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Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions : Resolving How the Epidermal Growth Factor Binds to Its ReceptorBjörkelund, Hanna January 2013 (has links)
Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now. This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates. In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.
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STRAIN-SPECIFIC PROTEIN INTERACTION AND LOCALIZATION OF TWO STRAINS OF POTATO YELLOW DWARF VIRUS AND FUNCTIONAL DOMAINS OF THEIR MATRIX PROTEINJang, Chanyong 01 January 2019 (has links)
Potato yellow dwarf virus (PYDV) is the type species of the genus nucleorhabdovirus which is typified by its nucleotropic characters of the members. The virus accomplishes its replication and morphogenesis in the nuclei of infected cells. Two strains, Constricta strain (CYDV) and Sanguinolenta strain (SYDV) have been described at the level of vector-specificity. CYDV is vectored by Agallia constricta and SYDV is transmitted by Aceratagllia sanguinolenta. The full-length genome of CYDV was sequenced. The 12,792 nt antisense genome encodes seven open reading frames in the order of, nucleocapsid protein (N), unknown protein (X), phosphoprotein (P), movement protein (Y), matrix protein (M), glycoprotein (G), and large polymerase protein (L). The features of each protein including a nuclear localization signal, isoelectric point, and transmembrane domain, were determined by predictive algorithms. The gene coding region was flanked by leader and trailer, and each ORF was separated by a conserved intergenic junction. In the intergenic junctions, the highly conserved cis-regulatory elements, polyadenylation signal, gene spacer, and transcription start site, were identified. The similarities of amino acid sequences between each cognate protein of SYDV and CYDV were higher than 80% except for X and P proteins. The protein localization and interaction assays of each CYDV protein identified strain-specific associations in comparison with those of SYDV and generated unique protein interaction and localization map compared to SYDV. Phylogenetic analysis using L protein identified that CYDV forms a clade with other leafhopper-transmitted rhabdoviruses. Protein sequence comparisons revealed that CYDV X has greater similarity to the cognate protein of Eggplant mottle disease virus than to SYDV X. The localization patterns of CYDV-N and -Y were different compared the cognate proteins of SYDV. The functional nuclear export domain of SYDV M was identified using c-terminal fragments of the Mwt(aa 211-243), MLL223AA(aa 211-243), and MKR225AA(aa 211-243). Based on the data, the functional domains M mediating membrane association, nuclear import and export were mapped for both strains and suggested a model whereby M mediates intra- and intercellular movement of PYDV nucleocapsid.
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