Optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom

master's thesis

• Title (english): Optimizing conditions for determination of the signaling molecules ERK and p38 MAPK by Western blot analysis
• Author: Dora Radić
• Mentor: Lada Rumora (mentor)
• Committee member: Lada Rumora (predsjednik povjerenstva)
• Committee member: Jasmina Lovrić (član povjerenstva)
• Committee member: Marija Grdić Rajković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2016-07-01, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Kronična opstrukcijska plućna bolest karakterizirana je snažnim upalnim odgovorom na inhalaciju štetnih čestica. Dolazi do nakupljanja imunosnih stanica koje luče citokine, kemokine i tkivne proteaze, posljedično tome dolazi do opstrukcije dišnih putova i izazito smanjenog protoka zraka. TLR receptori odgovorni su prvenstveno za urođenu imunost i za pojačavanje odgovora stečenog imuniteta. Nađena je veza između aktivacije TLR receptora, pokretanja kaskadne reakcije kojom
dolazi do aktivacije (foforilacije) MAPK kinaza (ERK, p38, JNK) i izlučivanja različitih citokina koji izazivaju upalni odgovor. Cigaretni dim te brojni drugi štetni čimbenici iz okoliša dovode do aktivacije TLR receptora. Agonist TLR4 (LPS) i agonist TLR2 (LTA) koriste se u svrhu ipitivanja upalnog odgovora potaknutog navedenim receptorima.Cilj ovog diplomskog rada bilo je
optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK iz THP-1 stanične linije Western blot metodom. Dobiveni rezultati pokazuju da su korištenjem protokola Cell Signaling Technology (CST) postignuti optimalni uvjeti i najbolji rezultati za sva četiri analizirana proteina: ERK, p-ERK, p38 i p-p38. Koncentracija proteina nanesenih na poliakrilamidni gel treba biti
između 30 i 40 μg po jažici. Signal pozadine značajno je smanjen korištenjem nitrocelulozne membrane u odnosu na PVDF membranu. Vizualizacija proteina kemiluminiscencijom uz korištenje detekcijske otopine svježe pripremljene u laboratoriju daje konačnu sliku proteina koje se ne razlikuje od slike dobivene uz korištenje komercijalno dostupne otopine (Amersham ECL
Prime; GE Healthcare Life Sciences).

Abstract (english)

Chronic obstructive pulmonary disease is characterized by a strong inflammatory response to inhalation of harmful irritating particles. Recruitment of immune cells that secrete cytokines, chemokines and proteases, consequently leads to airway obstruction and reduced air flow. TLR receptors are primarily responsible for innate immunity and they enhance the response of acquired immunity. There was a link between TLR receptors, activation (phosphorylation) of MAPK kinase (ERK, p38, JNK) and the secretion of various cytokines that cause an inflammatory response. TLR4 agonist (LPS) and TLR2 agonist (LTA) might be used for examination of the inflammatory response induced by these receptors. The aim of this study was the optimization of the conditions for determining the signaling molecules ERK and p38 MAPK from THP-1 cell lines by Western blot method. The results show that the optimal conditions and the best results for all the examined proteins ERK, p-ERK, p38 and p-p38 are achieved by using the protocol from Cell Signaling Technology (CST). The concentration of protein applied to the polyacrylamid gel should be between 30 and 40 μg per well. The background signal is significantly reduced by using a nitrocellulose membrane compared to PVDF membrane. Visualization of proteins with chemiluminescence detection solution freshly prepared in the laboratory, gives the final images of the proteins that do not differ significantly from the images obtained with a commercially available solution (Amersham ECL Prime; GE Healthcare Life Sciences).

• Keywords: kronična opstrukcijska plućna bolest; Western blot; TLR receptori; ERK; p38; lipopolisaharid; lipoteikoična kiselina
• Keywords (english): chronic obstructive pulmonary disease; Western blot; TLR receptors; ERK; p38; lipopolysaccharide; lipotheicoic acid
• Language: croatian
• URN:NBN: urn:nbn:hr:163:993535
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2016-10-27 09:58:58