Browsing by Subject "Western blot"

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  • Syrjä, Pernilla; Anwar, Tahira; Pääkkönen, Tarja; Kyöstilä, Kaisa; Hultin Jäderlund, Karin; Cozzi, Francesca; Rohdin, Cecilia; Hahn, Kerstin; Wohlsein, Peter; Baumgärtner, Wolfgang; Henke, Diana; Oevermann, Anna; Sukura, Antti; Leeb, Tosso; Lohi, Hannes; Eskelinen, Eeva-Liisa (2017)
    A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.
  • Lindelöf, Anna-Emilia (Helsingin yliopisto, 2019)
    Tiivistelmä - Referat - Abstract Background. Platelets are known to contain ample amounts of brain derived neurotrophic factor. Previous spectrophotometric studies carried out in Pia Siljander’s lab have shown that BDNF is secreted from activated platelets packed in extracellular vesicles. For this project we wanted to 1) confirm that BDNF really is secreted in extracellular vesicles (EVs)2) find out how the choice of agonist affected the BDNF cargo of the platelet derived EVs, and 3) find out if the BDNF is packed into EVs of certain densities rather than others. Methods. The platelets were isolated from platelet concentrates by size exclusion chromatography. The isolated platelets were then activated by thrombin and collagen co-stimulation (TC) and by Ca2+ionophore, respectively. The platelet activation produced extracellular vesicles (PEVs) which were isolated by differential ultracentrifugation. The isolated PEVs were then analysed by flow cytometry, ELISA and Western blot for EV typical membrane surface proteins and for their BDNF content. As we were interested finding out whether BDNF is enriched in PEVs to certain populations, density gradient centrifugation was performed. These samples were also analysed by Western blot and by ELISA. The size distribution and concentration of PEVs in all samples was analysed by Nanoparticle tracking analysis. Results and conclusions. This study confirmed that platelets secrete PEVs as a response to agonists. PEVs with higher BDNF concentration were produced using TC co-stimulation as compared to PEVs derived from the Ca2+ionophore. The result implies that BDNF is actively packed into PEVs for instance as a thrombogenic response. Based on the density gradient results it seems that BDNF was packed into certain population of PEVs with a density between 1.112 g ml-1 and 1.132 g ml-1 corresponding to a particle diameter of less than 500 nm. The finding that BDNF is actively packed into TC co-stimulation derived PEVs of a certain population is interesting from a theragnostic point of view, since EVs are likely to be key players in the development of new cell-based therapies. Had there been more time, it would have been interesting to optimize both the density gradient protocol and the ELISA analysis. This optimization of methods would make the process more efficient, less prone to sample loss, not to mention that there would be less intra-assay variation.
  • Tavakoli, Ali (Helsingin yliopisto, 2015)
    Recently, a new muscular disorder has been reported in chicken M. pectoralis major called Wooden Breast that might be linked to intensive growth selection. The purpose of this study was to find the influence of Wooden Breast condition on protein composition of the breast muscle with special emphasis on myofibrillar and sarcoplasmic protein fractions. For fulfilling the aims of the study, a total number of 34 pectoralis major muscles from both Wooden Breast and normal chickens (Ross 508, Aviagen Ltd, Scotland) were used to evaluate protein composition, protein profile of sarcoplasmic and myofibrillar proteins and their changes, by doing one-dimensional SDS-PAGE analysis. M. Pectoralis major defected by Wooden Breast demonstrated significant decline in sarcoplasmic (P?0.001) and myofibrillar (P?0.05) protein content, in comparison with normal breast muscles. Furthermore, gel electrophoresis indicated significant changes in the intensity of 18 sarcoplasmic protein bands and 7 myofibrillar protein bands. Majority of affected sarcoplasmic proteins were glycolytic enzymes. Moreover, impacted myofibrillar proteins play a role in muscle fibre integrity (e.g. desmin) or calcium signalling. Results of the present study also revealed desmin overexpression with coexistence of a desmin fragment in Wooden Breast which was confirmed by Western-blot technique. In conclusion, the findings of this study indicated defected chicken breast with Wooden Breast contained less protein and the composition differed compared to normal chicken breast muscle, which proves the influence of Wooden Breast condition on protein characteristics of chicken breast muscle. Further studies are needed to interpret the protein changes in the Wooden Breast and the possible role of these changes on metabolic and structural status of the muscle.
  • Roininen (os. Mertanen), Saara (Helsingfors universitet, 2016)
    Munuaisen kehitystä on tutkittu laajasti. Yksi tunnetuimmista munuaisen kehitykseen liittyvistä tekijöistä on gliasoluperäisen neutrotrofisen kasvutekijän (Gdnf) aktivoima Ret-reseptorityrosinikinaasi (Ret). Ret:n aktivaatio aloittaa hiirimalleilla tehdyissä tutkimuksissa uretersilmun haarautumisen. Ret:n puutoksen on taas havaittu aiheuttavan munuaisen täydellisen puutoksen. Toinen munuaisen kehitykseen liittyvistä keskeisistä tekijöistä on hiljattain löydetty pleksiini B2 – reseptori (pleksiini B2), jota aktivoi Semaforiini 4C (Sema 4C). Reseptoriaktivaatio aiheuttaa hiirimalleissa munuaisputken haarautumista – aktivaation puutos puolestaan johtaa munuaisputken haarautumisen vähentymiseen, munuaisen pienikasvuisuuteen ja sikiön kuolemaan. Tutkielmani tavoite on tutkia pleksiini B2:n ja Ret:n välistä vuorovaikutussuhdetta erilaisissa solumalleissa ko-immunopresipitaation (Co-IP) ja Western blot – menetelmän avulla. Käytin tutkimuksessani apinoista eristettyä COS7-munuaissolulinjan soluja, joihin siirsin valmistamiani pleksiini B2 – ja Ret-geenikonstrukteja sisältäviä DNA-plasmideja. Plasmidit sisälsivät aminohappo-tunnisteosan, jolla helpotettiin vasta-ainedetektiota. Tutkimustulokseni viittaavat siihen, että reseptorit vuorovaikuttavat keskenään mallissa, jonka solut yli-ilmentävä pleksiini B2:sta ja Ret:iä. Tulos kannustaa selvittämään jatkossa, millainen tämä vuorovaikutus on biokemiallisesti ja toiminnallisesti muissa solumalleissa ja fysiologisissa pitoisuuksissa hiirten alkioiden munuaissoluissa.