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Vzpostavitev metode za vzbujanje oksidativnega stresa v algi Chlamydomonas reinhardtii s svetlobo UV
ID Kudelić, Sumeja (Avtor), ID Klemenčič, Marina (Mentor) Več o mentorju... Povezava se odpre v novem oknu

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Izvleček
Regulirana celična smrt (angl. regulated cell death, RCD) je na nivoju genov uravnavan proces, ki pri rastlinah omogoča kontrolirano rast in prilagoditev na stresne pogoje. Pred kratkim je bilo pokazano, da tudi pri enocelični zeleni algi Chlamydomonas reinhardtii pod določenimi stresnimi pogoji pride do procesov, ki so podobni regulirani celični smrti v kopenskih rastlinah. Alga C. reinhardtii je modelni organizem za raziskovanje različnih fizioloških procesov kot so fotosinteza, respiracija, prilagoditev na različna sevanja, osmotski in oksidativni stres. Pri C. reinhardtii lahko regulirano celično smrt opazimo pri več stresnih pogojih, kot so dodatek vodikovega peroksida ali ocetne kisline, značilnosti RCD pa lahko opazimo tudi po obsevanju z UV-svetlobo. Izpostavljenost celic C. reinhardtii različnim sevanjem povzroči morfološko značilne spremembe, kot so povečanje volumna vakuol, lestvičenje DNA (angl. DNA laddering) in prisotnost fosfatidilserina na zunanji strani celične membrane. Ker mehanizmi, ki privedejo v regulirano celično smrt pri tem organizmu še niso jasni, smo želeli vzpostaviti novo metodo za vzbujanje oksidativnega stresa v C. reinhardtii. Celice C. reinhardtii smo zato različno dolgo obsevali z UV-svetlobo, da bi ugotovili pri kakšni izpostavitvi sevanja pride do procesov povezanih z RCD. Parametri, ki smo jih spremljali pri celicah, obsevanih z UV-svetlobo, so preživetje celic po obsevanju ter nukleazna in proteolitična aktivnost. Opazovali smo tudi morfološke spremembe s svetlobnim mikroskopom. Ugotovili smo, da pride do povečanja volumna vakuol v celicah, ki so bile daljši čas izpostavljene stresorju. Z agarozno gelsko elektroforezo smo preverjali aktivnosti nukleaz, ki razgrajujejo genomsko DNA na urejene fragmente enakih velikosti DNA. Nastanek urejenih fragmentov DNA je namreč značilen znak, da je prišlo do RCD. Z izvedbo encimskega testa smo preverjali aktivnost tripsinu podobnih proteaz, za katere sklepamo, da so ključne pri RCD. Na žalost smo izbrali precej kratek čas obsevanja celic z UV-svetlobo in nespecifičen substrat (Z-FR-AMC), zaradi česar razlik v aktivnosti proteaz nismo opazili.

Jezik:Slovenski jezik
Ključne besede:Chlamydomonas reinhardtii, regulirana celična smrt, oksidativni stres, UV-svetloba, osmotski stres, metakaspaze
Vrsta gradiva:Diplomsko delo/naloga
Tipologija:2.11 - Diplomsko delo
Organizacija:FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Leto izida:2021
PID:20.500.12556/RUL-130310 Povezava se odpre v novem oknu
COBISS.SI-ID:84692483 Povezava se odpre v novem oknu
Datum objave v RUL:13.09.2021
Število ogledov:740
Število prenosov:42
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Sekundarni jezik

Jezik:Angleški jezik
Naslov:Establishement of a method for induction of oxidative stress in a green alga Chlamydomonas reinhardtii using UV-light
Izvleček:
Regulated cell death (RCD) is a gene-regulated process that allows plants to grow and develop and adapt to stressful conditions. It has recently been shown that even in the unicellular green alga Chlamydomonas reinhardtii, processes similar to regulated cell death in plants are observed under certain stress conditions. Alga C. reinhardtii is a model organism for researching various physiological processes such as photosynthesis, respiration, adaptation to various radiations, osmotic and oxidative stress. In C. reinhardtii, regulated cell death may be observed under several stressful conditions, such as the addition of hydrogen peroxide or acetic acid, and the characteristics of RCD may also be observed after irradiation with UV light. Exposure of C. reinhardtii cells to various radiations, such as UV light, causes morphologically characteristic changes such as an increase in vacuole volume, DNA laddering, and the presence of phosphatidylserine on the outside of the cell membrane. Since the mechanisms that lead to regulated cell death in this organism are not clear yet, we wanted to establish a new method for inducing oxidative stress that would facilitate our study of the RCD process in C. reinhardtii. C. reinhardtii cells were therefore irradiated with UV light for different time periods to determine the radiation exposure associated with RCD-related processes. The parameters monitored in cells irradiated with UV light are cell survival after irradiation and nuclease and proteolytic activity. After irradiation of the cells with UV light, morphological changes were observed with a light microscope. We observed an increase in the volume of vacuoles in cells that have been exposed to the stressor for a long time. Agarose gel electrophoresis allowed us to verify the activity of nucleases that degrade genomic DNA into ordered fragments of equal DNA sizes. The formation of ordered DNA fragments is a characteristic feature of cells in which RCD has occurred. By performing proteolytic assay, we monitored the activity of proteases with tripsin-like activity, which were presumed to be crucial in RCDs. Unfortunately, we chose a rather short irradiation time of cells with UV light and a nonspecific substrate (Z-FR-AMC), which coved be the reason why no difference in protease activity was observed.

Ključne besede:Chlamydomonas reinhardtii, regulated cell death, oxidative stress, UV light, osmotic stress, metacaspases

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