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Doctor of Philosophy (PhD)
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This thesis combines research on PQN-75 expression, functional motifs of GLH-1/Vasa, and germ granule components in Caenorhabditis elegans to provide a comprehensive understanding of germline development, maintenance, and reprogramming, while also examining the role of pharyngeal gland cells in stress resistance and thermotolerance. In C. elegans, pharyngeal gland cells secrete mucin-like proteins, such as PQN-75, with similarities to human PRB2. The expression of PQN-75 in gland cells confers stress resistance and thermotolerance but does not affect fertility, instead it plays a role in the organism's ability to adapt to varying environmental conditions. While, GLH-1/Vasa, an ATP-dependent DEAD-box helicase, plays a critical role in safeguarding the germline by regulating translation and amplifying piwi-interacting RNAs. To elucidate the functions of GLH-1 and its role in germline development, CRISPR/Cas9 technology was employed to investigate its functional motifs in C. elegans by analyzing 28 endogenous mutant alleles. Results demonstrate that helicase activity is essential for GLH-1's association with P granules, and removing glycine-rich repeats diminishes P-granule interactions at the nuclear periphery. Additional, mass spectrometry reveals an affinity between GLH-1 and three structurally conserved PCI complexes, along with a reciprocal aversion for assembled ribosomes and the 26S proteasome. Suggesting that P granules compartmentalize the cytoplasm to exclude large protein assemblies, effectively shielding associated transcripts from translation, contributing to germline maintenance. Germ granules are essential for maintaining germline integrity and stem cell totipotency. Depletion of core germ granule components in C. elegans leads to germ cell reprogramming and sterility. To better understand the initiation of somatic reprogramming and the role of GLH-1 in this process, total mRNA (transcriptome) and polysome-associated mRNA (translatome) changes in a precision full-length deletion of glh-1 where examined. Here two significant changes were observed: first, GLH-1 suppresses the expression of neuropeptide-encoding transcripts, suggesting a role in repressing somatic reprogramming and maintaining germline integrity; second, GLH-1 promotes Major Sperm Proteins levels, repressing spermatogenic expression during oogenesis and promoting MSP expression to drive spermiogenesis and sperm motility, highlighting its importance in fertility. Our findings contribute to understanding the roles of PQN-75 and GLH-1/Vasa in C. elegans germline development, maintenance, and germline stem cell reprogramming, while also shedding light on the organism's stress resistance and thermotolerance mechanisms. With broader implications identifying early stem cell reprogramming processes and provides a platform for future research on germline biology in C. elegans.
Rochester, Jesse D., "Exploring the Impact of PQN-75 and GLH-1/Vasa on Germline Development, Maintenance, and GSC Reprogramming Using Caenorhabditis elegans as a Model" (2023). Electronic Theses and Dissertations. 3850.