Engineering metabolic pathways in bacteria requires regulation of cellular biology with predictable genetic control devices. Characterizing these controls is key to optimizing biochemical pathways for the sustainable production of high value chemicals. In prokaryotes, small RNA (sRNA) downregulate gene expression by antisense binding to messenger RNA (mRNA) transcripts. The relative ease of re-engineering sRNA targets by replacing its antisense targeting sequence makes sRNA an ideal candidate for implementing post-transcriptional controls in metabolic engineering. In previous work, we incorporated sRNA into metabolite-responsive RNA controllers, but in many cases did not observe the hypothesized trends in mRNA repression. However, we observed the lack of secondary structure within the antisense targeting sequence correlated with higher mRNA repression. To understand these effects and refine the functionality of our constructs, we built a range of new sRNA devices with additional sequence at the 5’ end. We targeted the sRNA to repress fluorescent protein expression, and used functional assays to measure fluorescence across a range of sRNA induction levels. We hypothesized that sRNA carrying a 5’ hairpin will result in greater repression than sRNA lacking structure at the 5’ end, and that base-pairing interactions between the 5’ sequence and the targeting sequence will lead to lower repression. This work aids in the development of rational design principles for using sRNA in RNA-based controls. By understanding the effects of sRNA structure, we will be better able to expand the post-transcriptional controls available to metabolic engineers. Ultimately, this extends our understanding of cellular biochemistry, while providing novel means by which to optimize engineered biochemical pathways.

Protein synthesis is a tightly regulated cellular process important for all of life. Specific diseases such as cancer can hijack the process of protein synthesis to drive specific disease states. My research focuses on determining if the protein making factories of a cancer cell can be secifically therapeutically targeted. I have focused my studies on prostate and bladder cancer. Prostate cancer is the second leading cause of cancer death in men. A well-studied signaling pathway that is altered in prostate cancer is the PI3K-AKT-mTOR pathway. This pathway converges on and activates the apparatus necessary to start protein synthesis through a protein called the eukaryotic initiation factor 4E (eIF4E). eIF4E is an mRNA cap-binding protein which recruits another translation factor to start protein synthesis in cells. Ribavirin is an mRNA cap-mimetic that can compete with eIF4E to inhibit mRNA translation. I will present my results from a preclinical trial using Ribavirin and a mouse model of prostate cancer. In addition to my work in prostate cancer I have also initiated preclinical studies in bladder cancer. Bladder cancer is the 5^th leading cause of cancer death in men and there have been no new theurapeutics for two decades. ARID1A is an ATP-dependent chromatin modifier that is one of the most mutated genes in bladder cancer. The Hsieh lab has discovered an intimate link between ARID1A levels and mRNA translation. I hypothesize that protein synthesis machinery represents a unique vulnerability, which can be therapeutically targeted in bladder cancer. I will present data on my findings using protein synthesis inhibitors such as puromycin and an ATP-site inhibitor of mTOR in ARID1A wild-type and mutant bladder cancer cell lines. Together, my work in the Hsieh lab points to a new treatment paradigm for targeting the protein synthesis machinery in human cancers.

Current chemotherapies exploit the larger pore size of tumor vasculature to reach cancer cells. However, this rudimentary form of targeting offers low selectivity and carries the risk of severe systemic toxicity. In addition, chemotherapy distribution to tumors is limited by the large distance between blood vessels in tumors, increased interstitial fluid pressure, and physiological resistance by the extracellular matrix (ECM). With the goal of addressing these limitations, researchers have developed drug-bearing polymers of various sizes and architectures that selectively target tumor cells. The Pun lab previously produced a novel drug delivery polymer, the sunflower polymer, that can be utilized for anticancer drug-delivery. The sunflower polymer is a macrocyclic brush polymer that can be functionalized with a folic acid targeting ligand and an anticancer drug, doxorubicin, that is released in the acidic tumor microenvironment. In order to maximize the potential of anti-cancer drug-delivery polymers such as this, the impact of polymer size and architecture on tumor penetration must be better understood. This is because the efficacy of any anticancer drug, regardless of its mechanism of action, is limited by its ability to navigate the tumor microenvironment and reach all viable cells in a tumor. This project seeks to discover how polymer size and architecture impacts tumor penetration by using a 3D in vitro perfusion chip model of the tumor microenvironment. The model consists of microfluidic channels seeded with KB carcinoma cells and ECM, and has been optimized using model polymers of polyethylene glycol (PEG) and dextran. Tumor penetration is studied by perfusing fluorescently labelled polymers of various morphologies through the seeded microfluidic channels. The information obtained from this project could potentially be used to optimize the size and architecture of drug-delivery polymers for tumor penetration. In turn, these optimizations could present a significant contribution to the development of anticancer drug-delivery polymers.

Optical coherence tomography (OCT) is an non-invasive imaging technique that is able to produce micrometer resolution images of human tissue. Building upon this imaging technique, the Wang lab has developed novel OCT based techniques to image the microvasculature of the human eye. With these high resolution OCT images, it would be possible to provide quantitiatve measurements of ocular features that can assess eye diseases non-invasively. These measurements would include blood flow rate, vessel density, vessel tortuosity, fractal dimensions, etc. The goal in this research is to determine which of these physical parameters in the eye can provide objective measures that correlate well with the severity of diabetic retinopathy (DR), the leading cause of blindness worldwide. Specifically, this research focused on five quantifiable metrics - vessel diameter, vessel area density, vessel skeleton density, vessel perimeter index, and vessel complexity index - and their correlations with the progression of the disease. The main hypothesis was that at least one of these parameters would show significant correlation with DR progression.Each of these metrics were quantified from OCT images of patients with DR using semi-automated computer software. Then, statistical analysis was done to calculate the correlations of each metrics with disease progression. Finally, an evaluation was performed to see which of these objective parameters can serve as sensitive biomarkers, possessing the potential to be used in diagnosing or monitoring the therapeutic treatments of diabetic retinopathy. 

Leigh Syndrome (LS), one of the most common childhood mitochondrial diseases, typically arises during the first year of life and results in a wide array of metabolic and motor deficiencies leading to death within a couple of years. LS is associated with mutations in any of several mitochondrially encoded genes, including NADH Dehydrogenase Subunit 2 (ND2). The ND2 gene codes for a subunit of complex I within the respiratory chain of the mitochondria. Among patients with the same ND2 mutation, the progression and pathology of LS varies widely. The variation might be due to the effects of variation at other genes that interact with ND2. To test this hypothesis, we have turned to a Drosophila model of LS, in which flies carry an ND2 loss-of-function mutation. Drosophila with the ND2 mutation experience LS-like symptoms, including decreased lifespan, metabolic dysfunction, and paralysis, when exposed to mechanical stress, termed ‘bang sensitivity’. To identify other genes that affect the ND2 mutation in Drosophila, we crossed female ND2 flies with males from the Drosophila Genetic Reference Panel (DGRP); a panel of 200 inbred, fully sequenced lines. The resulting offspring are aged 21 days and assayed for changes in bang sensitivity— the time it takes for individual flies to recover from paralysis. By comparing the effects of ND2 on the bang sensitivity phenotype in each DGRP background, we hope to identify specific genes that either worsen or ameliorate the phenotype. If successful, this study could help us identify genetic modifiers of this mitochondrial disease, and thus help us identify novel therapeutic targets.

The cultivation of tomatoes is currently a 58 billion dollar industry worldwide. Over the past few years, tomato plants have been subjected to more severe and abrupt climatic fluctuations than ever before. In-depth understanding of the tomato leaf anatomy and physiology can have significant consequences in agriculture. Fluxes (fluid flow rate) in leaf water and sugar-conducting tissues, xylem and phloem, are challenging to visualize in real time. It is important to attempt this measurement using intact leaves as excision interrupts control of normal flow paths. The possibility that vein density and flux influence photosynthetic rate is being investigated here.  Optical Coherence Tomography (OCT) is a non-invasive imaging modality that performs high-resolution cross-sectional imaging, allowing to visualize the structure and motion in biological samples in 3D.  In this study, we have used a state-of-the-art OCT system with 7 μm resolution, operating at 1300 nm wavelength, to investigate leaf structure, venation and vein flow in live tomato leaves for the first time. The low intensity imaging beam (3 mW) is in the near IR spectral region and imaging is performed at high speed (4 ms/frame) hence, does not interfere with the leaf’s natural physiological processes. We have used Rutgers’ tomatoes for this study. To improve the image contrast in the leaf veins, we are investigating using dye and lipid particles to be taken up by the leaves