Probiotics are found in numerous fermented foods such as yogurt, sauerkraut, and kombucha. Research shows that eating live probiotics are beneficial for the gut because it helps supplement the plethora of native bacteria. Now, these microorganisms are gaining popularity throughout the world—people are ingesting them in the form of food, drink, and even pills. Most probiotics are anaerobic. In fact, excess oxygen can damage organelles, create ionic imbalance and eventually even kill the cells. However, in the process of manufacturing these fermented foods, the probiotics in them often get exposed to oxygen, for example, due to leaks in packaging. Even the FDA does not have a rule about standard manufacturing processes regarding anaerobic conditions and yogurt. This could mean that the probiotics people eat are highly compromised: damaged or dead. I hypothesize that probiotics exposed to aerobic environments for extended periods of time will express more DNA repair enzymes such as DNA pol. 1 and 2, p53 or photolyase. This is because oxygen will cause probiotic cells to change function, and therefore, they will start to express enzymes, such as these DNA repair enzymes, to protect them from oxidative damage. In order to test this, I left probiotics out to oxidize for various amounts of time—0 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, and 1 hour. After oxidizing, I cultured the cells for 16 hours. I then pelleted the cells by centrifugation at 300 x g and washed and lysed them. Then, I tryptically digested cell proteins and analyzed them by LCMS (Liquid Chromatography Mass Spectrometry) for identification. In the future, these proteins can provide insights to how these oxidized probiotics impact the gut. Are they really the miracle microorganisms that benefit the gut, or could their compromised condition end up being harmful?

In the interstellar medium, molecules such as H₅⁺ have been proposed to exist. In order to detect molecules, understanding how a molecule absorbs radiation is key. There are relatively simple models that aid in predicting vibrational spectra such as the harmonic oscillator; however, H₅⁺ is not well described by these existing models because of its light central hydrogen atom, weak bonds, and large amplitude stretching. Thus, we are attempting to create a new simple model to describe the spectrum for H₅⁺ by exploring the coupling between vibrational states on the spectrum, and tracing their causes. Since it is difficult to understand high-dimensional models, a one-dimensional model was made and utilized in order to gather information about H₅⁺. To do this, DVR (discrete variable representation) was implemented, which is a method used to solve the Schrödinger equation. This aids us in getting energies and wave functions and finding the minimum energy pathway. The results show that coupling has allowed for tunneling to occur, explaining the differences in the spectra. By analyzing the coupling in H₅⁺, we are able to learn more about the vibrations of a complicated chemical system which can help us predict the location and intensity of the light absorbed by the molecule. Our findings could help contribute to one of our ultimate goals of developing a simple model that can be applied to other complex molecules that have similar trends and comparing the chemical theory to experimental results. Future work includes making the transition moment constant as a function of the distance between the outer H₂ groups, and using other approximations to look at intensities of the transitions.

Though renewed efforts in tuberculosis (TB) research have facilitated massive strides in treating Mycobacterium tuberculosis (Mtb), TB remains a global health problem with an estimated 10 million infections and 1.5 million deaths in 2018. The ability of the pathogen to sequester itself inside a granuloma, a mass of immune cells whose precise mechanism of regulation is unknown, prevents the simple study of Mtb pathogenesis and subsequent treatment discovery. Current in vivo models have been established to study TB infection using animal models or tissues, limiting biological relevance of human disease while current in vitro models lack components of the complex lung microenvironment during infection. We present the creation of a novel microscale infection model, which uses open and suspended microfluidic principles to enable spatial and temporal manipulation of cultures in suspended hydrogel plugs. Utilizing the ‘stacking’ feature of the device, we demonstrate the ability of a model granuloma consisting of M.bovis BCG (Mycobacterium bovis bacille Calmette-Guérin) and monocyte-derived macrophages to interact with a model vasculature layer consisting of endothelial cells. Analysis of soluble factors for proinflammatory cytokines and characterization of infection-dependent angiogenesis in the vasculature layer are used to verify crosstalk between cultures. In the future, we envision this model expanding to contain multiple immune cell types and to incorporate additional aspects of the lung anatomy to approach a more accurate pathophysiological model as a tool for other researchers’ studies.

The polycomb repressive complex 2(PRC2) is an important regulator of gene expression in developmental genes. The catalytic subunit histone-lysine N-methyltransferase (EZH2) binds to EED to precisely trimethylate H3K27 in the promoter loci to prevent transcription of a gene. We are using a novel EZH2 competitor protein, named EEDBinder-dCas9(EBdCas9) to prevent the PRC2 complex from forming and methylating H3K27me3 repressive marks on bivalent genes such as TBX18 and p16 in targeted regions. This will ultimately allow accessibility of the target gene to be transcribed without any permanent DNA modifications or chemical inducers in our induced pluripotent stem cells(iPSC) model. The goal of our study is to observe the capability of epigenetic memory in iPSCs independent of EBdCas9 once transcription is upregulated. Studying transcription levels of TBX18 and p16 iPSCs after EBdCas9/gRNA induction, allows us to identify functional H3K27me3 genomic location that are necessary for gene repression. In the case of downstream TBX18, we reveal that an upstream TATAbox is normally silenced by PRC2 complex to repress gene expression. Local disruption of PRC2 at targeted TBX18 promoter region using gRNA results in gene activation, local and neighborhood spreading towards TSS of reduced H3K27me3, EZH2, and JARID2 marks as well as recruitment of RNA Pol ll, and H2K27ac marks. To interrogate whether the cell acquires new epigenetic memory following EBdCas9/gRNA remodeling we let the cells proliferate for 2 days free of EBdCas9 or gRNA and measured transcript upregulation as well as local epigenetic memory by ChIPqPCR. We now show that iPSCs, free of EBdCas9/gRNA, maintain reduced levels of EZH2 and H2K27me3 2 days after epigenetic remodeling. Overall, EBdCas9 can eliminate PRC2 specific epigenetic regulation in a single locus allowing the dissection of key functional nucleation sites for PRC2 activity.

Probiotics are living organisms that when ingested have been linked with health benefits to the gut and improvement of conditions such as irritable bowel syndrome (IBS). The gut contains a plethora of microorganism populations that make up the microbiota. To understand how these populations communicate with each other, and the tissues surrounding them, it is imperative to identify and characterize the method of communication. Extracellular vesicles are one such possible method. Extracellular vesicles (EVs) are lipid-bilayer delineated sacks of material secreted from cells. It has been established that EVs are used as a waste disposal system. However, new research revealed that EVs can be used by the cell for methods of communication. Furthermore, EVs are now being linked to cell-to-cell and cell-to-organism communication. If EVs have been linked to communication, then characterizing them is one step closer to understanding how probiotic bacteria function. Previous studies have mainly characterized EVs by their size and divided them into 3 main groups: exosomes (40-150 nm), microvesicles (100-1000 nm), and apoptic bodies (>2000 nm). However, an analysis of proteins found in these EVs has not been performed yet. Here we compare EV proteins to proteins in the cell, to determine which protein fractions are secreted by cells through vesiculation for signaling purposes. To separate the cellular fraction from the EVs fraction, cell suspensions were centrifuged. First, the cells were pelleted and collected at 300x g. The leftover supernatant was spun at 16,000x g to pellet the EVs. Then, proteins from cells and EVs were solubilized and digested with trypsin. The tryptic peptides will be analyzed using liquid chromatography-mass spectrometry. A comparison of proteins in cells and EVs, and their relative concentrations can help us learn more about how probiotic EVs function in the gut.

There are an estimated 300 million people worldwide who are affected by asthma, a respiratory condition in which a person has inflammation and swelling in the airways. Asthma patients also experience increased vasodilation in their lungs, i.e. the widening of blood vessels, which causes increased blood flow and results in increased inflammation. The goal of this project is to create a device that makes free standing hydrogel rings, modelling the structure of blood vessels, offering a simple approach to better understand asthma and potential treatments. The device used to form the rings is 3D printed and can be printed in a range of sizes. The rings are composed of collagen I that has been seeded with smooth muscle cells. Once the hydrogel rings are cast, they can be transferred to a 96 well plate and be free standing of any rigid structures. The ability to be free standing allows us to measure the ring diameter and wall thickness, as well as measure any change in size when a vasodilator is added. Future steps to be taken with this project include optimizing the size for biological relevance, introduce endothelial cells to create multiple layers of cells that are involved in signaling for vasodilation, and increase the responsiveness that the rings have to constriction factors as well as dilators.

Scaffold proteins, which assemble enzymes and their substrates into multiprotein complexes, are critical for many cellular functions. By bringing enzymes and their substrates into close proximity, scaffold proteins are thought to enhance the rates of enzymatic reactions. For instance, Axin is a scaffold protein in the Wnt pathway that binds the transcription factor, ß-catenin and the kinase, GSK3ß. This tethering method is predicted to enhance the rate of ß-catenin phosphorylation. An outstanding question is whether a scaffold protein that binds to both GSK3ß and ß-catenin is sufficient to observe these effects or whether Axin has other structural properties that are not currently understood. Although there is no detailed structural information about Axin, it is predicted to be disordered. To address this gap, we have engineered a synthetic scaffold protein that can bind both GSK3ß and ß-catenin and contains a linker that is also presumed to be disordered. Previous work in the lab has reconstituted the reaction between Axin, ß-catenin and GSK3ß in vitro and found that Axin enhances the rate of ß-catenin phosphorylation. Using enzyme kinetics, I have compared the rate of ß-catenin phosphorylation of the engineered scaffold to that of Axin. Identifying the quantitative kinetic comparisons between the engineered scaffold protein with the natural scaffold protein could tell us more about what aspects of a tethering system make it effective at enhancing the rates of reactions. We are expecting to see similar tethering effects between the two scaffold proteins, as their linkers are both disordered and equivalent in length. 

Exhibiting high photoluminescence quantum yield, tunable surface functionalization and well-defined emission linewidths, colloidal semiconductor nanostructures are promising materials for a wide range of applications from lignocellulosic depolymerization to low power nonlinear optics. In past decades, synthesis of zero-dimensional (quantum dots) and one-dimensional (nanorods) systems has attracted much interest. In contrast, the potential of two-dimensional (nanoplatelets) and three-dimensional (nanotetrapods) structures remains to be tapped. As a consequence of their anisotropic morphology, the tunable emission frequency and linewidth of nanoplatelets and nanotetrapods renders them excellent candidates for single-photon emitters in devices such as hybrid photonic integrated circuits. Integrated photonics are devices that utilize light-matter coupling with embedded light-emitting media to achieve state-of-art engineering processes like ultralow threshold lasing and quantum many-body simulations. However, it remains a challenge to find suitable photoluminescent agents with outstanding desirable features. The subject of this research presents a solution to this problem—nanotetrapods and nanoplatelets used for coupling to the nanocavities in the integrated circuits. Owing to their tunable surface chemistry, nanotetrapods and nanoplatelets have the potential to be optimized for efficient processing with photonic cavities, with clear pathways for deterministic positioning. Moreover, the structures’ adjustable sizes and dimensions allow for maximization of single-photon behavior, including high quantum yield and narrow emission linewidth. Herein, I report the seeded-synthesis of several II-VI and III-V nanotetrapods and two approaches to growing nanoplatelets via a solution-phase decomposition procedure and the colloidal atomic layer deposition pathway. To understand both the spectral and structural properties of the nanostructures, I characterize the products by UV-vis and fluorescence spectroscopy as well as transmission electron microscopy. By exploring different routes to synthesizing the highly absorbing and emissive anisotropic colloidal nanostructures and investigating strategies for modifying the materials’ surface chemistry, it will be possible to achieve specialized spectral functionalities with these nanostructures.