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Proteomic analysis of lysozyme and lysozyme-like proteins of synanthropic mites
Chum, Tomáš ; Erban, Tomáš (advisor) ; Mikeš, Libor (referee)
This diploma thesis was focused on the study of lysozyme and lysozyme-like proteins, either of similar function (antibacterial) or molecular weight (14 - 17 kDa), of synanthropic acaroid mites. In general, animals utilize lysozymes for defensive (antimicrobial) or digestive purposes but also as a digestive enzyme. Some chitinases or other enzymes that act similarly to lysozyme can be utilized for similar purposes. Chitinases belong to house dust mite allergens. One of major mite are historically named lysozyme-like proteins which name relates to their size similar to lysozyme. Bacteriolytic activity has also 14.5 kDa (UniprotKB Q8MWR6) protein. The species selected for the study were domestic mites Dermatophagoides farinae, D. pteronyssinus and Lepidoglyphus destructor. Presence of lysozyme was detected by direct detection with polyclonal antibody using immunohistochemistry and dot blots. Immunohistochemistry proved presence of lysozyme epitopes in the feces of D. farinae, D pteronyssinus a L. destructor. Dot blot analysis demonstrated the presence of imunoreactivity of antibody in spent growth medium extracts (SGME) of all three species. This implies that lysozyme is synthesized in the midgut. The presence of lysozyme and lysozyme-like proteins was proved using 2D electrophoresis and MALDI TOF/TOF...
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In-vitro analysis of amoeboid-mesenchymal transition of A375m2 melanoma cells
Kasalová, Lenka ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee)
The invasion of cancer cells is an important aspect of cancer progression. Single tumor cells exhibit at least two types of invasion in 3D environment, mesenchymal and amoeboid invasion. Tumor cells can switch between these two modes of movement depending on cellular status and surrounding environment. Amoeboid-mesenchymal transition (AMT) is less explored then mesenchymal-amoeboid transition (MAT). We performed a proteomic analysis of amoeboid-mesenchymal transition of human melanoma cell line A375M2. We have induced amoeboid-mesenchymal transition by treatment with a ROCK inhibitor Y27632 in 3D matrigel matrices and in 2D environment. Induction of the amoeboid-mesenchymal transition has changed a level of expression of 92 proteins and a level of phosphorylation of 15 proteins. Expression of only 17 proteins and phosphorylation of 8 proteins was identically changed in both of these environments. We found that PKCα regulates amoeboid migration and that treatment of cells with a PKCα inhibitor Gö6976 induces amoeboid-mesenchymal transition. Analysis of the proteomics data have further shown that induction of AMT by the ROCK inhibitor Y27632 leads to activation of antiapoptotic signals and activation of signaling pathways involved in regulation of actin cytoskeleton especially regulation of focal...
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