HSP72 is a chaperone protein induced by cellular stress and physical activity. HSP72 protein is reduced in aging and obesity, and genetic overexpression of HSP72 in skeletal muscle improves metabolic health in male mice and human muscle cells – however limited research has been performed in females. HSP72 knockout (KO) increased body fat, decreased insulin sensitivity, and impaired mitochondrial quality control in male mice. In contrast to findings in male mice, female KOs showed the opposite phenotype of reduced body weight and gonadal fat, improved insulin sensitivity, and increased voluntary wheel running compared WT controls. Skeletal muscle mitochondria from female KO mice were smaller and more spherical than WT and these alterations in morphology were paralleled by increased fission signaling. Enhanced complex I, II, and IV activity was observed in skeletal muscle mitochondria of female KO vs WT mice. Collectively the metabolic phenotype of female HSP72 KO mice recapitulated that of animals harboring a muscle-selective overexpression of estrogen receptor alpha (ERα). Indeed, muscle ERα transcript and protein were induced in female KOs over WT, but this response was not observed in male animals. Our findings show that, in contrast to male HSP72KO mice, the improvement in mitochondrial function and metabolic health observed in female KO mice is underpinned enhanced ERα action. Our research points to a sex-biased role for HSP72 in regulating estrogen action and in governing metabolic health in male and female mice.

The target of the rapamycin complex subunit lst8, also known as MLST8, is an integral portion of the mechanistic target of rapamycin growth pathway that is conserved across eukaryotic species. This pathway regulates cell proliferation and metabolism, via the protein subunits, and activates biosynthesis. By utilizing the presence of the MLST8 gene in the Strongylocentrotus purpuratus genome, this project explores the experimental procedure necessary to efficiently produce isolated MLST8 plasmids for further experimental analysis to study the roles of the growth pathway and protein more closely. Identification and characterization of the MLST8 gene sequence was determined using BLAST, Echinobase, InterPro, and phylogenetic analysis with MEGA and Multiple Align Show. In order to clone the gene, the MLST8 region of the genome was PCR amplified, ligated into a vector, and transformed into competent DH5-ɑ bacteria so the resulting MLST8 plasmids could be isolated for experimental analysis of cloning success. The results showed confirmation of successful cloning using restriction enzyme digestion and Sanger sequencing. Knowledge of effective MLST8 cloning procedure provides the supplementary groundwork for future research in cancer genetics. Specifically, it provides guidance when studying overactive proliferation and metabolism pathways, or mechanisms of drug resistance for advancements in cancer immunotherapy.

Stronglyocentrotus purpuratus is a valuable model organism for exploration in developmental and evolutionary biology because of its close relationship to chordates. This experiment aims to clone and characterize the nucleolar protein 56 (NOP56) gene of the S. purpuratus genome to gain insight into gene function. Research focused on successful amplification of NOP56, and successful transformation of competent cells. Sequence analysis, including BLAST, InterPro, and Echinobase, helped confirm gene identity. A phylogenetic tree was constructed to analyze evolutionary relationships. To clone NOP56 we used PCR and gel electrophoresis. Purification and spectrophotometry were used to isolate our PCR product, and ligation and bacterial transformation were performed to set up colony PCR and inoculation. Colony PCR and inoculation helped amplify the transformed colonies. Transformed colonies were screened using blue-white selection and gel electrophoresis to confirm the insert’s presence. One positive colony for NOP56 was produced. The colony was cultured, and plasmid DNA was quantified after isolation. Downstream experiments, like restriction digest and sequencing were not performed. As such, the results only confirm successful amplification, cloning and possible propagation of a recombinant plasmid. The outcomes demonstrate the use of key techniques that establish a strong foundation for future identification and functional analysis, thereby potentially contributing to the understanding of gene regulation in echinodermata.

Understanding gene identity and function in Strongylocentrotus purpuratus is crucial for expanding our understanding of echinoderm genomics and development. This project aimed to identify and clone an unknown Strongylocentrotus purpuratus gene, sp20463. The research question centered on whether sp20463 could be successfully cloned and identified. Initial sequence analysis via BLAST and Interpro suggested sp20463 encodes CCA tRNA nucleotidyltransferase 1 (TRNT1), a highly conserved mitochondrial enzyme that adds the 3’ terminal CCA to tRNAs which is necessary for protein synthesis. To further support gene identification, a phylogenetic analysis was conducted, which revealed strong evolutionary conservation of sp20463 with TRNT1 orthologs across diverse species. Both genes were amplified using PCR, ligated into plasmid vectors, and introduced into bacteria through transformation. Blue/white colony screening was utilized to identify potential recombinant colonies, followed by colony PCR and gel electrophoresis to verify insert presence. Verified colonies underwent plasmid isolation, restriction digest, and Sanger sequencing to verify the presence of the insert and confirm evidence of cloning success. This experiment demonstrates the utility of standard cloning protocols and highlights challenges in isolating mitochondrial genes, informing future strategies for gene identification in echinoderms.

The invention of induced pluripotent stem cells (iPSCs) created novel opportunities for disease modeling and personalized medicine, but may be unsuitable for XX individuals. During human female embryonic development, one X chromosome is silenced by the XIST long non-coding RNA, which recruits proteins to create a condensed chromatin structure. This process is called X-chromosome inactivation (XCI). When female fibroblasts are reprogrammed into iPSCs, XIST expression is often lost, leading to a breakdown of XCI known as X-chromosome erosion (Xe). Xe genes are reactivated, therefore disrupting dosage compensation and rendering the iPSCs unusable. De novo DNA methyltransferases DNMT3A and DNMT3B contribute to XIST repression by methylating its promoter, but the upstream triggers and full regulatory mechanism behind erosion remain unclear. Transcriptomic analyses have revealed downregulation of various zinc finger proteins (ZNFs) in eroded cells. KLF4, a core pluripotency factor, is known to transcriptionally upregulate ZNFs, potentially downregulating activity of transposable elements such as Long Interspersed Element-1 (LINE1). Here, we will investigate the effects of KLF4 overexpression on the rate of erosion in hiPSCs and further dissect the interplay between KLF4, ZNFs, LINE1 and XIST in regulating the stability of the inactive X chromosome. These findings will clarify mechanisms of XCI erosion and support more effective use of iPSCs in women’s health research.