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LINC complex: The link between chromatin integrity and sperm motility
Šanovec, Ondřej ; Komrsková, Kateřina (advisor) ; Lánská, Eva (referee)
The LINC complex (Linker of the Nucleoskeleton and Cytoskeleton) is a protein structure located in the nuclear membrane that connects the cytoskeleton with the nucleoskeleton. This complex can be found in every mammalian cell including the gametes. However, here the LINC complex is more diverse and less studied than in the somatic cells. In this thesis, the LINC complex and its role in spermiogenesis have been studied in wild-type and Protamine 2 knockout (Prm2-/- ) mice. Protamines are small proteins that replace histones during spermiogenesis. The mouse model generated by the group of prof. Hubert Schorle has a deletion in Prm2 in exon 1 and its sperm possess a surprising phenotype including complete loss of motility. Therefore, it was hypothesized that the LINC complex might be responsible for miscommunication between the sperm head and tail which leads to the loss of sperm motility. Results from this study suggest that the LINC complex is not influenced by Prm2 deletion, however, actin dynamics, cytoskeletal motor proteins and tubulin acetylase/ histone deacetylase activity might be impaired. Prm2-/- sperm have a significantly higher abundance of β-actin compared to the wild type. Next, Prm2-/- sperm also show a different pattern of acetylation of α-tubulin but no change in the abundance of...
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Optimization of electrophoretic determination of protamine and insulin
Molnárová, Katarína ; Křížek, Tomáš (advisor) ; Hraníček, Jakub (referee)
This work deals with optimization of a method for separation and detection of protamine and insulin using capillary zone electrophoresis. The composition of background electrolyte, the solution pH and the injection method were optimized. Citric acid in a concentration range of 80 to 240 mmol L-1 and chloroacetic acid ranging from 50 to 150 mmol L-1 were tested sequentially. The optimized method uses a fused silica capillary with inner diameter of 50 μm. The total length of capillary is 50.0 cm, effective length is 8.5 cm. The injection of the sample is performed on the short end of the capillary. The method uses chloroacetic acid of 100 mmol L-1 concentration as the background electrolyte. Driving voltage is 20 kV. Sample is injected hydrodynamically with a pressure of 5 kPa for 3 s. The analytes are detected spectrophotometrically at wavelength of 200 nm. The method allows for determination in case of protamines in concentration range between 4 μg mL-1 and 300 μg mL-1 and insulin from 5 μg mL-1 to 300 μg mL-1 . The limits of detection are 1 μg mL-1 for protamine and 2 μg mL-1 for insulin. Repeatability of migration times and peak areas tested at 30 μg mL-1 and 200 μg mL-1 concentration levels using hydrodynamic injection showed values of relative standard deviation lower than 6 % suggesting...
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Study of interaction between protamine and heparin and its applicability in capillary electrophoresis
Martínková, Eva ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Heparin is an acid mixture of glycosaminoglycans with high negative charge density which naturally occurs in human body. Due to its ability to bind antithrombin III and thus accelerate inhibition of thrombin it has anticoagulant effect. This is abundantly used in clinical practice for operations, in case of embolia or heart-attacks. Protamine is a mixture of small basic peptides, which is used in clinical practice as a heparin antidote. The interaction between heparin and protamine is electrostatic and is also used for determination of heparin in human plasma or blood using affinity methods. In my study it was found that if protamine and heparin are mixed in one vial, a complex is formed. Its resulting charge depends on concentration ratio of protamine and heparin. On the other hand, in case the protamine is injected as a sample and heparin is added to background electrolyte as a protein-binding ligand, it is possible to determine heparin from decreasing protamine peak area. Because of the complexity of protamine-heparin interaction, tetraarginine was used as structurally close model of protamine to increase repeatability of measurements. The method for determination of heparin was optimalised. It uses 20 mM or 60 mM ortho-phosphoric acid as background electrolyte, 1 mg/mL solution of tetraarginine...
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