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Document Type |
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Thesis |
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Document Title |
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Expression and Characterization of G-protein Coupled Receptor Proteolytic Site of Polycystic Kidney Disease-1 Gene Product التعبير الجيني وتوصيف منطقة مستقبلات ج-بروتين المقترنة الخاصة بجين-1 لداء تكيس الكلى المتعدد |
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Subject |
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Faculty of Science |
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Document Language |
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Arabic |
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Abstract |
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Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent hereditary disorder primarily affecting the kidneys. It is caused by mutations in either the PKD1or PKD2 genes, encoding the proteins polycystin-1 and polycystin-2, respectively. ADPKD is characterized by aberrant cell proliferation, perturbed extracellular matrix dynamics, and disrupted cellular polarity, resulting in the formation of renal cysts. The PKD1 gene mutation accounts for approximately 85% of ADPKD cases. This thesis focuses on investigating the role of the G-proteolytic site domain (GPS) within polycystin-1, a membrane protein with distinct peptide domains implicated in cell-cell and cell-matrix interactions. The primary objective is to elucidate the interactions between the GPS domain and various components of the extracellular matrix (ECM), aiming to uncover their significance in healthy kidney development and the underlying mechanisms driving ADPKD pathogenesis. To accomplish this, the GPS region of polycystin-1 was amplified from genomic DNA and subsequently cloned into the pET-21a(+)-MBP(TEV) expression vector. The resulting fusion protein, MBP-His-GPS, was expressed in Escherichia coli and purified utilizing an immobilized metal affinity chromatography (IMAC) column. Functional characterization of MBP-GPS was conducted by examining its impact on the proliferation of human embryonic kidney epithelial cells (HEK-293). The findings revealed a significant dose-dependent reduction in cell proliferation rates upon exposure to the fusion protein. Furthermore, pull-down experiments employing MBP-GPS were performed on HEK-293 cells to identify putative interacting proteins associated with the GPS domain. The resulting cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the peptides resulting from tryptic digestion were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.
Key words: Polycystin-1, ADPKD, G-proteolytic site (GPS) domain. |
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Supervisor |
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Dr. Hala Salim Sonbol |
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Thesis Type |
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Doctorate Thesis |
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Publishing Year |
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1445 AH
2023 AD |
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Added Date |
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Wednesday, December 27, 2023 |
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Researchers
| الاء مقبل السرحاني | Al-sirhani, Alaa Muqbil | Researcher | Doctorate | |
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