In response to acute genotoxic stress, such as chemoradiation therapy, stem cells undergo temporary cell cycle arrest at the G1/S phase transition. This state, called quiescence, is reversible once stress-free conditions allow reentry into the cell cycle. We have previously identified the underlying mechanism behind quiescence in Drosophila Germline Stem Cells (GSCs) and human-induced pluripotent stem cells (hiPSCs). Mitophagy, or autophagy of the mitochondria, is required to enter quiescence. Surprisingly, we have observed a reserve of cyclin E (CycE) associated with the outer mitochondrial membrane that’s present in normal GSCs and hiPSCs but is reduced in quiescent stem cells. The role of CycE in quiescence remains unclear. Previously we have shown that reduced levels of CycE via inhibition of mTOR have driven cells toward mitophagy-dependent quiescence. This reveals that mitophagy serves as an alternative mechanism of CycE inhibition in contrast to the typical p21-mediated inhibition. Additionally, Parkin, a ubiquitin ligase activated by a serine/threonine kinase PINK1, is a key protein involved in mitophagy required for quiescence, and it has been found that CycE is a degradation target of this protein complex. Our hypothesis is that CycE degradation is necessary for entry into quiescence. To test this we upregulated CycE with a deleted portion of its PEST domain, which is a target for ubiquitination, under UAS-GAL4 control and used the GSC spectrosome morphology to observe quiescence. We observed a six-fold reduction of quiescent GSCs with overexpressed CycE, and hence concluded that CycE degradation is necessary for entry into quiescence. Determining the mechanism of CycE in stem cell quiescence is critical to understanding how cancer stem cells can avoid chemoradiation therapy. This project allows us to characterize the role of CycE within mitophagy and strengthen our understanding of the mechanisms that govern the cell cycle and quiescence.

Signaling of fibroblast growth factor receptors (FGFR) is critical for the development of vascular cell types. FGFR exists as two alternative splice variants: the b and c isoforms. Previous experiments have shown that activation of the c isoform leads to arterial endothelial cell development and inhibition of the c isoform is critical to perivascular development. These results were found using a c isoform-specific computationally designed protein. The goal of my project is to replicate these isoform specific results in an endogenous context. Our hypothesis is that induced pluripotent stem cells (IPSCs) overexpressing the b isoform will develop into pericytes and IPSCs overexpressing the c isoform will develop into arterial endothelial cells. I used the Gibson assembly method to create b/c isoform overexpression plasmids that can be inserted into the AAVS safe harbor site and used bacterial transformation to increase the amount of DNA. I am using stable transfection to create IPSC overexpression cell lines and adapting a previously verified 14-day protocol for creating endothelial cells from IPSCs to monitor each cell line’s differentiation. I am performing assays such as qPCRs, Western Blots, and immunofluorescence to quantify perivascular and endothelial markers in the cell lineages. Our findings should agree with our isoform specific hypothesis. In future experiments, we plan to engraft the overexpression cell lines into immunodeficient mice and assay how varying ratios of the two cell types affect their regenerative potential in vivo.

In the pursuit of higher density integrated circuits and the increased use of stacked die chips, the importance of wafer die preparation plays a crucial role in the semiconductor manufacturing process, turning silicon wafers into individual dies. An important step of die preparation is wafer backgrinding, which enables wafers to be thinned significantly, thus allowing for higher density packaging of chips. Backgrinding occurs near the end of a chip’s manufacturing process, thus it is important to quantify the surface quality of the wafers after the grinding process to avoid potential damage to the finished product. The most relevant process parameters include chuck speed, wheel speed and vertical feed rate. By systematically varying the process parameters and analyzing their impact on key output metrics including surface roughness, thickness variation and surface damage allows us to study each parameter in greater detail. We expect the tool to preform best when following the default given recipes supplied by the tool manufacturer, however to better understand the affect of each parameter we must vary each variable in a controlled manner. By performing a parameter-based design of experiment (DOE) we can study how each parameter affects our key metrics. At the Washington Nanofabrication Facility, where this project will take place, there are a variety of different processes and products all with different requirements. Understanding the relationship that each parameter of grinding has on the surface of the wafer allows us to develop different recipes for specific use cases depending on the requirements of each process.

Angiogenesis, or the formation of new blood vessels, is crucial for normal bodily function but is especially important in diseases that cause blood vessel breakdown such as diabetic vasculopathy. Angiogenesis is regulated by activation of the Tie2 receptors in endothelial cells, which have two main ligands: angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 binding has been shown to stabilize blood vessels and inhibit vascular leakage, while Ang2 antagonizes these effects. We have previously shown that a computationally designed Tie2 super-agonist which presents eight copies of the Ang1 F-domain strongly activates Ang1-like signaling in human umbilical vascular endothelial cells (HUVECs). In this project, we hope to assess the Tie2 super-agonist’s ability to rescue diabetes induced blood vessel defects in a diabetic blood vessel organoid (BVO) model. To model diabetic conditions, a three-dimensional blood vessel organoid model has been cultured in a high glucose media along with inflammatory cytokines associated with the diabetic phenotype. Western blotting and immunofluorescence staining will be used to assess the relative quantities and localization of proteins involved in vascular stability and inflammations upon treatment with the Tie2 super-agonist. Vascular degeneration is a very harmful condition associated with many prevalent diseases including diabetes, so the Tie2 super-agonist could potentially be a new therapeutic drug candidate for treating blood vessel dysfunction in patients with these conditions in the future.

Titanium dioxide is a compound that has been used in the realm of nanotechnologies for decades. Titanium dioxide is used as a protective and high-refractive index optical coating. It also has strong mechanical and chemical stability. This is also true of other ceramics throughout the Washington Nanofabrication Facility with compounds such as aluminum oxide, titanium nitride, and aluminum nitride. With this in mind, the goal is to explore the best conditions to reactively sputter titanium dioxide. Thus, I developed a series of experiments including a screening for significant factors, and a following set of experiments using previous results to find an optimum point. In order to produce titanium dioxide in a physical vapor deposition environment such as the sputter tool utilized, the chamber holding the designated surface was pumped down and exposed to a chamber filled with argon, whose function was to hit the target titanium and a percentage of oxygen designed to react with the titanium in order to form titanium dioxide. By generating experiments designed to better understand how titanium and oxygen would react within the sputter tool, there was an aim to better screen for factors and understand the surface composition of the titanium dioxide. The desired outcome of this research is to build a working titanium dioxide recipe that optimizes deposition. Based on preliminary testing, the ideal outcome is likely produced under low pressure and higher power conditions. Future goals may include uniformity and accuracy within titanium dioxide, but also with other materials. With the implementation of this recipe, which is both optimized for the deposition of the material as well as its applicability for the lab, other recipes utilizing similar methods are desired. With a working titanium dioxide recipe, titanium and aluminum nitride recipes can better be developed.