자료유형  

 학위논문

Control Number  

0017164843

International Standard Book Number  

9798346380740

Dewey Decimal Classification Number  

571.6

Main Entry-Personal Name  

Yang, Xue.

Publication, Distribution, etc. (Imprint  

[S.l.] : Stanford University., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

106 p.

General Note  

Source: Dissertations Abstracts International, Volume: 86-05, Section: B.

General Note  

Advisor: Khavari, Paul.

Dissertation Note  

Thesis (Ph.D.)--Stanford University, 2024.

Summary, Etc.  

요약The epidermis, as a critical barrier, relies on a fine-tuned balance between keratinocyte proliferation and differentiation to maintain its integrity and functionality. This process is tightly regulated by different signaling pathways initiated from various cell membrane receptors like Notch and EGFR. G-protein-coupled receptors (GPCRs), with over 800 members, are the largest family of cell membrane receptors, targeted by approximately 34% of all FDA-approved drugs. However, a systematic study of skin-specific GPCRs in human epidermal homeostasis is lacking. To investigate this, we applied two advanced screening approaches. The first, a Fluorescence Activated Cell Sorting (FACS)- based pooled CRISPR Screen, enables the isolation of cells exhibiting specific gene signatures, to pinpoint genes influencing the differentiation phenotype. The second technique, single cell Perturb-Sequencing, allows us to map the transcriptomic landscape of individual cells undergoing various genetic manipulations. By integrating these two screenings, we identified adhesion GPCR (aGPCR) ADGRL2, as a novel epidermal prodifferentiation regulator among all epidermal GPCRs. By applying the TRUPATH BRET2 assay, we observed ADGRL2 dominantly activating the Gα13 subtype.At the molecular level, we applied nanodisc purification of the ADGRL2/Gα13 complex followed by Cryo-Electron Microscopy (Cryo-EM) and identified several binding interface between ADGRL2's intracellular loops 2 and 3 (ICL2, ICL3) with Gα13, with ICL3 especially interacting with Gα13's conserved and unique GTPase domain. This represents the first documented instance of an aGPCR ICL3 engaging with a Gα protein from a structural study. In summary, our findings highlight that ADGRL2's signaling through Gα13, mediated by interactions with its ICL2 and ICL3 loops, as crucial for regulating keratinocyte differentiation.The second part of my thesis is focusing on the function of RNA EXOSOME component 2(EXOSC2/rrp4) in newly identified disease SHRF, characterized by Short stature, Hearing loss, Retinitis pigmentosa, and distinctive Facial features (#OMIM 617763). To understand the pathogenic mechanisms of SHRF, we studied the effects of EXOSC2 mutations in patient-derived lymphoblasts, CRISPR-engineered mutant fetal keratinocytes. We identified that EXOSC2 is a critical gene and that the G198D mutation disrupts its interaction with other RNA exosome components, such as EXOSC3/10, leading to protein and complex instability, further altering the autophagy pathway related genes. To futher study the EXOSC2 gene's function in vivo, we created several CRISPR knockouts of the Drosophila rrp4 gene and analyzed mutants with a PiggyBac transposon insertion in the 3'UTR and RNAi-mediated knockdown. Our findings show that Drosophila rrp4 is essential, and its phenotypes can be rescued by wild-type human EXOSC2, but not by the pathogenic variant from the patients. Meanwhile, we observed that Drosophila rrp4 is crucial for eye development, muscle ultrastructure, and wing vein formation. Overexpression of the transcription factor MITF, which regulates the autophagy genes ATG1 and ATG17, was sufficient to rescue the small eye phenotype and adult lethality caused by rrp4 inhibition. Furthermore, pharmacological activation of autophagy with rapamycin alleviated the lethality associated with rrp4inactivation. Our findings suggest that defective autophagy contributes to SHRF pathogenesis and indicate potential therapeutic approaches.

Subject Added Entry-Topical Term  

Cells.

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Homeostasis.

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CRISPR.

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Antibiotics.

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Mutation.

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Microscopy.

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Genetic engineering.

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Insects.

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Autophagy.

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Flow cytometry.

Subject Added Entry-Topical Term  

Keratin.

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Lipids.

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Genomics.

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Penicillin.

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Genes.

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Protein expression.

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Bioengineering.

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Bioinformatics.

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Cellular biology.

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Genetics.

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Pharmaceutical sciences.

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Physiology.

Added Entry-Corporate Name  

Stanford University.

Host Item Entry  

Dissertations Abstracts International. 86-05B.

Electronic Location and Access  

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Control Number  

joongbu:656201