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Browsing by Subject "western blot"

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  • The effect of NETO2 on synaptosomal kainate receptor subunit levels in fear-related brain regions 
    Rydgren, Emilie (2018)
    Kainate receptors (KARs) are glutamate receptors that modulate neurotransmission and neuronal excitability. They assemble from five subunits (GRIK1-5 or GluK1-5) present at both pre- and postsynaptic membranes. KAR function is regulated by neuropilin and tolloid-like (NETO) proteins, which also regulate postsynaptic GRIK2 abundance. Some KAR subunit gene variants associate with psychiatric disorders. Moreover, Grik1, Grik2 and Grik4 knock-out (KO) mice display changes in anxiety- and fear-related behaviours. In previous work, Neto2 KO mice expressed higher fear and impaired fear extinction in the fear conditioning paradigm. We hypothesised that this phenotype could be due to reduced KAR subunit abundance in fear-related brain regions, i.e. ventral hippocampus, amygdala and medial prefrontal cortex (mPFC). We specifically investigated GRIK2/3 and GRIK5 levels in the subcellular synaptosomal (SYN) fraction using western blot. We did not observe any difference between genotypes in any of the brain regions. However, our statistical power may have been insufficient, particularly for amygdala and mPFC. Also, an effect on synaptic KAR subunit abundance might be specific to either pre- or postsynaptic compartment, and thus more difficult to detect in SYN fractions. Alternatively, NETO2 absence may affect KAR actions instead of their subunit levels in fear-related brain regions, which could be examined through electrophysiological recordings. Ultimately, unravelling how a molecular system without NETO2 gives rise to fear behaviour in mice may lead to a better understanding of fear-related disorders in human and to new therapeutic strategies.
  • The presence of glycoprotein VI on platelet-derived extracellular vesicles 
    Pessi, Emilia (2024)
    Platelets originate from megakaryocytes and therefore contain the same receptors. This also applies to the extracellular vesicles (EVs) they release into the bloodstream. Glycoprotein VI (GPVI) is an activating collagen receptor on platelets. It plays an essential role in platelet biology by binding to collagen and activating platelets, leading to generation of EVs. Regulation of hemostasis involves shedding of GPVI from activated platelets, leading to a soluble fragment of GPVI. Soluble GPVI is used as a biomarker for diseases. According to current literature, GPVI is present on megakaryocyte-derived EVs but not on platelet-derived EVs (pEVs), as it is considered that activation of platelets leads to proteolytic cleavage of GPVI. Research on the presence of GPVI on pEVs is so far limited and the results are inconsistent. Based on alternate finding on the presence of GPVI on pEVs using proteomics (Palviainen et al. 2024), the aims of this project were to investigate the presence of GPVI on pEVs and to compare the presence of GPVI on megakaryocyte-derived EVs and pEVs. The presence of GPVI on pEVs was investigated by using multiple set of samples which could express GPVI differently. Platelets from platelet concentrate were isolated, activated, removed after activation and the samples were analysed with flow cytometry. Isolated pEVs were analysed with dot blot and western blot. To obtain megakaryocyte-derived EVs, K562 cell line was differentiated to megakaryocyte-like cells and EVs were isolated from cell conditioned media. The presence of GPVI on pEVs and megakaryocyte-derived EVs was compared with western blot. GPVI was found on pEVs. However, an expected difference in the presence of GPVI between pEVs from activated and unstimulated platelets was not observed. The results also indicated a higher amount of GPVI in megakaryocyte-derived EVs compared to pEVs, but further optimization of the methods used is required for more reliable results. GPVI, previously thought to exist only on megakaryocyte-derived EVs in the circulation or in soluble form cleaved from activated platelets, may actually be present on pEVs. Distinguishing the presence of GPVI between megakaryocyte-derived EVs and pEVs, is relevant when using the receptor as a biomarker. The results of this study are a foundation for further investigation of GPVI on pEVs to elucidate this exciting discrepancy.

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