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The extremely radiation and desiccation resistant bacterium Deinococcus radiodurans possesses three genes encoding Endonuclease III-like enzymes (DrEndoIII1, DrEndoIII2, DrEndoIII3). In vitro enzymatic activity measurements revealed that DrEndoIII2 is the main Endonuclease III in this organism, while DrEndoIII1 and 3 possess unusual and, so far, no detectable EndoIII activity, respectively. In order to understand the role of these enzymes at a cellular level, DrEndoIII knockout mutants were constructed and subjected to various oxidative stress related conditions. The results showed that the mutants are as resistant to ionizing and UV-C radiation as well as H2O2 exposure as the wild type. However, upon exposure to oxidative stress induced by methyl viologen, the knockout strains were more resistant than the wild type. The difference in resistance may be attributed to the observed upregulation of the EndoIII homologs gene expression upon addition of methyl viologen. In conclusion, our data suggest that all three EndoIII homologs are crucial for cell survival in stress conditions, since the knockout of one of the genes tend to be compensated for by overexpression of the genes encoding the other two.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

The development of a lipid nano-delivery system was attempted for three specific poly (ADP-ribose) polymerase 1 (PARP1) inhibitors: Veliparib, Rucaparib, and Niraparib. Simple lipid and dual lipid formulations with 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1′-glycerol) sodium salt (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phosphocoline (DPPC) were developed and tested following the thin-film method. DPPG-encapsulating inhibitors presented the best fit in terms of encapsulation efficiency (>40%, translates into concentrations as high as 100 µM), zeta potential values (below −30 mV), and population distribution (single population profile). The particle size of the main population of interest was ~130 nm in diameter. Kinetic release studies showed that DPPG-encapsulating PARP1 inhibitors present slower drug release rates than liposome control samples, and complex drug release mechanisms were identified. DPPG + Veliparib/Niraparib presented a combination of diffusion-controlled and non-Fickian diffusion, while anomalous and super case II transport was verified for DPPG + Rucaparib. Spectroscopic analysis revealed that PARP1 inhibitors interact with the DPPG lipid membrane, promoting membrane water displacement from hydration centers. A preferential membrane interaction with lipid carbonyl groups was observed through hydrogen bonding, where the inhibitors’ protonated amine groups may be the major players in the PARP1 inhibitor encapsulation mode.

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase with specificity for a broad range of oxidized DNA lesions. The genome of an extremely radiation- and desiccation-resistant bacterium, Deinococcus radiodurans, possesses three genes encoding for EndoIII-like enzymes (DrEndoIII1, DrEndoIII2 and DrEndoIII3), which reveal different types of catalytic activities. DrEndoIII2 acts as the main EndoIII in this organism, while DrEndoIII1 and 3 demonstrate unusual and no EndoIII activity, respectively. In order to understand the role of DrEndoIII1 and DrEndoIII3 in D. radiodurans, we have generated mutants which target non-conserved residues in positions considered essential for classic EndoIII activity. In parallel, we have substituted residues coordinating the iron atoms in the [4Fe-4S] cluster in DrEndoIII2, aiming at elucidating the role of the cluster in these enzymes. Our results demonstrate that the amino acid substitutions in DrEndoIII1 reduce the enzyme activity without altering the overall structure, revealing that the residues found in the wild-type enzyme are essential for its unusual activity. The attempt to generate catalytic activity of DrEndoIII3 by re-designing its catalytic pocket was unsuccessful. A mutation of the iron-coordinating cysteine 199 in DrEndoIII2 appears to compromise the structural integrity and induce the formation of a [3Fe-4S] cluster, but apparently without affecting the activity. Taken together, we provide important structural and mechanistic insights into the three EndoIIIs, which will help us disentangle the open questions related to their presence in D. radiodurans and their particularities.

I denne studien undersøkes betydningen av den koronarelaterte nedstengningen av norsk arbeidsliv for opplevelsen av jobbfellesskap. Dette gjøres ved å undersøke 1) om det har vært noen endring i opplevd jobbfellesskap fra tiden like før nedstengningen 12. mars 2020 til slutten av juni 2021 og 2) betydningen av koronarelaterte endringer i arbeidssituasjonen. I analysene av endring over tid benyttes data fra spørreskjemaundersøkelsene Medbestemmelsesbarometeret 2020 (N=3575) og Pandemiundersøkelsen 2021 (N=3067). Endringene i arbeidssituasjonen studeres i pandemiutvalget, og vi ser mer konkret på betydningen av hjemmekontor, permitteringer, endringer i arbeidsoppgaver og arbeidstid. Resultatene viste ingen endring i opplevelsen av jobbfellesskap fra før pandemien til juni 2021. Utviklingen over tid var imidlertid forskjellig på Sør- og Vestlandet sammenliknet med i Oslo og omegn. Når det gjelder endringene i arbeidssituasjonen, fant vi ingen sammenheng for hjemmekontor eller permitteringer. Sammenhengen mellom endring i arbeidsoppgaver og opplevd jobbfellesskap kunne heller ikke påvises med sikkerhet. Analysen viste videre at både reduksjon og økning i arbeidstid var negativt korrelert med opplevelsen av jobbfellesskap. Funnene diskuteres opp imot ulike perspektiver på fellesskapsdannelse og organisatoriske endringer. Studien bidrar til å belyse hvordan opplevelsen av jobbfellesskap kan være på det samme nivået til tross for store endringer i sosiale og materielle strukturer.