Date of Graduation

5-2025

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Evans, Timothy A.

Committee Member

Pinto, Ines

Second Committee Member

Sakon, Jason

Keywords

CRISPR; Development; Drosophila; Genetics; Lipin

Abstract

Regulation of energy expenditure and energy storage is a key characteristic of life. Numerous enzymes play a crucial role in regulating energy metabolism. Lipins are a highly conserved family of proteins that function in both lipid synthesis and gene regulation. In the glycerol-3 phosphate pathway, lipins act as a phosphatidate phosphatase (PAP) producing diacylglycerol. In gene regulation, lipins act as transcriptional co-regulators of metabolic genes. The enzymatic PAP activity occurs in the cytoplasm; however, for lipins to function as gene regulators, they must translocate into the nucleus. Nuclear translocation is facilitated by the nuclear localization signal (NLS) within the protein. While the Lipins family is conserved throughout many organisms, Drosophila melanogaster possess a single Lipin ortholog, making it an ideal model organism to study the function of this protein.

This research aimed to achieve three main goals: observe effects of RNAi knockdown of Lipin in the ring gland, characterize a mutant fly stock lacking the NLS sequence, and create a Lipin null mutant for use in future studies. It was observed that the knockdown was successful, but did not show a biologically significant delay. These findings suggest that Lipin may not be essential in the ring gland for successful development. While characterizing the LipinΔNLS mutant (lacking the NLS), it was confirmed that the form of Lipin in these animals could not enter the nucleus in starvation conditions, while nuclear translocation was observed in control animals. Additionally, these LipinΔNLS animals did not show significant pupariation or eclosion delays, but a higher fatality rate. Lastly, using the CRISPR/Cas-9 system, a “LipinNull” mutant was created which would produce truncated, nonfunctional Lipin. This mutant stock could have applications in further research to better understand the functions and mechanisms of this multi-functional protein.

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