time intervals (24, 48, 72 and 96 hours) were investigated. Acetate, fructose, hexanoate and malate served as a carbon source. The second part of this work was focused on the selection of a suitable fluorescent probe for the bacterium Rhodospirillum rubrum (propidium iodide, SYTOXTM, fluorescein diacetate), which was used to determine the viability of bacterial cells using flow cytometry. This method was used to determine the viability of bacterial cells after their exposure to various stress factors (thermal stress, freezing and thawing, ethanol stress, osmotic stress, peroxide stress, pH stress and UV stress). Finally, the growth curve of bacteria Rhodospirillum rubrum was determined by measurement of the optical density.
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Entry of bacteria Mycobacterium bovis BCG into B lymphocytes
Šamajová, Marianna ; Konečná, Klára (advisor) ; Kubelková, Klára (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...
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Entry of bacteria Mycobacterium bovis BCG into B lymphocytes
Šamajová, Marianna ; Konečná, Klára (advisor) ; Kubelková, Klára (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...
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Microvesicle and exosome detection in immune-related diseases
Šťastná, Evelína ; Drbal, Karel (advisor) ; Fabišik, Matej (referee)
Exosomes (ES) and microvesicles (MV), collectively called extracellular vesicles (EV), are submicroscopic vesicles encapsulated by a phospholipid bilayer. Smaller ES (40 - 100 nm) originate in endosomal compartment, while larger MV (50 - 1000 nm) shed from cell plasma membrane. EV are secreted by all types of cells. They consist of lipids and proteins, but their composition varies according to the cell they originate from. In addition, they differ in the cargo they transport (DNA, RNA and proteins). They occur in every bodily fluid in much higher amounts compared to the original cells themselves, what makes them an attractive and accessible biomarker of autoimmunity diseases, cardiovascular diseases or tumours. For detection of EV, sensitive flow cytometry (FCM) is used, which I am going to compare to alternative methodologies. Part of this work will be description of EV biogenesis and then I will focus on the role of EV in coagulation and inflammation related to autoimmune diseases, more specifically in rheumatoid arthritis (RA).
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Entry of bacteria Mycobacterium bovis BCG into B lymphocytes
Šamajová, Marianna ; Konečná, Klára (advisor) ; Kubelková, Klára (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...
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The use of molecular-biology methods (QRT-PCR) and immunocytological methods (flow cytometry and immunocytochemistry) for the detection of minimal residual disease in neuroblastoma
Grüncveigová, Veronika ; Vícha, Aleš (advisor) ; Daňková, Pavlína (referee)
With a continuous development of molecular-biology methods more attention has been paid to molecular detection of minimal residual diseases in solid tumors. In our study we focused on detection of MRD in neuroblastoma. Neuroblastoma is one of the peripheral neuroblastic tumors (pNTs) that accounts approximately for10 percent of all childhood cancers. The question raised however not answered until this day is whether evidence of MRD in bone marrow may be used as independent prognostic factor in diagnosis of neuroblastoma. Furthermore, it is important to establish what kind of testing technique should be used and what values to look at. There exist various methodologies in detection of MRD evidence in neuroblastoma. These methods differ in cost and complexity, but mainly some of them are more specific and sensitive than the other. Cancer cells may be detected in the blood as well as in the bone marrow. Very often it is the bone marrow that is affected by the metastasis in neuroblastoma, therefore 85% of all high risk neuroblastomas show positive results in the standard cytomorphology tests of bone marrow. Low numbers of cancer cells in bone marrow or peripheral blood (especially during or after the end of treatment) are below the standard values of detection limit in most of the classic methodologies...
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Stress resistance of bacterial pure cultures and mixed cultures
Chorvátová, Michaela ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
The purpose of this bachelor thesis was to investigate a stress resistance of bacterial monocultures and defined mixed culture. The theoretical part contains a literary review, which deals with the general stress resistance of bacteria and the dynamics of their growth. Furthermore, polyhydroxyalkanoates are introduced, as well as their involvement into stress response of bacteria. In this review was also elaborated the flow cytometry, significant modern method, which was important part of the experiment. In the experimental part, the resistance to osmotic and temperature stress was studied. For this purpose, monocultures of the PHA accumulating strain Cupriavidus necator H16 and the mutant strain Cupriavidus necator H16/PHB-4 unable to accumulate PHA were used. The defined mixed culture was prepared by cocultivating these two strains. Overall, the accumulation and utilization of PHA, namely poly (3-hydroxybutyrate), increased the resistance of bacteria. The most notable was the increase in the case of osmotic stress, when salt concentrations did not significantly reduce the viability of Cupriavidus necator H16 against a mutant strain which viability decreased with growing intensity of stress. On the other hand, experimental temperatures did not have a significant effect on cell viability. The highest growth of bacteria was recorded, in most cases, at their temperature´s optimum of 30 °C.
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