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Analysis of substrate specificity and mechanism of GlpG, an intramembrane protease of the rhomboid family.
Peclinovská, Lucie ; Stříšovský, Kvido (advisor) ; Konvalinka, Jan (referee)
Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...
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Mixed lipids and their interaction with biopolymers
Cigánková, Hana ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of interactions between hyaluronan and lipid DOTAP and its mixtures with DPPC in water. First, the aggregation behavior of the lipid itself and its mixtures was measured, afterwards the influence of hyaluronan in different concentration was studied. Further the fluorescence anisotropy of fluorescence probe DPH in these mixtures and influence of cholesterol was measured. These properties were investigated by fluorescence spectoscopy using pyrene and DPH as fluorescence probes. The measurements revealed that the addition of hyaluronan has observable influence on the aggregation behavior of the lipid DOTAP and its mixtures with DPPC and that cholesterol influences liposomes fluidity.
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Phospholipids as the basis of biodegradable delivery systems
Burdíková, Jana ; Čeppan, Michal (referee) ; Mravec, Filip (advisor)
This thesis is focused on investigation of phospholipid-hyaluronan system. First, appropriate method for preparation of bulk solution of phospholipid/lipid and suitable fluorescence probe were chosen. Sonification was selected as a method for preparation of bulk solution and pyrene was chosen as a fluorescence probe. From the group of phospholipids lecithin was selected. Next to phospholipid, lipid with no phosphate group (DPTAP) was utilized for comparison, alternatively a mixture of lipid (DPTAP) and phospholipid (DPPC). Instead of hyaluronan another polyelectrolytes (sodium polystyrene sulfonate, sodium alginate) were used too. Measurements were performed in water environment and in phosphate buffer saline (PBS). All investigation was accomplished by fluorescence spectroscopy and dynamic light scattering.
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Study of the interaction of hyaluronan and biocompatible amphiphilic substances
Cigánková, Hana ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of the interactions between hyaluronan and selected phospholipids and their mixtures in water or buffer. First, the aggregation behavior of these lipids was measured, afterwards the influence of hyaluronan was studied. This behavior was investigated by fluorescence spectoscopy using pyrene as fluorescence probe. The Critical Agregation Concentration of the lipids before and after addition of hyaluronan was determined. The measurements revealed that the addition of hyaluronan has an observable influence on the aggregate behavior of the selected lipids and their mixtures.
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A role of adipokinetic hormones in insect lipid metabolism
BÁRTŮ, Iva
This PhD. thesis summarizes the effect of adipokinetic hormones (AKHs) on a spectrum of mobilized lipids in model insect species the locust Locusta migratoria and the firebug Pyrrhocoris apterus. The results revealed that mobilization of diacylglycerols and fatty acids from the fat body into the haemolymph is not uniform and suggested there is partial specificity of individual AKHs. This could contribute to the answer of the question why some insect species have more than one AKH. The results of the thesis are supplemented by studies of AKH effect on lipid metabolism in stress conditions elicited by insecticide treatments. Co-application of AKH and insecticide enhanced the effect of insecticide
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Analysis of lipids mobilized by adipokinetic hormones in the firebug Pyrrhocoris apterur (Heteroptera: Pyrrhocoridae)
BÁRTŮ, Iva
An effect of two adipokinetic hormones (Pyrap-AKH and Peram-CAH-II) on distribution of diacylglycerol (DG) molecular species and their fatty acid (FA) constituents were investigated by liquid chromatography (HPLC) - electrospray ionization mass spectrometry (ESI-MS) in haemolymph of the firebug Pyrrhocoris apterus. The results show that DGs with characteristic FAs are preferentially mobilized from fat body (FB) by the action of both AKHs produced by P. apterus. Both the macropterous and brachypterous morphs show very close DG and FA profiles. A separate action of the Pyrap-AKH and the Peram-CAH-II, however, exerts distinct differences in FA distribution at the macropterous morph. It seems that partially C16 and mainly unsaturated C18 FAs play a dominant role in the AKH action with an exclusive position of linoleic acid (18:2) which represents 50 - 60% of the total DG mobilization. The metabolically active C16 and C18 FAs are preferentially absorbed from the linden seeds and accumulated in the FB.
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