Browsing by Subject "actin"
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(2016)Actin is known as abundant cytoplasmic protein, which functions as a component of the cytoskeleton and in cell motility together with motor protein myosins. However, actin is also present in the nucleus, where it has been shown to take part in the control of gene expression, both independently and as part of chromatin remodeling complexes. An important aspect in the study of nuclear actin is to identify other nuclear proteins interacting with actin and to confirm these interactions in biochemical experiments. Through these interactions actin can be linked to various nuclear processes. The aim of this master’s thesis study was to express and purify five nuclear proteins that have been suggested to bind actin and to study the binding in detail both with actin filaments and monomers. The proteins of interest include four actin-related proteins (ARPs) Arp4, Arp5, Arp6 and Arp8 as well as RNA polymerase II subunit Rpb8. Out of the proteins selected for this study, the expression and purification of Arp4 and Rpb8 was successful. Neither one did bind monomeric actin with high affinity, but interestingly Rpb8 did bind actin filaments. On the other hand, Rpb8 did not have any effect on actin polymerization. These results provide new insights into nuclear actin function. It has been suggested earlier that Arp4 would form a heterocomplex with actin, but the results of this study do not support this. The binding between Rpb8 and actin in RNA polymerase II complex has not been extensively studied before, so the results provide new information about the function of actin in the polymerase complex.
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(2010)Suomalaisen perinnöllisen gelsoliiniamyloidoosin syntymekanismit ovat vielä epäselviä. Tässä tutkimuksessa pyrittiin selvittämään, mitkä muut syyt voisivat johtaa gelsoliiniamyloidoosissa ilmenevien oireiden syntyyn amyloidisäikeiden muodostumisen lisäksi. Potilaasta eristettyjä sileälihassoluja kasvatettiin ja niistä eristettiin proteiinit. Näiden solujen proteiiniekspressiota verrattiin kontrollisileälihassolujen proteiiniekspressioon. Ekspressio erot selvitettiin SDS-PAGE-analyysillä ja Coomassie-värjäyksellä sekä anti-FAF-, alfa-aktiini- ja beeta-aktiini-vasta-ainevärjäyksillä. Proteiinit tunnistettiin massaspektrometrillä. Tässä tutkimuksessa tunnistetut kaikki viisi proteiinia (alfa-aktiini, anneksiini A1, anneksiini A2, anneksiini V ja vimentiini), joiden ekspressiot erosivat gelsoliiniamyloidoosia sairastavan potilaan- ja kontrolli-sileälihassolujen välillä, ovat yhteyksissä solun tukirankaan ja erityisesti aktiinisäikeiden muokkaukseen. Lisäksi solut värjätiin alfa-aktiinivasta-aineella. Aktiinitukiranka oli rikkonainen verrattuna kontrollisolujen aktiinitukirankaan. Tämän tutkimuksen perusteella voidaan siis olettaa gelsoliiniamyloidoosin oireiden johtuvan amyloidin kertymisen lisäksi viallisesta aktiinitukirangan homeostasiasta. Amyloidin muodostuminen on kuitenkin yksi tärkeimmistä oireiden syistä. Koska gelsoliiniamyloidoosiin ei ole vielä olemassa spesifistä hoitoa, olisi sellainen tärkeää löytää. Siksi tässä tutkimuksessa selvitettiin ryhmämme kehittämän amyloidi-inhibiittori kykyä estää amyloidisäikeiden muodostuminen Tioflaviini-T-mittauksilla (ThT). Inhibiittori osoittautui tehokkaaksi amyloidisäikeiden muodostumisen estäjäksi. Amyloidi-inhibiittori voisikin olla tulevaisuudessa lääke gelsoliiniamyloidoosiin.
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(2022)Abstract Background. Faba bean (Vicia faba L.,) is a protein-rich grain crop and a good source of many nutrients. However, the productivity and composition of this crop can be limited by several biotic and abiotic stresses. Acid soil leads to a reduction in water uptake, and influences on major biological and chemical processes. However, some plants can manage stress. Mostly, low pH reduces plant vegetative organs and a has a negative effect on physiological processes such as gas exchange, photosynthesis, chlorophyll concentration, and transpiration. The aim of this study was to find homolog genes in close spp. and o test selected gene response as a factor in resilience to rootzone acidity in Vicia faba L. Methods. Four faba bean genotypes were selected for our experiment based on germplasm screening in aquaponic conditions from a previous study. According to obtained results of morpho-physiological measures from a previous experiment, IG11204 and Bourdon showed tolerance to acid only. DULUC is tolerant to acid and Al treatments. In contrast, Nora38 demonstrated susceptibility to both stresses. Selected genotypes were grown under the same experimental conditions in order to proceed with samples for further analysis (total RNA extraction, cDNA synthesis, RT-PCR, gel-electrophoresis, validation of selected gene by RT-qPCR). Results. Selected genes have been identified as hub genes in A. thaliana (alternative oxidase 1A, glutamate dehydrogenase 1, zinc fingers superfamily protein), which demonstrated an increase in expression levels under acidic pH stress. We have found homolog genes in the Hedin cultivar. Selected homolog gene were used to check its expression in four genotypes under Al3+ and acidic stresses using RT-qPCR. Finally, up-regulation of the GDH1 was detected by acid and also occurred by Al3+ treatments. Discussion. Our analysis of gene expression revealed that GDH1 was expressed differently between control and treated groups. However, its activity was detected for both stresses. The expression of GDH1 transcripts in this study, also concurs with findings where GDH1 in A. thaliana were expressed under Al3+ stress. Furthermore, it was confirmed that they are novel Al3+ resistance genes, which are regulated by the transcription factor STOP1 (sensitive-to-proton-rhizotoxicity1). GDHs are involved in stress tolerance, and pH-regulated metabolic pathways – GABA shunt. It participates in the regulation of cytosolic pH, carbon fluxes into the TCA cycle, nitrogen metabolism, deterrence of insects, protection against oxidative stress, osmoregulation and signaling in plant cells under different stress conditions. The GABA shunt is enhanced by stresses, which cause cytosol acidification. Genes expressed in conditions of low pH partly overlap with those expressed in plants exposed to Al3+ stress. Generally, acidic soil is associated with the co-occurrence of Al3+ and H+ rhizotoxicities.
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(2019)Epithelial cells form a barrier between the tissue and the external environment. Epithelial morphogenesis refers to the shaping of epithelial layers and is a key step in the development of organisms. The actin cytoskeleton provides the cell its form and during epithelial morphogenesis, produces force to shape the cells. To achieve this, the actin cytoskeleton is organized into protrusive and contractile networks. In a living cell, these actin networks are dynamic, as the filaments are constantly undergoing assembly and disassembly. Actin-binding proteins regulate the turnover of actin filaments, but in epithelial morphogenesis, the regulatory role of most of these proteins is still relatively unknown. In all multicellular organisms, actin disassembly is controlled by ADF/cofilin. ADF/cofilin activity is furthermore enhanced by other actin-binding proteins, one of which is cyclase-associated protein (CAP). CAP promotes actin turnover by accelerating ADF/cofilin mediated actin disassembly and in recycling actin monomers to sites of actin polymerization. Unlike ADF/cofilin that regulates actin disassembly throughout the whole cell, CAP could be subject to more specific spatial regulation, as loss of CAP leads to F-actin accumulation on the apical side of epithelial cells. However, the role of CAP in morphogenetic cell rearrangements remains poorly known. In addition, the in vivo role of the biochemical functions of CAP has not been elucidated. The aim of this master’s thesis is to describe the role of CAP in regulating the actin cytoskeleton in the follicular epithelium of the fruit fly Drosophila melanogaster. For this purpose, chimeric mutant flies with homozygous CAP loss of function mutation were generated. Subsequently, the effect of the CAP loss of function was observed in follicle cell populations undergoing morphogenetic changes. In addition, CAP loss of function was rescued with different transgenes producing mutant CAP proteins to identify the protein domains of CAP with in vivo significance. In addition, a Drosophila CAP specific antibody was purified to be used in immunostaining. The ovaries were imaged using confocal microscopy. In this thesis, it is shown that CAP loss of function caused accumulation of filamentous actin in all observed follicular cell populations. Surprisingly, the actin turnover was rescued by all of the used CAP rescue transgenes, but the mutant transgenes exhibited phenotypes resembling the CAP loss of function in other epithelial tissues. Moreover, CAP loss of function caused defects in the follicle cell movement and cell spreading. The loss of function also caused expression changes in other actin-binding proteins. The findings of these thesis support the current knowledge of CAP importance for functional actin turnover in the follicle cells, even though the protein domain necessary for in vivo function could not be deciphered. Moreover, this project provides indication that CAP has an indispensable role in dynamic morphogenetic processes in the epithelium. Together with other actin-binding proteins, CAP could regulate epithelial actin turnover in spatially directed manner, providing force for epithelial cell adhesions or protrusions.
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(2019)Epithelial cells form a barrier between the tissue and the external environment. Epithelial morphogenesis refers to the shaping of epithelial layers and is a key step in the development of organisms. The actin cytoskeleton provides the cell its form and during epithelial morphogenesis, produces force to shape the cells. To achieve this, the actin cytoskeleton is organized into protrusive and contractile networks. In a living cell, these actin networks are dynamic, as the filaments are constantly undergoing assembly and disassembly. Actin-binding proteins regulate the turnover of actin filaments, but in epithelial morphogenesis, the regulatory role of most of these proteins is still relatively unknown. In all multicellular organisms, actin disassembly is controlled by ADF/cofilin. ADF/cofilin activity is furthermore enhanced by other actin-binding proteins, one of which is cyclase-associated protein (CAP). CAP promotes actin turnover by accelerating ADF/cofilin mediated actin disassembly and in recycling actin monomers to sites of actin polymerization. Unlike ADF/cofilin that regulates actin disassembly throughout the whole cell, CAP could be subject to more specific spatial regulation, as loss of CAP leads to F-actin accumulation on the apical side of epithelial cells. However, the role of CAP in morphogenetic cell rearrangements remains poorly known. In addition, the in vivo role of the biochemical functions of CAP has not been elucidated. The aim of this master’s thesis is to describe the role of CAP in regulating the actin cytoskeleton in the follicular epithelium of the fruit fly Drosophila melanogaster. For this purpose, chimeric mutant flies with homozygous CAP loss of function mutation were generated. Subsequently, the effect of the CAP loss of function was observed in follicle cell populations undergoing morphogenetic changes. In addition, CAP loss of function was rescued with different transgenes producing mutant CAP proteins to identify the protein domains of CAP with in vivo significance. In addition, a Drosophila CAP specific antibody was purified to be used in immunostaining. The ovaries were imaged using confocal microscopy. In this thesis, it is shown that CAP loss of function caused accumulation of filamentous actin in all observed follicular cell populations. Surprisingly, the actin turnover was rescued by all of the used CAP rescue transgenes, but the mutant transgenes exhibited phenotypes resembling the CAP loss of function in other epithelial tissues. Moreover, CAP loss of function caused defects in the follicle cell movement and cell spreading. The loss of function also caused expression changes in other actin-binding proteins. The findings of these thesis support the current knowledge of CAP importance for functional actin turnover in the follicle cells, even though the protein domain necessary for in vivo function could not be deciphered. Moreover, this project provides indication that CAP has an indispensable role in dynamic morphogenetic processes in the epithelium. Together with other actin-binding proteins, CAP could regulate epithelial actin turnover in spatially directed manner, providing force for epithelial cell adhesions or protrusions.
Now showing items 1-5 of 5