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Monitoring of mitochondria in living cells using fluorescence methods
Taliánová, Eliška ; Čejková, Darina (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the topic of monitoring mitochondria and evaluating their activity. The structure and function of cellular mitochondria are briefly discussed. Contains a list of commonly available fluorescent dyes. It investigates the possibilities of influencing mitochondria in vitro. It deals with the influence of mitochondrial membrane potential by means of various concentrations of glucose and subsequently evaluates mitochondrial activity using the dye JC-10.
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Monitoring of mitochondria in living cells using fluorescence methods
Taliánová, Eliška ; Čejková, Darina (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the topic of monitoring mitochondria and evaluating their activity. The structure and function of cellular mitochondria are briefly discussed. Contains a list of commonly available fluorescent dyes. It investigates the possibilities of influencing mitochondria in vitro. It deals with the influence of mitochondrial membrane potential by means of various concentrations of glucose and subsequently evaluates mitochondrial activity using the dye JC-10.
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Application of Capillary Electrophoresis in Forensic Molecular Genetic Investigation
Grafnetterová, Anna ; Doubková, Klára (advisor) ; Coufalová, Pavla (referee)
5 Abstract The capillary electrophoresis (CE) is an analytical method based on the principle of the traditional plate electrophoresis. Modern ways of fluorescence marking and the detection result in the higher reliability of analyses as well as in more precise results. The utilization of this method is wide within a lot of science fields. In special cases this method can even replace methods used so far, i.e. HPLC. The CE is mainly used in the forensic investigation in particular for person identification based on the DNA analysis of the even minimal sample amount. The STR analysis is an example of such examination. It uses short tandem repeats with high variability within the population. Commercial companies e.g. Promega Corporation, Applied Biosystems etc., develop corresponding instrumentation, software and kits which are important for receiving correct data. As a final result it is obtained DNA profile evaluated by comparison of samples from the "crime scene" with the reference samples. Besides the using in the human domain the DNA profiles are often used in the veterinary practice, in animal (cats, dogs, horses, cattle etc.) raising or breeding, for improving the particular characteristics. Key words: capillary electrophoresis fluorescent labeling STR STR analysis DNA profil
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Development of model system for study of bacterial adhesion on lung epithelium of CF patients
Nosková, Libuše ; Hodek, Petr (advisor) ; Švédová, Martina (referee)
Cystic fibrosis is an inherited disease bearing a number of health difficulties. The main complication is a chronic colonization and infection of respiratory tract with specific microorganisms - especially Pseudomonas aeruginosa. The lung infection with this microorganism is the most common cause of all of death in these patients. The colonization of respiratory tract is mediated by the series of adhesive structures such as lectin PA-IIL. Currently, the most widely used therapy is an antibiotic treatment. Due to the increasing resistance to antibiotics another methods for treatment are being searched. One possibility is a passive immunization of patients with chicken antibodies. For this purpose, we prepared antibodies against one of the adhesive structures of P. aeruginosa - recombinant lectin PA-IIL. These antibodies be able to recognize a native lectin PA-IIL, expressed by P. aeruginosa. To test the ability of antibodies to prevent adhesion of bacteria on the lung epithelial cells a suitable model system was necessary to develop. The basal components of this system include epithelial cells and P. aeruginosa. Epithelial cells from airways of cystic fibrosis patient were isolated by two methods. One method is based on the direct isolation from the dissected tissue and the second one is a brushing...
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Monitoring of mitochondria in living cells using fluorescence methods
Taliánová, Eliška ; Čejková, Darina (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the topic of monitoring mitochondria and evaluating their activity. The structure and function of cellular mitochondria are briefly discussed. Contains a list of commonly available fluorescent dyes. It investigates the possibilities of influencing mitochondria in vitro. It deals with the influence of mitochondrial membrane potential by means of various concentrations of glucose and subsequently evaluates mitochondrial activity using the dye JC-10.
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Monitoring of mitochondria in living cells using fluorescence methods
Taliánová, Eliška ; Čejková, Darina (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the topic of monitoring mitochondria and evaluating their activity. The structure and function of cellular mitochondria are briefly discussed. Contains a list of commonly available fluorescent dyes. It investigates the possibilities of influencing mitochondria in vitro. It deals with the influence of mitochondrial membrane potential by means of various concentrations of glucose and subsequently evaluates mitochondrial activity using the dye JC-10.
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Preparation of recombinant forms of the extracellular part of mouse leukocyte receptors from NKR-P1 family.
Adámek, David
Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.
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Effect of binding of a fluorescent label on the protein structure and function
Petrovová, Gabriela ; Kavan, Daniel (advisor) ; Martínek, Václav (referee)
Fluorescent labeling is a method used for visualization of various types of biomolecules including proteins and protein complexes. However, the effect of protein labeling on protein structure and functions has not been investigated so far. The goal of the diploma thesis was to examine an influence of NHS-fluorescein binding on structure and function of human carbonic anhydrase I (hCA-I). The particular aims of this work were to prepare recombinant 15N-hCA-I which was used for NMR structure analysis of carbonic anhydrase upon fluorescent labeling. Furthermore, enzyme activity was measured in order to find out a correlation between the concentration of NHS- fluorescein and protein function. In addition, the reaction mixtures were systematically analyzed by ESI FT-ICR mass spectrometry. The analysis revealed experimental conditions for fluorescent labeling of human carbonic anhydrase I with minimal effect on protein structure and function. The results of this study show that the calculation of molar excess of NHS-fluorescein cannot rely on a simple procedure provided by manufacturer. However, due to decrease of enzyme activity upon fluorescent labeling, it is better to take into count the influence of NHS-fluorescein concentration on the relative enzymatic activity. Moreover, the calculation of molar...
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Preparation of recombinant forms of the extracellular part of mouse leukocyte receptors from NKR-P1 family.
Adámek, David
Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.
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Preparation of recombinant forms of the extracellular part of mouse leukocyte receptors from NKR-P1 family.
Adámek, David ; Man, Petr (advisor) ; Brdička, Tomáš (referee)
Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.
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