Title Promjena ekspresije metionin sulfoksid reduktaze B3 u uzorcima bubrega štakora u ovisnosti o dobi i spolu Title (english) Change in the expression of methionine sulfoxide reductase B3 in rat kidney samples depending on age and sex Author Ana Sever Mentor Marija Ljubojević (mentor)Mentor Domagoj Đikić (komentor)Committee member Domagoj Đikić (predsjednik povjerenstva)Committee member Duje Lisičić (član povjerenstva)Committee member Dubravka Hranilović (član povjerenstva)Committee member Marija Ivković (član povjerenstva)Granter University of Zagreb Defense date and country 2024-02-27, Croatia Scientific / art field, NATURAL SCIENCES Abstract Oksidativni stres predstavlja stanje narušene oksido-redukcijske ravnoteže unutar stanice uzrokovano neravnotežom nastajanja i uklanjanja reaktivnih vrsta kisika (ROS). U fiziološkim uvjetima ROS predstavlja važnu kariku u održavanju života stanice, dok su povišene koncentracije ROS jedan od preduvjeta starenja jer se narušava integritet svih makromolekula. Senescencija ili starenje nastupa nakon oštećenja kao stanje postupnog neuravnoteženja staničnih fizioloških i metaboličkih funkcija. Stanica ulazi u senescenciju poglavito zbog oštećenja molekule DNA koja se, ako nisu pretjerana, popravljaju. Inače, pokreću se mehanizmi stanične smrti. Djelovanjem ROS dolazi do oksidacije aminokiseline metionina (Met, M), koja sadrži sumpor, unutar proteina. Oksidacijom Met nastaje spoj metionin sulfoksid (MetO) u dva epimera – S i R, koje reduciraju enzimi članovi obitelji metionin sulfoksid reduktaze (Msr), MsrA i MsrB. Stereospecifičnost uzrokuje da MsrA reducira S-epimer, a MsrB R-epimer MetO. Cilj ovog rada bio je utvrditi postoje li dobne i spolne razlike analogne promjenama ekspresije metalotioneina koji dominira u ženkama, raste starenjem i koji je potencijalno uključen u regeneraciju MsrB3. Na nivou ekspresije enzima MsrB3 u bubrezima štakora odredili smo ciljanu mRNA RT-PCR tehnikom, prisutnost proteina u tkivnim homogenatima western blot analizom te raspodjelu proteina u tkivnim narescima metodom imunofluorescencije. Granične pozitivne spolne razlike u korist ženki na razini nukleinskih kiselina potvrđene su i western blot analizom. Izrazito snažan intenzitet vakuoliziranog imunofluorescencijskog obojenja u mužjaka unio je nedosljednost u rezultate te zahtjeva provjeru samog protutijela do konačne interpretacije dobivenih rezultata, koji su suprotni od očekivanih i za sada ne potvrđuju povezanost promjena ekspresije metalotioneina sa MsrB3 enzimom u bubrezima.
Abstract (english) Oxidative stress is a state of an altered redox balance caused by unequal production and depletion of reactive molecular species. In physiological conditions, reactive oxygen species (ROS) serve as important signaling molecules and are involved in cell viability. When their concentration is heightened, they are considered to be the main drivers of the aging process due to their ability to disrupt the integrity of all macromolecules. Senescence or aging is a state in which a cell progressively falls into metabolic and physiological disbalance caused by extensive damage to its components. A cell usually enters senescence due to irreparable DNA damage. If the damage is not extensive, the cell is subjected to repair. Otherwise, the damage will lead to the activation of mechanisms involved in cell death. The effect of ROS molecules on proteins is observed through the sulfur-containing amino acid methionine (Met, M), which is readily oxidized in said conditions. Methionine sulfoxide (MetO) is present in two epimers - S and R. The two epimers are reduced by enzymes, part of the methionine sulfoxide reductase (Msr) family, Msr A and Msr B. Because of stereospecificity, MsrA reduces the S-epimer while MsrB reduces the R-epimer of MetO. The main goal of this master's thesis was to elucidate the existence of possible sex and age differences consistent with the changes found in methalotionein expression. Methalotionein expression is dominant in females, heightens with age and is potentially involved in MsrB3 regeneration. On the basis of MsrB3 expression in rat kidneys, we produced the target mRNA using the RT-PCR method, established the presence of proteins in tissue homogenates with western blot analysis and the overall distribution of proteins in tissue samples via imunnofluorescent staining. Borderline positive differences were confirmed in females following the nucleic acid analysis and western blot analysis, but the immunofluorescent staining produced inconsistent results with intense vacuolar staining in males, so antibody specificity has to be reviewed.. The final results were contrary to the ones expected and at present do not confirm the correlation between metalothionein and MsrB3 expression in the kidneys.
Keywords
MsrB3
metalotionein
oksidativni stres
bubreg
senescencija
Keywords (english)
MsrB3
metallothionein
oxidative stress
kidney
senescence
Language croatian URN:NBN urn:nbn:hr:217:209045 Project Number: IP-2013-11-1481 Project Number: 533-03-23-0006 Project Number: KK.01.1.1.02.0007 Study programme Title: Experimental Biology-University graduate programme Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2024-03-13 12:02:19
