Regulation of Autophagy Related Gene ATG1 by Ecdysone Signaling During Metamorphosis in Drosophila melanogaster
Student name: Bineeta Debnath
Project advisor: Craig Woodard
In order to maintain homeostasis in animals, cell growth, death, and nutrition release must be coordinated. The steroid hormone 20-hydroxyecdysone (ecdysone) controls developmental changes during metamorphosis in Drosophila melanogaster. Ecdysone also induces autophagy, the self-degradation of cellular proteins and organelles. The larval fat body, a nutrient reservoir that undergoes tissue remodeling during metamorphosis, stores the energy and nutrients that sustain the pupa throughout this developmental phase. The Drosophila larval fat body uses autophagy to release stored molecules (Lőrincz et al., 2017). It is understood that autophagy controls the release of nutrients in fat bodies during metamorphosis, but the signaling pathways that activate autophagy are not fully known. A leading hypothesis is that insulin signaling represses autophagy by activating the protein, target of rapamycin (TOR), and during metamorphosis, ecdysone represses insulin signaling, resulting in the activation of autophagy (Nassel et al., 2015). TOR blocks the autophagy process by inhibiting an autophagy-related gene ATG1, which is required in inducing autophagy. As ecdysone signaling is hypothesized to increase autophagy by repressing insulin signaling, it is also possibly inducing the expression of ATG1 to induce autophagy. In this study, transgenic flies were created by using the Gal4 - UAS system, which are deficient in ecdysone signaling-deficient. Fat body was dissected from the F1 progeny larvae to isolate RNA and synthesize cDNA. The cDNA was amplified using ATG1 and ß-actin gene-specific primers in a conventional PCR reaction. The PCR results were examined using gel electrophoresis. To determine the relative gene expression of ATG1, Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR) reactions were carried out using ATG1 primers in ecdysone signaling-deficient and wild-type control flies.

Evaluating a Revised Protocol for the Champer-Specific Differentiation of iPSC-Derived Cardiomyocytes
Student Name: Isabel DiBiasio-Hudson
Project Advisor: Dr. Todd Herron, University of Michigan; Dr. Rebeccah Lijek, Mount Holyoke College
The prospective utility of cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) is largely dependent on their ability to be efficiently and precisely produced as subtype-differentiated (chamber-specific) tissues. Chamber- specific monolayer and three-dimensional tissues have the potential to revolutionize the study of cardiac disease and drug development as in vitro models of disease and accurate, ethical models for testing the chamber-specific cardiotoxicity of clinical drugs. However, the majority of differentiation approaches to date produce a mixture of atrial and ventricular CMs which, without further treatment, progress to predominantly (but not exclusively) ventricular cardiomyocytes (VCMs) over 15-30 days1 . In 2018, Cyganek et al. established that treating cardiac progenitors with retinoic acid (RA) produces sub- specified atrial cardiomyocytes (ACMs) showing strong atrial protein markers when these cells are further treated on RPMI 1640 media with no glucose and high levels of lactate to select for exclusively metabolic CMs2 . The goal of this study was to determine whether healthy atrial cardiomyocytes could be produced with high specificity through treatment with RA but without the use of metabolic CM selection media. Four batches of ACMs and two of VCMs, all derived from iPSC line 19-9-11, and one human VCM sample were evaluated. Successful ACM differentiation was determined based on functional response (quantified through calcium flux imaging) to cardiac drugs with known effect and Western Blotting analysis of β-myosin heavy chain protein abundance. It was found that while this protocol effectively produces cells in high number with low functional variation and which respond as expected to drug treatment, most ACM/VCM samples tested continue to demonstrate a degree of mixed ACM/VCM protein characteristics. More research is needed to determine and reduce the subtype variability between and within cell lines and determine the timeline of progression of ACMs differentiated by RA addition towards exhibiting VCM characteristics.

Coordinated Regulation of Tissue Remodeling in Drosophila melanogaster
Student Name: Ayesha Binte Khalid
Project advisor: Craig Woodard
Tissue remodeling plays a critical role in regulating growth and development in multicellular organisms, such as wound healing and tumor metastasis. Proteases such as matrix metalloproteinases (MMPs) play a role in tissue remodeling by degrading the extracellular matrix (ECM) and cell-cell junctions to increase the mobility of individual cells (1). Drosophila melanogaster is a very good model organism in which to study tissue remodeling processes. Fat body remodeling (FBR) is an essential process for successful metamorphosis in D. melanogaster, involving the transformation of polygonal larval fat body cells (FBCs) into more spherical cells with actomyosin dependent locomotion (1). We hypothesize that Actin cytoskeleton defects could potentially impair the migration of FBCs, resulting in problems such as nutrient deprivation and delayed wound healing. In Drosophila pupae, FBCs can migrate rapidly to wound sites through adhesion-independent peristaltic swimming, powered by waves of cortical actomyosin that propel them forward and contract at the rear. This process helps FBCs attach to the wound, release antimicrobial peptides, and seal the epithelial gap to fight infection (2).
In this study, I build on previous research examining FBR by categorizing mutant fly lines into "no FBR" or "partial FBR" phenotypes through larval dissections and scoring (3). I also analyzed the actin cytoskeleton organization of the mutant flies. I have examined fat bodies from mutant and control genotypes by staining them with Rhodamine-Phalloidin to observe the actin cytoskeleton organization, and assessed its correlation with the "no FBR" or "partial FBR" phenotypes. Most of the mutant lines exhibited "no FBR" or "partial FBR" phenotypes, but we observed no visible actin cytoskeleton structural defects in these mutants. The results of staining with Rhodamine-Phalloidin sheds light on whether defects in actin cytoskeleton organization are linked to "partial FBR" or "no FBR" formation in cells. This study provides insight into the specific role of each identified gene in the FBR process in D. melanogaster, paving the way for future research in this area.

Investigating how Depo Provera impacts CEACAM1 expression in the genital tract
Student Name: Alex Taylor
When studying Chlamydia trachomatis in mice models, the mice need to be treated with Depo Provera, a routine human contraceptive, to be able to contract this pathogen. While this is an accepted practice within the Chlamydia trachomatis research field, not much is known about why this contraceptive makes mice susceptible to infection. In order to investigate the mechanisms behind this practice, mice were treated with Depo Provera, and the gene expression within their genital tracts' was quantified. One gene that had a significant change in gene expression was CEACAM1. CEACAM1 is a transmembrane carcinoembryonic antigen cell adhesion molecule that acts as a biological rheostat in numerous biological processes. This gene is of particular interest as two of those processes include the regulation of T lymphocytes and barrier function maintenance: two important factors involved in the pathogenesis of Chlamydia. Over the past year, literature research has been conducted to fully understand this gene's inner workings and how its expression might affect the immunoregulation of the genital tract. Though no experimentation has been completed yet, RTqPCR is in the process of being done.