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Attempts to Elucidate the Role of SNAP23 in Regulated and Pathological Exocytosis in Pancreatic Acinar Cells Using an Inducible SNAP23 Knockout MouseFernandez, Nestor Alejandro 31 December 2010 (has links)
One contentious issue regarding pancreatic acinar exocytosis concerns which SNAP25 isoform (SNAP23/29/47) mediates the various fusion events in this cell type. Based on dominant-negative over-expression studies, SNAP23 was hypothesized to be the putative isoform mediating apical exocytosis, basolateral exocytosis, and ZG-ZG fusion. Unfortunately, using a SNAP23 KD mouse model, 80% SNAP23 KD was insufficient to manifest any secretion phenotype. A novel syncollin-pHluorin exocytosis imaging technique initially meant to assess which fusion events are perturbed by SNAP23 KD was successfully developed and displayed improvements over previous imaging techniques. The syncollin-pHluorin imaging enabled visualization of apical and basolateral exocytosis as well as sequential ZG-ZG fusions. Combined with spinning disk microscopy, this assay allows 3D live exocytosis imaging with high temporal and spatial resolution. This novel imaging assay will be useful in visualizing apical, basolateral, sequential, and lateral fusion events for future acinar studies.
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Attempts to Elucidate the Role of SNAP23 in Regulated and Pathological Exocytosis in Pancreatic Acinar Cells Using an Inducible SNAP23 Knockout MouseFernandez, Nestor Alejandro 31 December 2010 (has links)
One contentious issue regarding pancreatic acinar exocytosis concerns which SNAP25 isoform (SNAP23/29/47) mediates the various fusion events in this cell type. Based on dominant-negative over-expression studies, SNAP23 was hypothesized to be the putative isoform mediating apical exocytosis, basolateral exocytosis, and ZG-ZG fusion. Unfortunately, using a SNAP23 KD mouse model, 80% SNAP23 KD was insufficient to manifest any secretion phenotype. A novel syncollin-pHluorin exocytosis imaging technique initially meant to assess which fusion events are perturbed by SNAP23 KD was successfully developed and displayed improvements over previous imaging techniques. The syncollin-pHluorin imaging enabled visualization of apical and basolateral exocytosis as well as sequential ZG-ZG fusions. Combined with spinning disk microscopy, this assay allows 3D live exocytosis imaging with high temporal and spatial resolution. This novel imaging assay will be useful in visualizing apical, basolateral, sequential, and lateral fusion events for future acinar studies.
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Biochemistry and physiological role of otoferlin / Biochemie und physiologische Funktion von OtoferlinReuter, Kirsten 10 October 2011 (has links)
No description available.
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Regulace vnitřního pH kvasinek - vliv vybraných transportních proteinů / Regulace vnitřního pH kvasinek - vliv vybraných transportních proteinůZalom, Peter January 2011 (has links)
Intracellular pH affects nearly all biochemical processes in yeast, the processes regulating the cytosolic pH includes function of many transport proteins. In this work, the impact of selected sodium transporters on cytosolic pH has been studied in two yeast species: Saccharomyces cerevisiae and Zygosaccharomyces rouxii including wild-type and mutants with affected sodium transport. Measurements of cytosolic pH and buffering capacity have been performed using fluorescent protein probe pHluorin - a pH sensitive derivate of green fluorescence protein. Several procedures for calibration of pHluorin fluorescence response have been compared and the importance of a proper correction of the calibration curve has been demonstrated. It has been shown that cytosolic pH is influenced by the function of Nha1 transport protein in S. cerevisiae as well as in Z. rouxii but not by Sod2-22 transporter in Z. rouxii. It has been demonstrated that the buffering capacity of cytosol decrease in the presence of glucose in all strains studied.
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Změny vnitrobuněčného pH kvasinek za stresových podmínek / Changes of intracellular pH in yeast cells under stress conditionsDivín, Radek January 2015 (has links)
Title: Changes of intracellular pH in yeast cells under stress conditions Author: Radek Divín Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jaromír Plášek, CSc. Abstract: Specific values of intracellular pH (pHi) can affect all biochemical processes in a cell and this phenomenon is closely connected with the degree of importance of changes in the intracellular pH under the stress conditions. In the Master Thesis, the yeast cells Saccharomyces cerevisiae were used as a model of organism eukaryotic cells. Monitoring of intracellular pH of the cells was performed by the method of synchronous fluorescence scan technique of genetically encoded fluorescent probes pHluorin which was located in the cytosol of the cells. The cells were exposed to stress conditions due to the chemical changes in the environment. Consequently, their ability to maintain a stable value of the intracellular pH in various acidic environments was studied in more detail. The attention was also focused on the impact on optimizing of glucose cytosolic pH. The work was centered on the changes in intracellular pH under the influence of the presence of KCl in suspension. Furthermore, the decrease of cytosolic pH of monitored cells by protonophore CCCP was investigated. The effect of stress environment on the...
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Měření vnitrobuněčné koncentrace iontů v mikroorganismech / The monitoring of intracellular ion concentrations in microbial cellsVodáková, Adéla January 2013 (has links)
The Master Thesis focuses on monitoring of intracellular ion concentrations in bacteria Escherichia coli and yeast Saccharomyces cerevisiae using genetically encoded fluorescent probes with green fluorescent protein (GFP). Aquired knowledge about this protein and its spectral characteristics is summarized in the introduction. For experimental study a pH-sensitive sensor which displays a ratio change of two excitation fluorescence peaks - pHluorin - was chosen. This probe was tested in bacteria and yeast cells. The experiments concentrated on the ability of the cell to maintain a constant cytosolic pH under various conditions like different pH values of the suspension, addition of glucose or KCl to the suspension. Another topic discussed in the thesis is the elimination of the cell autofluorescence from the GFP signal. For this purpose the synchronous fluorescence scan technique was succesfully used. I have found out that by using this method the measurements of cytosolic pH values are even more accurate thanks to the improved signal to noise ratio.
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Analýza zotavování membránového potenciálu kvasinek za stresových podmínek vyvolaných protonoforem CCCP / The analysis of membrane potential recovery in yeast under CCCP-induced stressBabuka, David January 2016 (has links)
The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.
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Synaptic vesicle recycling <i>in Vivo</i> / Das Recycling synaptischer Vesikel <i>in Vivo</i>Denker, Annette 02 November 2011 (has links)
No description available.
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