diplomsko delo
Anže Jenko (Author), Marina Klemenčič (Mentor)

Abstract

Regulirana celična smrt (RCD) je pri živalskih celicah zelo jasno definiran in dobro opisan proces, ki je ključen pri razvoju in odzivu na različne okoljske stresorje. Pri tem procesu imajo ključno vlogo encimi kasapaze. Podobno velja tudi za rastline, kjer kaspaze niso prisotne, so pa prisotni njihovi strukturni homologi, metakaspaze. Metakaspaze naj bi imele pomembno vlogo pri poteku RCD pri rastlinah, a ta vloga do zdaj še ni točno poznana in okarakterizirana. Celično smrt smo pri modelnem enoceličnem organizmu Chlamydomonas reinhardtii inducirali z dvema različnima stresorjema, in sicer z vodikovim peroksidom (H2O2) ter soljo (NaCl), ter spremljali in kvantificirali različne parametre odziva, ki jih je dodani stresor v celičnih populacijah povzročil. Parametri, ki smo jih spremljali, so bili sprememba rasti kultur, preživetje celic, stanje genomske DNA ter metakaspazna aktivnost celic. Rast v kulturah in preživetje celic sta bila pri obeh stresorjih negativno korelirana s časom izpostavljenosti in koncentracijo stresorja. Pri celicah, ki so bile izpostavljene H2O2, smo opazili postopno in urejeno fragmentacijo DNA. Temu pojavu pravimo lestvičenje (angl. ''laddering''). Pri dodatku NaCl kulturam je na gelu prišlo do pojava razmaza DNA (angl. ''smear'') ali pa je le-ta ostala intaktna. Celice, ki so bile izpostavljene NaCl, so pokazale očitno znižanje metakaspazam podobne encimske aktivnosti, medtem ko so tiste celice, ki so bile izpostavljene vodikovemu peroksidu, pri različnih koncentracijah pokazale metakaspazno aktivnost, a ta pojav ni bil koncentracijsko odvisen. Na podlagi znanih podatkov iz literature smo sklepali, da je pri izpostavitvi celic H2O2 prišlo do RCD, pri dodatku NaCl pa do nekroze. RCD pri enoceličnem organizmu efektivno povzroči samomor. Takšno vedenje ima torej v logiki sebičnega posameznika smisel zgolj na ravni celotne populacije. Zanimalo nas je, če lahko RCD doprinese k preživetju populacije kot celote, zato smo po tretiranju kultur celice prenesli na sveže gojišče ter spremljali njihovo rast oz. pojavnost kolonij. Ker je prišlo do kontaminacij kultur, so tam rezultati nezanesljivi, a v splošnem s katerimkoli stresorjem tretirane celice niso bile sposobne znova vzpostaviti zdrave kulture. Rast na trdnem gojišču je dala bolj konkretne rezultate; do rasti je prišlo tudi pri tistih celicah, ki so bile dlje časa izpostavljene vodikovemu peroksidu, ne pa soli.

Keywords

zelene alge;Chlamydomonas reinhardtii;regulirana celična smrt;oksidativni stres;nekroza;osmotski stres;encimi;metakaspaze;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [A. Jenko]
UDC: 577.15(043.2)
COBISS: 1538396099 Link will open in a new window
Views: 733
Downloads: 204
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Other data

Secondary language: English
Secondary abstract: In animal cells, regulated cell death (RCD) is a well defined and molecularly characterised process that is orchestrated by proteolytic action of various types of caspase enzymes. RCD plays a vital role in organism's development and in response to a vast array of different environmental stress conditions. Similar also applies to plants, despite the fact that these organisms lack caspase enzymes. Instead, plants in their genomes carry genes which encode metacaspases. Like caspases these proteases are cysteine proteases as well but they cleave different types of substrates. Metacaspases are believed to be also involved in plant RCD, however their precise role has not been fully characterised yet. We induced cell death in the unicellular algal model organism Chlamydomonas reinhardtii using two different stress agents, hydrogen peroxide (H2O2) and salt (NaCl). After treatment we monitored different parameters of cell response such as: changes in growth, cell viability, genomic DNA stability and metacaspase protease activity. Cell growth and viability were both negatively correlated with concentration and time of exposure to stress agents. H2O2-treated cells exhibited orderly and continuous fragmentation of DNA. This occurence is termed laddering. DNA of cells exposed to NaCl displayed DNA smear on gel or it remained intact. Cells exposed to NaCl also gradually exhibited decline of metacaspase-like enzyme activity, whereas those that were exposed to H2O2 displayed some metacaspase-like activity, however without any obvious correlation to concentration. Based on literature we deduced that NaCl-treated cells underwent necrotic cell death, whereas H2O2-treated cells experienced RCD. When a unicellular organism undergoes RCD this leads to what is effectively suicide. Since such behaviour is not in line with the idea of selfish individual, it therefore makes sense only if it is beneficial to the survival of the population as a whole. After treatment of cell cultures with stress agents we transferred cells into fresh TAP medium and monitored their growth or formation of colonies. Since our cultures got contaminated, the results are inconclusive, however regardless of the stress agent, Chlamydomonas cells were not able to reestablish a healthy culture. Growth on TAP plates displayed more definite results, enabling the growth of cells that were treated with H2O2 but not of those that were treated with salt.
Secondary keywords: Chlamydomonas reinhardtii;regulated cell death;oxidative stress;necrosis;osmotic stress;metacaspases;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 47 str.
ID: 11363448