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Browsing by study line "Molecular and analytical health biosciences study track"

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  • Puutio, Johanna (2020)
    Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles that are secreted by eukaryotic and prokaryotic cells. EVs carry macromolecules and signalling molecules to adjacent cells and play an important role in intercellular communication under both pathologic and homeostatic conditions. Therefore, they have become of significant interest for their therapeutic, diagnostic and prognostic potential. EVs are small and highly heterogeneous in size, shape, cargo and membrane composition, posing several challenges for establishing analytical and clinical guidelines. Therefore, EV research requires standardized and robust methods for their separation and characterization. In this study physical and immunochemical methods were employed to characterize human platelet-derived EVs (pEVs) generated from platelets activated with different external biochemical stimuli. The platelet-activating effect of the pro-inflammatory S100A8/A9 protein complex and a combination of thrombin and collagen were studied with nano flow cytometry. The size distribution of pEVs was studied with nanoparticle tracking analysis (NTA) and asymmetrical flow field-flow fractionation (AF4), which represents a newly emerging method on the EV field. Finally, fluorescent labelling and co-localization analysis were employed to characterize membrane marker composition of pEVs and assess its usefulness as an analytic tool for EV research. We succeeded in providing new hints towards meaningful discoveries in platelet biology by characterizing the way platelets respond to inflammatory and hemostatic signals by shedding pEVs. When platelet activation markers are characterized with flow cytometry, the S100A8/A9 protein appeared to cause a shift in membrane activation markers when compared to the thrombin- collagen mix and the baseline control. Increased TLT-1 translocation and decreased integrin αIIbβ3 expression on pEV surfaces suggests that S100A8/A9 induced pEV secretion through differently packed platelet α-granules, rather than from the plasma membrane. An increase in TLT-1 expression compared to decreased P-selectin and αIIbβ3 suggests that S100A8/A9 stimulation shifts platelet phenotype towards secretion rather than aggregation. A protocol for small pEV separation with AF4-MALS was set up. With this method, subtle differences between small pEV populations were seen that were not distinguishable with NTA or flow cytometry. When investigated with AF4-MALS, S100A8/A9 induced pEVs appeared larger than those produced with thrombin- collagen activation. The mean particle sizes of the pEV populations obtained from activated platelets were generally also larger than those produced without an activator. We tested novel methods to detect subtle differences in small EV population sizes that are easily missed with conventional methods due to their technical limitations. A well-optimised AF4 protocol can detect different pEV subpopulations and is a promising tool for EV. In the future, when AF4 is combined with a MALS detector and a fraction collector, nanoimaging of fluorescently labelled EVs could be combined with it as a downstream application to obtain information on their versatile biological functions.
  • Saarela, Sanna (2020)
    Ischemic heart failure is the leading cause of death in the world. The disease is caused by coronary heart disease, in which the narrowed coronary arteries limit oxygen- and nutrient-rich blood from reaching the myocardial tissue. Obstructed arterial blood flow can cause myocardial necrosis and scarring. Scar tissue is non-contractile and poorly elastic. It can thus compromise the pumping capacity of the heart. Current medical and interventional therapies have only very limited efficacy to reduce myocardial scarring. Preclinical and clinical research efforts are underway to generate myocardial scar-reducing and regenerative therapies. In the field of cardiac cellular therapies, the delivery of cells has conventionally been based on intramyocardial injections. However, epicardial patches have been demonstrated to reduce scarring and promote myocardial healing. In addition to merely being a carrier or cover for the cellular transplant, the biomembrane of the patch can also be considered as an active element for the patch’s therapeutic activity. Thus, the properties of the biomembrane can have a major impact on both the cellular and the therapeutic tissue response. The aim of this Master's thesis was to build a standardized test set up to study the properties of the biomembrane. Biomembrane permeability to small (glucose, lactate) molecules and different size proteins was investigated. In addition, the set up was modified to enable the investigation of biomembrane properties on the survival of the grafted cells. Finally, the test set up was evaluated by studying the properties of ProxiCorTM, the biomembrane currently used together with autologous atrial micrografts (AAMs) in epicardial patch. As a result, the set up was successfully constructed and characterized. The ProxiCorTM membrane demonstrated permeability to both small molecules and proteins, and a stable pH was maintained across the membrane. ProxiCorTM enabled traverse serum-induced proliferation of cells compared to the control impermeable membrane. Taken together, these results prove the functionality of the test set up and thus support its further development.
  • Pająk, Daria Anna (2020)
    Influenza A viruses are pathogens infecting birds and selected mammals. They are responsible for around 500 000 human deaths each year and pose a substantial economic burden to the healthcare system. The most important pathway in influenza virus detection is a retinoic acid-inducible gene I pathway, which recognizes the 5’-triphosphate in viral RNA. Its activation leads to the production of interferons: a group of cytokines important in overcoming viral infection. In order to replicate successfully, viruses had to develop mechanisms to overcome host defences. They include, among others, regulation of interferons and interferon stimulated genes expression. During influenza A virus infection, this function is performed by viral non-structural protein 1 (NS1). The aim of this study was evaluating the effect of NS1 of five different avian influenza strains and one seasonal influenza strain on activation of type I and III interferon gene promoters. The NS1 of seasonal virus H3N2 shown the highest suppression of both interferon I and III promoters, while NS1 originating from avian H9N2 and H7N7 strains had limited effect on interferon promoter activation. NS1 of H5N1/04, H5N1/97 and H7N9 was very effective at suppressing interferon type I promoter, which correlates with the severity of the infection in humans. When it comes to interferon type III promoter, H7N9 was very efficient at the suppression, while NS1 of H5N1/04 had little impact on promoter activation. The study has provided more information on the efficiency of potentially pandemic avian influenza strains at inhibition of interferon response and may be a base for further research. The project was conducted at the Finnish Institute of Health and Welfare.