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Directed Migration Achieved in Mamma...

Qudrat, Anam.

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Directed Migration Achieved in Mammalian Cells via Ca 2+ Signal Rewiring in Synthetic Protein Chimeras.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Directed Migration Achieved in Mammalian Cells via Ca 2+ Signal Rewiring in Synthetic Protein Chimeras./
作者:	Qudrat, Anam.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
面頁冊數:	120 p.
附註:	Source: Dissertations Abstracts International, Volume: 80-10, Section: B.
Contained By:	Dissertations Abstracts International80-10B.
標題:	Biomedical engineering. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13426657
ISBN:	9781392016862

Directed Migration Achieved in Mammalian Cells via Ca 2+ Signal Rewiring in Synthetic Protein Chimeras.
Qudrat, Anam.

Directed Migration Achieved in Mammalian Cells via Ca 2+ Signal Rewiring in Synthetic Protein Chimeras. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 120 p.

Source: Dissertations Abstracts International, Volume: 80-10, Section: B.

Thesis (Ph.D.)--University of Toronto (Canada), 2019.

This item must not be sold to any third party vendors.

Synthetic biology achieves control over cellular behavior by endogenous re-wiring. Ca2+ signals allow cells to regulate diverse processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g. VEGFR2), Ca2+ signals are generated upon ligand binding (e.g. VEGF-A). Here, firstly we engineered fusion proteins that generate a Ca2+ signal upon ligand binding by creating fusions of domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we re-wired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Next, we validated this design strategy in two systems, i.e., inflammatory cytokines such as TNFα and antibodies. A system of proteins was used: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs allowing migration towards the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, post-ganciclovir treatment, cells undergo death via the thymidine kinase suicide mechanism. Hence, this forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low pH microenvironment. Lastly, we exploited antibodies to bind their antigens exclusively in combination with the ability of the cytoplasmic domain of VEGFR2 to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e. nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumour necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state but instead, only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.

ISBN: 9781392016862Subjects--Topical Terms:

535387
Biomedical engineering.

Directed Migration Achieved in Mammalian Cells via Ca 2+ Signal Rewiring in Synthetic Protein Chimeras.
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520 $a Synthetic biology achieves control over cellular behavior by endogenous re-wiring. Ca2+ signals allow cells to regulate diverse processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g. VEGFR2), Ca2+ signals are generated upon ligand binding (e.g. VEGF-A). Here, firstly we engineered fusion proteins that generate a Ca2+ signal upon ligand binding by creating fusions of domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we re-wired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Next, we validated this design strategy in two systems, i.e., inflammatory cytokines such as TNFα and antibodies. A system of proteins was used: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs allowing migration towards the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, post-ganciclovir treatment, cells undergo death via the thymidine kinase suicide mechanism. Hence, this forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low pH microenvironment. Lastly, we exploited antibodies to bind their antigens exclusively in combination with the ability of the cytoplasmic domain of VEGFR2 to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e. nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumour necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state but instead, only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.
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