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Browsing by master's degree program "Magisterprogrammet i farmaceutisk forskning, utveckling och säkerhet"

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  • Juntunen, Maiju (2020)
    Cancer immunotherapy refers to therapy strategies that utilise the mechanisms of the immune system to treat cancer patients. The benefits of the approach include the possibility for specific targeting and utilisation of the host immune system. The treatment methods include cancer vaccines, oncolytic viruses (OVs), cell-based immunotherapies and antibodies. The interplay between the cancer and the immune system has been observed crucial for the progress of the cancer and the success of immunotherapies. An immune inflamed tumour microenvironment has been observed beneficial for the success of several therapy methods. Many immunotherapy methods rely on detecting tumour specific antigens that are used to guide the therapy agent to the target site. This strategy poses challenges when considering tumour immune evasion mechanisms, which can cause downregulation of target antigens, and heterogeneity of tumour cells and patients. OVs have the advantage of not requiring predetermined target structures to exert their effect to the tumour cells. They cause direct tumour cell lysis and induce immune responses, and may be modified to express additional genes, including immunostimulatory agents. However, virus-related immunosuppressive mechanisms and a rapid viral clearance may limit their effects. A Western Reserve (WR) Vaccinia virus (VACV) is a highly oncolytic virus strain but the virus has been observed to suppress the function of the cyclic guanosine monophosphate adenosine monophosphate synthase – stimulator of interferon genes (cGAS STING) innate immune pathway which has been shown to have a significant role in anti-tumour immune responses. The aim of this study was to create a WR VACV encoding a dominantly active (D A) STING and to determine whether the virus is capable of activating the cGAS STING pathway. The effects were compared to a corresponding virus vvdd tdTomato that does not have the STING encoding gene. The pathogenicity of viruses was controlled by a double deletion of the thymidine kinase and vaccinia growth factor genes which restricts the virus replication to tumour cells. Transgene fragments were cloned from template plasmids by polymerase chain reactions (PCRs) and joined together in a Gibson Assembly (GA) reaction to form a STING-P2A-eGFP gene insert. The insert was attached to a shuttle vector pSC65-tdTomato by restriction enzyme digestion, ligation and transformation in Escherichia coli. The correct transgene plasmid construct was verified by Sanger sequencing and PCRs. The transgene was inserted to a modified WR VACV vvdd-tdTomato-hDAI by a homologous recombination. The newly created VVdd STING-P2A-eGFP virus was purified by plaque purification. The STING protein expression was studied by an immunocytochemistry (ICC) assay. The immune signalling pathway activation was examined by testing nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation in RAW-Blue cells and dendritic cell activation and maturation in JAWS II cells. The cell viability after iinfection was studied with four cell lines; A549, B16-F10, HEK293 and MB49. The D-A STING expressing virus was produced successfully. The ICC experiment verified the capability of the VVdd STING-P2A eGFP to produce the STING protein in the infected cells. The preliminary findings indicate that the VVdd STING-P2A-eGFP virus activates the NF-κB signalling in the RAW-Blue cells and that the activation is dependent on the STING expression. The activation level is relative to the infection concentration at MOI range 0,001 to 0,1. The findings suggest that the VVdd-STING-eGFP virus can induce innate immune signalling via the STING pathway. The reference virus did not activate the signalling. The in vitro experiments also indicated that the STING virus may induce DC activation and maturation. We observed a trend of CD86 and CD40 expression upregulation on the JAWS II DCs. The effects to the cell viability were inconclusive. More studies should be conducted to verify the results. The effects of the virus should be studied in more advanced cancer models that take into account the complexity of the immune system. These preliminary results indicate the that the VVdd-STING-P2A-eGFP virus could stimulate the immune signalling through the STING pathway.
  • Parviainen, Heli (2020)
    Statins are a commonly used group of drugs that reduce the cholesterol levels in blood and have been shown to reduce cardiovascular morbidity and mortality. However, a considerable percentage of patients experience adverse effects during statin treatment. Statin adverse effects have been associated with genetic polymorphisms and drug-drug interactions that affect the elimination and active transport of these drugs. A more comprehensive knowledge of statin metabolism may be a step towards better management of statin treatments. Statin metabolism both in vivo and in vitro has been subject of study for years. In vitro incubation conditions may considerably affect the observed clearance, and results obtained with different methods or in different laboratories may not be directly comparable to each other. No single in vitro study on a wide panel of statins has previously been conducted. Six statins and some of their metabolites, fourteen compounds in total, were included in the study. The intrinsic clearance (CLint) of these molecules was investigated in vitro on human liver microsomes (HLM) and a panel of eleven cytochrome P450 (CYP) enzymes recombinantly expressed in E. coli. Observed CLint values for each compound in HLM and for each compound-CYP pair with observed depletion were calculated. The percentual contributions of each CYP enzyme to the metabolism of the compounds was calculated. The results obtained with recombinant CYP enzymes (rcCYP) were complemented with studies on HLM with specific chemical inhibitors of CYP enzymes. In this study the metabolism of statin lactones seemed to be faster than the metabolism of the corresponding statin acids. Atorvastatin lactone, 2-hydroxy atorvastatin lactone, 4-hydroxy atorvastatin lactone and simvastatin were extensively metabolized. Atorvastatin, 2-hydroxy atorvastatin, 3R,5S-fluvastatin, 3S,5R-fluvastatin, pitavastatin lactone and simvastatin acid showed intermediate metabolism. 4-hydroxy atorvastatin, pitavastatin, pravastatin and rosuvastatin rates of metabolism were below quantification limit. CYP3A4 had a major role in the metabolism of atorvastatin and its metabolites, simvastatin and simvastatin acid. CYP3A4 also had activity towards pitavastatin lactone. CYP2C9 had a high activity towards both 3R,5S-fluvastatin and 3S,5R-fluvastatin. CYP2D6 may play a part in the metabolism of pitavastatin lactone. CYP2C8 may have some activity towards simvastatin and simvastatin acid. The data is mostly in agreement with previous in vitro and in vivo studies regarding both the metabolism rate of statins and the contributions by different CYP enzymes to the metabolism of statins. Due to the screening nature of the study and some methodological constraints, these data should be considered as preliminary and require confirmation in further studies.
  • 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.
  • 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.
  • 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.
  • Eriksson, Veronica (2020)
    Migraine was ranked as the second largest cause of disability in 2016 in the Global Burden of Disease (GBD) study. People with migraine have a greater disability and a lower health-related quality of life than those of the general population. Many migraine patients experience functional and emotional impairment due to their disease. Migraine can limit their daily activities and impact their private, professional and social life. Migraine affects the patient also in between the attacks and can impact their education, career and their family and loved ones. Comorbid diseases and failed treatment lines add to the burden of migraine. Furthermore, migraine also imposes an economic burden. Stigma is described as the hidden burden of disease. Chronic migraine patients have been found to have higher stigma than episodic migraine patients. Even though migraine is one of the most common disabling headache disorders, it is still both under-recognised, under-diagnosed and under-treated. The objectives of this study were to determine the extent of the burden and the stigma of migraine in adult Finnish migraine patients. This study aimed to produce comprehensive and current information about migraine and its severity in Finland, highlighting the burden it poses on the migraine patients as well as on society. Migraine is most prevalent among the working aged population, which increases the societal burden of the disease. This study was conducted as a cross-sectional electronic survey amongst adult Finnish migraine patients. The participants were contacted through the Finnish Migraine Patient Advocacy Group. The questionnaire consisted of the already existing and validated Migraine Disability Assessment (MIDAS) Questionnaire and of measures developed by the author. The final data consisted of 608 responses. Of all respondents with 8 or more headache days a month, over 90% were categorised in the severe disability group (MIDAS grade IV), thus having similar disability to those with 15 or more headache days a month (i.e. respondents with probable chronic migraine). The proportion of respondents with severe disability (MIDAS grade IV) was greater in the present study (65.0%) than in a study conducted in Finland in 2000 (47%), indicating that migraine disability in Finland might have become more severe during the past two decades. The mean level of headache pain in the present study was 6.2 (on a scale of 0-10) and pain was the aspect that most respondents viewed as the worst aspect of migraine. This highlights the importance of proper pain management in migraine care. Many of the respondents were also at risk for medication overuse, which highlights the importance of monitoring medication use and informing the patients about possible risks. Stress was reported as the most common migraine trigger, and reducing stress at the workplace was also reported as the most important way of how migraine could better be managed at the workplace. Almost half (44.4%) of all respondents felt stigmatised due to their migraine. Reasons for this stigma and suggested solutions on how to reduce/manage the stigma were quite similar. The ignorance of others was the most reported reason for their migraine stigma, and increasing awareness and correct information about migraine was the most reported way of reducing the stigma. Many of the respondents had faced, due to their migraine, belittlement at work, from family and friend and from healthcare professionals. Facing belittlement from healthcare professionals was reported to have happened often by 11.5% and sometimes by 34.7% of all respondents. Of all respondents, 55.6% worried often and 29.8% worried sometimes about the onset of the next migraine attack. The majority of the respondents had severe disability based on their MIDAS grades. Many other aspect of the burden were reported as well, inculding stigma, reported by almost half of the respondents. Further and future studies need to be conducted to get an even better understanding of the burden and stigma of migraine experienced by adult Finnish migraine patients. This includes further and more intricate quantitative and qualitative analyses of the data from this study, as´well as studies with new perspectives based on the results found in this study.
  • 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.
  • 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.