magistrsko delo
Rebeka Podgrajšek (Author), Kristina Sepčić (Reviewer), Marjetka Podobnik (Mentor), Marjetka Podobnik (Thesis defence commission member), Apolonija Bedina Zavec (Thesis defence commission member), Kristina Sepčić (Thesis defence commission member), Tom Turk (Thesis defence commission member), Apolonija Bedina Zavec (Co-mentor)

Abstract

Listeriolizin O (LLO) je porotvorni protein bakterije Listeria monocytogenes. Njegovo delovanje je odvisno od pH. Na Kemijskem inštitutu so pripravili mutanta LLO Y406A, katerega aktivnost je pri sobni temperaturi še bolj odvisna od pH kot aktivnost divjega tipa (LLO wt). LLO Y406A je zato zanimiv za biotehnološke aplikacije. Namen naše raziskave je bil opredeliti učinek LLO wt in LLO Y406A na krvnih celicah pri fiziološki temperaturi (37 °C). Primerjali smo odziv različnih rakavih krvnih celic (K-562, Jurkat, Raji) z odzivom zdravih celic, izoliranih iz krvi. Stanje celic smo določali prek umiranja, sledenja izločenih zunajceličnih veziklov, nastajanja reaktivnih kisikovih spojin in količine ATP. Ugotovili smo, da je toksičnost LLO Y406A za krvne celice pri fiziološki vrednosti pH (7,4) zelo nizka. Tretiranje z LLO Y406A je povzročilo 20-odstotno umrljivost celic pri 300–7000-krat višjih koncentracijah kot tretiranje z LLO wt. Poleg tega smo ugotovili, da sta LLO wt in LLO Y406A pri fizioloških pogojih bolj toksična za rakave kot za zdrave krvne celice. Tretiranje z LLO wt je povzročilo 20-odstotno umrljivost zdravih celic pri 1,7–300-krat višjih koncentracijah kot pri rakavih celicah, LLO Y406A pa pri 3–30-krat višjih koncentracijah kot pri rakavih celicah. Z nižanjem vrednosti pH se aktivnost LLO Y406A še poveča. Pri vrednosti pH 6,5 je povzročil 20-odstotno umrljivost zdravih celic pri 4–40-krat višjih koncentracijah kot pri rakavih celicah. Povečano vezikulacijo kot odziv na LLO wt ali LLO Y406A smo zaznali že pri 10–100-krat nižji koncentraciji proteina kot umiranje celic. LLO wt in LLO Y406A nista povzročila povečanja reaktivnih kisikovih spojin, je pa LLO wt povzročil povišanje ATP, LLO Y406A pa ne. Pri višjih koncentracijah proteinov opazimo padec ATP.

Keywords

celična biologija;vezikulacija;listeriolizin O (Y406A);ATP;reaktivne kisikove spojine;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [R. Podgrajšek]
UDC: 577.2(043.2)
COBISS: 77507843 Link will open in a new window
Views: 216
Downloads: 22
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Other data

Secondary language: English
Secondary title: ǂThe ǂresponse of human cells to Listeriolysin O and its mutants
Secondary abstract: Listeriolysin O (LLO) is a pore-forming protein of the bacteria Listeria monocytogenes. Its activity depends on pH. At the Institute of Chemistry, an LLO Y406A mutant was prepared. Its activity at room temperature is more dependent on pH than the activity of its wild-type (LLO wt). LLO Y406A is therefore interesting for biotechnological applications. The purpose of our study was to define the effects of LLO wt and LLO Y406A on blood cells at a physiological temperature (37 °C). We compared the response of different cancer blood cells (K-562, Jurkat, Raji) with the response of healthy cells isolated from blood. Cell condition was determined through dying, tracking secreted extracellular vesicles, observing the formation of reactive oxygen species (ROS) and the amount of ATP. LLO Y406A toxicity to blood cells, at physiological pH (7.4), was found to be very low. Cell treatment with LLO Y406A resulted in 20% cell mortality at 300–7000 times higher concentrations than with LLO. In addition, we found that LLO wt and LLO Y406A are more toxic to cancerous blood cells than to healthy blood cells in physiological conditions. LLO wt treatment resulted in 20% mortality of healthy cells at 1.7–300 times higher concentrations than of cancer cells, and LLO Y406A at 3–30 times higher concentrations in healthy cells than in cancer cells. By lowering pH, the activity of LLO Y406A further increases. At pH 6.5, it causes 20% mortality of healthy cells at 4–40 times higher concentrations than of cancer cells. Increased vesiculation in response to LLO wt or LLO Y406A was detected at 10–100 times lower protein concentration than cell death. LLO wt and LLO Y406A did not cause the increase of ROS, but LLO wt caused an increase in ATP, while LLO Y406A did not. At higher concentrations, we noticed a decrease in ATP.
Secondary keywords: cell biology;vesiculation;listeriolizin O (Y406A);ATP;reactive oxygen species;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. Ljubljana, Biotehniška fak.
Pages: XIII, 79, [1] f.
ID: 13403732