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Faculty of Pharmacy


Recent Submissions

  • Venäläinen Birgitta (2024)
    Antimicrobial resistance (AMR) is a growing global health concern, and the development of new antibacterial agents is crucial to addressing this issue. Commercial antibiotics are not as effective as they used to be to combat infections. Previous studies have demonstrated the promising antimicrobial activity of etrasimod and one of its derivatives, compound 24f, against Gram-positive species. Therefore, as part of this study, we modified the carboxylic acid functional group to produce new derivatives. We synthesized derivatives of etrasimod and 24f, in order to generate a variety of compounds for evaluation of their antimicrobial effectiveness. Furthermore, the study evaluates the compounds' in vitro antibacterial activity and in vivo efficacy using Caenorhabditis elegans worms as an infection model. C. elegans is a widely used model organism in biological research, and it is particularly useful for studying host-pathogen interactions and drug efficacy. In addition, the cytotoxicity on mammalian cells (HeLa) was determined. Compound 18 showed the lowest cytotoxicity level (CC50 = 75.71±14.4 µM) of tested compounds. The antibacterial activity of new etrasimod derivatives was tested against Gram-positive (Staphylococcus aureus, including methicillin-resistant strains (MRSA)) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli). The tested compounds showed activity against Gram-positive bacteria but not against any of the Gram-negative strains. Compounds 9 and 18 showed to be the most active compounds, having a minimum inhibitory concentration of 5–6 and 8–10 μM, respectively. Moreover, both compounds showed promising activity in vivo, being able to significantly reduce the bacterial load in infected worms and improve their survival rates in survival experiments. The study provides insights into the development and assessment of potential antibacterial agents, addressing the contemporary challenge of AMR. The study's findings suggest that compounds 9 and 18 could be potential candidates for further development as novel antimicrobial agents.
  • Pagès Guitart, Mireia (2024)
    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.
  • Hossain, Md Alamgir (2024)
    Vascular endothelial growth factor C (VEGF-C) is a key lymphangiogenic protein and potential therapeutic target, but its production in bioactive form from bacteria has been challenging. This study focused on the purification method of VEGF-C from Escherichia coli (E. coli) using three-step chromatographic approaches involving anion exchange, hydrophobic interaction, and size exclusion chromatography. While the major purified species migrated at the 35 kDa size, it lacked biological activity in a Ba/F-3-VEGFR-3 bioassay, likely due to interference from the partially cleaved maltose binding protein fusion tag. The maximum yield of 0.311 mg of VEGF-C per liter of bacterial culture represented a modest 3.11-fold increase over previous Ni-affinity chromatography but a 1.93-fold decrease compared to amylose affinity chromatography purification. Further work is needed to optimize tag removal and purification to enable larger-scale production of bioactive VEGF-C for research and potential therapeutic applications. While this study demonstrates conventional chromatography can purify VEGF-C, but highlights challenges in obtaining high yields of the bioactive VEGF-C.
  • Tiainen, Hanna (2024)
    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.
  • Baramaki, Iman (2024)
    This study aimed to optimize the iDISCO+ tissue clearing method for studying neural networks and proteins expression in intact brain tissue and to establish an efficient analysis pipeline, particularly for analyzing c-Fos-stained brain samples. Additionally, our objective was to utilize iDISCO for investigating the effects of the rapid-acting antidepressant nitrous oxide (N2O), an N-Methyl-D-Aspartate Receptor (NMDAR) antagonist, using c-Fos expression as a biomarker of neural activity. We conducted two experiments: initially, a small-scale study inducing myoclonic seizures with 10% flurothyl (N=6). The cleared samples, immunolabeled with an anti-c-Fos antibody, were imaged using light-sheet microscopy. Despite a good signal-to-noise ratio, we observed limitations in antibody penetration as well as issues with autofluorescence loss and high signal artifacts within the brain ventricles. These findings indicated the need for protocol modifications and optimization. Next, a larger-scale experiment (N=12) with 50% N2O treatment was conducted to investigate its impact on brain activity. DELiVR, a deep learning-based analysis pipeline, was used for cell detection and atlas alignment. Preliminary analysis with DELiVR showed promising results, highlighting the need for further refinement in optimizing the algorithm tailored to the specific datasets. Additionally, an observation suggesting heightened activity in the hippocampal formation and retrosplenial area following 50% N2O administration was noted, consistent with previous literature. While the optimized iDISCO+ protocol successfully addressed earlier challenges, additional research is required to fine-tune the analysis pipeline for reliable quantitative conclusions.