Browsing by Subject "KYP-2047"

Autophagy is a pathway for cells to degrade intracellular components that are no longer needed or are detrimental for the cells. It is essential for cell homeostasis and survival and has been related to various diseases and pathophysiology. Autophagy is a complex process and there are still several unclear und unknown aspects to it. Regulation of autophagy is essential to prevent unwanted and escess activation, and several pathways and molecules, both stimulatory and inhibitory, are included. Different signaling pathways are sensitive to a variety of environmental clues. Two main autophagy pathways are mTOR-dependent pathway and mTOR-independent pathway. Induction of autophagy in the latter pathway is dependent on the interaction of Bcl-2 and Beclin 1. Prolyl oligopeptidase (PREP) is a peptidase enzyme that has several substrates. PREP-inhibition by KYP-2047 can reduce aggregation of α-synuclein in two ways: by increasing rate of autophagy and by decreasing dimerization. The aim of this study was to find out how PREP affects the interaction between Bcl-2 and Beclin 1 and how this affects autophagy. Based on previous studies, PREP-inhibition seems to increase the amount of Beclin 1 and to affect the phosphorylation of Bcl-2 and Beclin 1, leading to dissociation of the complex. Hypothesis was to see differences in colocalization of Bcl-2 and Beclin 1 in cells treated with different PREP-modifications and for PREP-inhibition to decrease the colocalization. Human embryonic kidney cells 293 (HEK-293) and hPREP knockout cell line created from them by using CRISPR/Cas9-silencing were used in the experiments. Two experiments were performed on regular HEK-cells: inhibitor experiment with KYP-2047 (1 or 10 µM) and overexpression experiment (transfection with either active or inactive hPREP plasmid). After immunofluorescence staining, cells were analysed with confocal microscope and colocation analysis of Bcl-2 and Beclin 1 was performed. The intensity of Beclin 1 in the nuclei was stronger than in other parts of the cell in all samples, which could indicate a stronger activity of its nuclear tasks compared to autophagy. However, the antibody used for immunofluorescence has most likely caused this staining pattern. Based on previous knowledge, it was expected to see differences in colocalization of Bcl-2 and Beclin 1 in cells treated with different PREP-modifications. However, there were no significant differences in colocalization of Beclin 1 and Bcl-2 in any of the experiments but it was nearly 100 percent in all treatments. Since rate of autophagy in cells was not detected, it is impossible to determine, if there were changes in autophagy that were not reflected as changes in colocalization of these two proteins. It is possible that even a small change in colocalization can affect the rate of autophagy or there might be subpopulations where the interaction is interrupted and these changes are so small that they are not detectable with the methods used in this experiment. Both Bcl-2 and Beclin 1 also have functions not related to autophagy, which could be one reason behind the results gained in this study.

Angiogenesis may be regarded as one of the most important phenomena involved in basic physiology as well as in numerous pathological conditions. Angiogenesis is a multistep process involving the balance of pro- and con-angiogenic factors. Several studies have suggested that angiogenesis is regulated in vitro and in vivo by peptides thymosin ȕ4 (Tȕ4) and tetrapeptide Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline). There are also studies supporting the view that Ac-SDKP, a peptide fragment is released from the proline-containing C-terminus of Tȕ4 (43-mer) by hydrolyzing prolyl oligopeptidase (POP). POP is a widely existing serine protease cleaving oligopeptides of no longer than 30 amino acids. Thus, Tȕ4 should first be cleaved into a shorter peptide by some other, yet unknown peptidase. POP has been mostly studied in memory and learning disorders as well as in neurodegenerative diseases. The true physiological character of POP is still unresolved. In this Master's thesis, the associations of the factors involved in angiogenesis are reviewed in the literature part as well as the character, presence and function of the angiogenic molecules 7ȕ4, Ac-SDKP and POP. In the experimental part attempts were made to find whether POP and Tȕ4 increase Ac-SDKP formation and capillary tube network and consequently, whether the POP activity, tetrapeptide and capillary formation could be inhibited by the proline-spesific POP inhibitor KYP-2047. The study had two phases. The first phase included POP activity and Ac-SDKP measurements(time period 0-180 min) with Wistar rat kidney homogenates. Study groups were 0,1 and 0,5 µM KYP-2047 (+2 µM Tȕ4), 1:20 (0.625 µM) human recombinant POP (+ 2 µM Tȕ4), 2 µM 7ȕ4 (pos. control) and raw homogenate (neg. control). The second phase involved the study of capillary formation (time period 0-180 min) with primary endothelial HUVECs on a 48-well plate seeded with 50 000 cells/well on an extracellular membrane mimicking MatrigelTM Matrix dissolved in DMEM. Study groups treated with fetal bovine serum and antibiotics were 5 and 10 µM KYP-2047 (+4 µM Tȕ4), 1:20 (0.625 µM) human recombinant POP (+4 µM Tȕ4)4 µM Tȕ4 (pos. control) and DMEM (neg. control). The wells were cultured and capillary formation photographed with a light microscope using a digital camera. All experiments were repeated four times, and each study group in wells was measured in triplicate. Enclosed capillaries were counted manually and statistical tests were performed. 7ȕ4 along with POP participated in the formation of AC-SDKP in the kidney homogenates. Cultures of primary endothelial cells on Matrigel resulted in clear capillary formation in Tȕ4 and POP groups. KYP-2047 had a strong POP-inhibitory activity on antiangiogenesis throughout the study resulting. Obviously, underlying mechanisms of angiogenesis and the function of the interaction between POP, Tȕ4 and Ac-SDKP in capillary formation require further studies.