Učinak DMAG-a i utišavanja Hsp90 na vijabilnost THP-1 stanica

master's thesis

• Title (english): Effect of DMAG and Hsp90 silencing on the viability of THP-1 cells
• Author: Dubravka Živković
• Mentor: Karmela Barišić (mentor)
• Mentor: Jozsef Petrik (mentor)
• Committee member: Karmela Barišić (predsjednik povjerenstva)
• Committee member: Jozsef Petrik (član povjerenstva)
• Committee member: Sandra Šupraha Goreta (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2017-06-02, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Apoptoza je poseban oblik stanične smrti nužan za razvoj organizma i održavanja stanične homeostaze, tj. ravnoteže između gubitka i stvaranja stanica normalnog tkiva tijekom života. Hsp su proteini toplinskog šoka koji sudjeluju u više procesa u stanici koji su nužni za normalni rast i razvoj same stanice. Inhibicijom Hsp90 u stanici se nakupljaju nepravilno smotani proteini i stanici nedostaju proteini kako bi normalno funkcionirala. Time se potiče ulazak stanice u apoptozu. DMAG inhibira i α i β izomer, dok je siRNA dizajnirana za utišavanje samo α izomera. Cilj ovog rada je utvrđivanje apoptoze u THP-1 stanicama, od kojih su neke tretirane sa DMAG-om u različitim koncentracijama, a nekima je RNA interferencijom utišana mRNA za Hsp90 iz citosola. Opseg stanične smrti i apoptoze određuje se dvjema metodama, a to su bojanje stanica tripanskim plavilom i TUNEL reakcijom. TUNEL reakcijom nisu identificirane apoptotične stanice, niti nakon ponovljenog mjerenja. U uzorku THP-1 stanica, bojanih sa tripanskim plavilom, koje su tretirane siRNA molekulama i koji je fiksiran nakon 24 h, utvrđeno je 77,6 % mrtvih stanica. Postotak mrtvih THP-1 stanica bojanih tripanskim plavilom raste sa koncentracijom DMAG-a kojim su stanice bile izložene.

Abstract (english)

Apoptosis is a special form of cell death, necessary for the development of an organism and maintenance of cellular homeostasis, that is the equilibrium between the loss and generation of normal-tissue cells during life. Hsp are the heat shock proteins which participate in several cellular processes, which are necessary for the normal growth and development of the cell itself. In the case of inhibition of Hsp90 the incorrectly folded proteins accumulate in the cell and the cell lacks the proteins to function normally. The transition of a cell to apoptosis is thereby induced. DMAG inhibits both α and β isomers, while siRNA is designed to destroy only α isomers. The aim of this thesis is to establish the apoptosis in THP-1 cells, of which some were treated with DMAG in various concentrations, and for some the mRNA level of cytosolic Hsp90 was silenced by RNA interference. The scale of cell death and apoptosis is determined by two methods, and those are cell coloration by Trypan Blue and TUNEL reaction. The TUNEL reaction has not identified any apoptotic cells, even not after repeated measurement. In the sample of THP-1 cells, coloured by Trypan Blue, which were treated by by siRNA molecules, and which was fixed after 24 hours, 77.6% dead cells were found. The percentage of dead THP-1 cells coloured by Trypan Blue increases with the DMAG concentration to which the cells had been exposed.

• Keywords: apoptoza; siRNA; DMAG; tripansko plavilo; TUNEL metoda
• Keywords (english): apoptosis; siRNA; DMAG; Trypan blue; TUNEL method
• Language: croatian
• URN:NBN: urn:nbn:hr:163:608725
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-13 16:42:36