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The role and function of stromal enzymes in keratoconus pathogenesis
Ďuďáková, Ľubica ; Jirsová, Kateřina (advisor) ; Svozílková, Petra (referee) ; Ardan, Taras (referee)
Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...
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Study of phosphorylation of inorganic pyrophosphatase from Streptococcus pneumoniae
Štechová, Michaela ; Svobodová, Jaroslava (referee) ; Doubravová, Linda (advisor)
The human patogen Streptococcus pneumoniae encodes a single copy of eukaryotic-like Ser/Thr protein kinase StkP. StkP regulates virulence, competence, stress resistence, gene expression and plays a role in the regulation of cell division cycle. Analysis of phosphoproteome maps of the wild type and stkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including Mn-dependent inorganic pyrophosphatase PpaC. Mass spectrometry analysis identified two phosphorylation sites in an active site of the protein. Pyrophosphatases are essential enzymes that catalyze hydrolysis of inorganic pyrophosphate produced during various biosynthetic reactions that utilize ATP. Changes in pyrophosphatase activity have been described to have global effects on cell metabolism, growth and division of bacteria. The aim of this thesis was to investigate the phosphorylation of inorganic pyrophosphatase PpaC in S. pneumoniae. Gene ppaC was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of PpaC by StkP was examined in a kinase assay but we did not confirm that PpaC is a direct substrate of StkP in vitro. Further we prepared a set of mutants in ppaC gene. We replaced two presumable phosphoaminoacids identified by mass-spectrometry with...
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