Browsing by study line "ei opintosuuntaa"

Antibiotic-resistant bacterial infections are a silently spreading pandemic that endangers public health and the healthcare system globally. Common infections may become more life-threatening, and hospital-acquired multi-drug-resistant infections soon compromise all medical procedures, such as surgeries and chemotherapy treatments. Two major players affecting the effectiveness of antimicrobial therapy against polymicrobial infections are interactions between bacterial species and biofilm formation. Bioiflm-embedded cells are protected from various threats, such as the host immune system and antibiotic interventions in the commensal polymicrobial community of increased virulence. Given the increasingly limited options of antibiotics against biofilm-associated antimicrobial-resistant infections, novel therapeutic strategies are needed. Phage therapy has regained interest as a promising strategy for treating antibiotic-resistant bacterial infections and limiting the evolution of resistance. In particular, phage-antibiotic combination therapy has been shown to be more efficient in treating pathogenic bacteria than using either one alone. In this study, I aimed to find a phage-antibiotic combination therapy against the formation of single and dual-species biofilm of S. aureus and P. aeruginosa and demonstrate the therapeutic potential of phages in combination with antibiotics by using a simple but clinically relevant in vitro biofilm model that supports the concomitant growth of P. aeruginosa and S. aureus. I found out that using phage Stab21 with ciprofloxacin or vancomycin alone or in combination was more effective in preventing the biofilm formation of S. aureus than using phage or antibiotic therapy alone. This was observed in the single-species biofilm of S. aureus and the dual-species biofilm in coculture with P. aeruginosa. Even though Stab21 alone could not infect S. aureus in liquid culture, adding ciprofloxacin or vancomycin at sublethal concentrations increased phage production.

The orexin system is an important regulator of the sleep/wake cycle, and small molecule agonists of the orexin receptors could be beneficial for patients suffering from type 1 narcolepsy. To develop new therapeutics, understanding the interactions between the orexin peptides and their cognate receptors is crucial. The three-dimensional arrangement of the orexin-A peptide complexed to its cognate orexin-2 receptor has so far remained elusive. Here, we identify structurally conserved regions at the predicted binding site and conserved residues of the orexin-A peptide by comparing orexin 2 receptor sequences from nine diverse species as well as with insect allatotropin receptor, a distant relative, and orexin-A sequences from 10 different species. We also visualized the conservation of interaction networks in the orexin 2 receptor binding site by building homology models of the putative orexin receptor of Ciona intestinalis and the allatotropin receptor of Manduca sexta. Structural conservation in the binding site is concentrated on transmembrane segments 2-3-7, in particular the salt bridge between D2.65 and H7.39, and a hydrogen bond network between Q3.32-T2.61-Y7.43. Conservation in orexin-A is concentrated on the C-terminus, while the most conserved individual residues of the peptide are L20, G29, I30, and L31. Applying our conservation results into the analysis of two previously suggested binding modes for orexin-A shows that one of the two demonstrates better mirroring of the conserved residues between the peptide and the binding site. Finally, our data shows that the hydrophobic side of helix 1 of the amphipathic orexin-A should be seriously considered in the analysis of binding modes.

In recent years, animals have been recognized as promising next-generation protein production systems. Animal transgenesis has been achieved primarily in insect cells infected by recombinant baculoviruses. Baculovirus Expression Vector Systems (BEVS) transform the DH10Bac strain of Escherichia coli with the shuttle vector to produce recombinant baculovirus carrying the cargo of interest. The cargo includes at least one promoter driving the expression of at least one protein. PGK is a strong promoter that is naturally active in almost all species where it has been tested, including invertebrates like Drosophila. The PiggyBac transposon-based system is a known strategy for genome integration of foreign genes to create transgenic animals. Nevertheless, nobody has used baculoviruses to deliver genes and produce proteins in earthworms nor to create transgenic earthworms. There is also no information on the sequence of any endogenous E. fetida (earthworm) promoter yet. This project aimed to pilot a novel gene delivery method by creating baculoviruses through the BEVS, carrying the PGK promoter and the GFP reporter gene, and to assess the promoter activity in both Sf9 insect cells and E. fetida through evaluation of GFP fluorescence. Another target was to test the fluorescence after the addition to the baculovirus of the PiggyBac-based inverted terminal repeats (ITRs), flanking the PGK-GFP transcriptional unit. The secondary objective was to develop a non-lethal method for live worm imaging. Conventional restriction enzyme cloning was used to create the shuttle vectors, and restriction digest and Sanger Sequencing were used to identify the positive clones. The Bac-to-Bac BEVS was followed to create baculovirus particles carrying the cargo (PGK-GFP and PGK-GFP-ITR), infect Sf9 insect cells and monitor the PGK activity. Prior to in vitro transfection, the bacmid DNA was confirmed by PCR. These baculoviruses were also used to infect E. fetida and monitor the PGK activity in vivo. E. fetida autofluorescence was assessed before infection. PGK resulted in being much weaker in Sf9 than expected. The flanking of the transcriptional unit of GFP with the ITRs improved the GFP expression. 16% ethanol was shown to anaesthetize E. fetida for 10 to 15 minutes safely. Wild-type and starved E. fetida were shown to have very mild autofluorescence in their digestive system and setae. The coelomic fluid was shown to have strong autofluorescence. Thus, its excretion is crucial before imaging GFP. Likely, all the in vivo fluorescence after infection was due to the worm’s autofluorescence. Therefore, PGK and GFP were unlucky choices for E. fetida.