Category: College of Science

Spectroscopic characterization of newly designed fluorescent probes for biomedical research

Author(s): Amira Anwar

Mentor(s): Ozlem Dilek, Chemistry & Biochemistry

Abstract

The development of fluorescent probes is crucial for advancing cellular imaging and disease diagnostics, particularly in detecting oxidative stress, a key driver of cancer, neurodegenerative diseases, and fibrosis. A hallmark of oxidative stress is the carbonylation of biomolecules, which occurs when reactive oxygen species (ROS) modify proteins and other biomolecules, leading to cellular dysfunction. In this project, we designed and synthesized hydrazine-based small-molecule fluorophores to selectively target biological aldehydes, particularly those generated by lysyl oxidases (LOXs) during collagen oxidation. These aldehydes serve as important biomarkers of fibrosis and metastatic progression. The fluorophores react with aldehydes to form hydrazones, resulting in a fluorescence turn-on response that enables real-time monitoring of oxidative stress in cellular systems. To characterize probe performance, we analyzed their photophysical properties using UV-visible and fluorescence spectroscopy. Additionally, reaction kinetics were evaluated via UV-visible spectrophotometry, and structural validation was conducted using NMR and mass spectrometry. By refining probe design for enhanced fluorescence sensitivity and specificity, this work contributes to the development of next-generation imaging tools for studying disease mechanisms and advancing biomedical diagnostics.

Audio Transcript

Hi everyone, and thank you for checking out my presentation. I’ll be sharing my research on the spectroscopic characterization of newly designed fluorescent probes for biomedical research. These probes are designed to detect biologically relevant aldehydes—aldehydes are highly reactive molecules that can build up in the body during oxidative stress. This kind of cellular stress is involved in many major health conditions like cancer, fibrosis, and neurodegenerative diseases. By creating tools to visualize these aldehydes in real time, we hope to provide a better way to study early-stage disease and potentially improve how we diagnose and/or monitor these conditions. So, why is this important? Our bodies naturally produce molecules called reactive oxygen species, or ROS. But when there’s too much ROS and not enough defense, it leads to oxidative stress. This damages important parts of our cells—proteins, DNA, lipids, and nucleic acids. One of the results of this damage is something called carbonylation, an irreversible modification to proteins that disrupt its normal function which serves as a biomarker for disease progression. By detecting this early and accurately could give us new ways to track disease or catch it early—especially in conditions like cancer or metabolic disorders. Right here is an image of a normal cell being attacked by free radicals, elevated levels of ROS leads to oxidative stress. The role of LOX. A group of enzymes called lysyl oxidases, or LOX are important in building collagen, the main protein in connective tissues. But during that process, they produce aldehydes.These aldehydes, especially when overproduced, are closely tied to tissue damage and disease. So if we can track them, we can better understand what’s going on in the body during early disease stages.So this ia relaly good image on how LOX cross linking collagen occurs and potential way to image LOX. To tackle this, I designed a small molecule—called fluorescent probe—that glows when it detects aldehydes. The idea is simple: no aldehyde, no glow. But when an aldehyde is present, the probe reacts and lights up.This happens via click chem mechanism Shown right here. This kind of “turn-on” fluorescence lets us track where and when aldehydes are forming, even in real-time, which is incredibly useful for research and diagnostics. Here’s a detailed view of how it works: We start with an amine which converts into a hydrazine molecule—Molecule 5. This hydrazine reacts specifically with aldehydes. Once it does, the product becomes a fluorescent hydrazone molecule. We tested this with two model aldehydes: formaldehyde and butyraldehyde. And we saw that when the reaction happens, there’s a clear increase in fluorescence.To study the probe’s behavior, we used tools like UV-Vis and fluorescence spectroscopy. These techniques let us measure how much light the molecule absorbs and emits. Our data showed that after reacting with butyraldehyde, there was a strong increase in fluorescence. That confirmed the probe was working just as we hoped. We also wanted to know how fast this reaction happens and in what environments it works best.In our kinetic studies, we found that the probe reacted more quickly with butyraldehyde than with formaldehyde, and it performed better in methanol than in pbs which is similar to human body ph 7. Even better, the reaction happens in seconds, which is perfect for real-time tracking. Here’s a visual. In these three vials, you can see how the probe behaves: The first vial just has the probe in methanol—no fluorescence. The second has methanol, butyraldehyde and the probe—strong fluorescence. The third has methanol, formaldehyde and the probe—some fluorescence, but not as much.So, in summary, we’ve developed a fluorescent probe that’s small, fast-reacting, and selective for aldehydes. It gives a clear signal when aldehydes are present, which opens up exciting possibilities for detecting biologically relevant aldehydes, with an emphasis on those generated by lysyl oxidases (LOX). This tool could help researchers track disease progression or even assist in diagnostics in the future. Our next steps include:Testing the probe in live cells to see how it performs in biological systems. Making more versions of the probe, including ones that work in the near-infrared range for deeper imaging in tissues. And eventually, applying it in 3D printed tissue models or even live animal studies.Thank you so much for watching!

College of Science OSCAR

Synthesis and spectroscopic characterization of drug-fluorescent probe conjugates for live cell imaging

Author(s): Maryam Baig

Mentor(s): Ozlem Dilek, Chemistry and Biochemistry

Abstract

Recent advancements in technology and medicine have brought attention to fluorescent probes due to their non-invasive, highly sensitive capabilities in live cell imaging, especially in biomedical fields. These probes are particularly valuable for visualizing cellular signaling processes and tracking drug activities at the cellular level, making them essential tools for studying cell functions and drug delivery systems. In this study, we focus on the development and characterization of a coumarin-based fluorescent probe conjugated with omaveloxolone (OMA), a drug used to treat rare genetic disorders. The resulting drug-fluorophore conjugate provides a means to explore organelle-specific drug activation and monitor cellular signaling processes. The synthesis begins with the preparation of a fluorescent hydrazone compound, which is then conjugated to OMA to create the final drug-fluorophore construct. The conjugation process was monitored using kinetics and UV-vis spectroscopy, with detailed studies of reaction kinetics, including solvent choice and concentration, aimed at optimizing conditions for further investigations. The drug-fluorophore conjugate was characterized through thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), and UV-vis spectroscopy. Various optimization steps were implemented to enhance the conjugate’s stability and efficiency, ensuring maximum fluorescence while preserving the biological activity of OMA. The fluorescent probe is designed to possess strong fluorescence and stability, making it ideal for cellular labeling when conjugated with the OMA drug. This conjugate is expected to demonstrate high fluorescence efficiency, stability within the cellular environment, low toxicity, and sufficient cell permeability. With the integration of fluorescent imaging technologies and molecular designs, drug visualization will have the potential to redefine our understanding of drug action in cellular systems, enabling the drug-conjugate dynamics in subcellular environment from new perspectives.

Audio Transcript

My name is Maryam Baig. I am a senior undergraduate at George Mason University, and I am presenting my project on the Synthesis and spectroscopic characterization of drug-fluorophore conjugates for live cell imaging. I had the opportunity to work on this project through Dr.Ozlem Dilek, who is part of the Department of Chemistry and Biochemistry located in the Institute for Advanced Biomedical Research at SciTech campus.

To provide a background for this project, I’d like to begin by explaining what fluorophores are. Fluorophores are chemical molecules that absorb Ultraviolet Visible light and project the emission in the form of light, and they help make up fluorescent probes. Fluorescent probes are molecular tools that allow scientists to visualize and observe live cell processes using highly sensitive, non-invasive and safe detection in biological cells. Omaveloxolone (OMA) is a drug being developed to treat Frederick’s ataxia, a rare and worsening disease that affects the nervous system. The fluorophore we are using for this project is a coumarin, and we have found that coumarin-based fluorophores have low inherent toxicity and can be readily internalized and washed out from cells, making them ideal for cell studies. In this project, we will focus on developing the fluorescently labeled OMA to monitor the delivery of fluorophore-OMA drug probes inside cells.

On this slide, we have two molecules that we will be using for our project. On the left side, you can see the OMA drug. It is a big molecule with multiple ketones present. Those are the double bonds with the oxygen molecules. On the right side, we have our CF3 coumarin. This is a published molecule and it is the coumarin that will we will be synthesizing and then conjugating with the OMA.

On this slide we have the synthesis procedure of our starting material, which is an amine, into the CF3 hydrazine that we will be using to conjugate to the OMA. As you can see, we will be adding an NH2 group, which is in amine group, to the existing amine.

To begin, we started by doing a thin layer chromatography between the drug, the dye and the conjugate after we had made the three. We diluted our samples in methanol, and then our TLC chamber solutions included various ratios of solvents that allowed us to visualize the mobility and composition of molecules on the TLC plates.

These are images from our TLC experiment. The samples on the left on the TLC plates are the CF3 dye, while the samples on the right are the product. As you can see, we observed a slight difference in shift between the lowest dots on the TLC samples. This indicates that we may have something new in our product.

From here we moved onto kinetics experiments, and after doing absorbance and emission data collection and nuclear magnetic resonance tests, we were able to make some conclusion. The data from the absorbance and emission graphs show that the drug-dye conjugate is fluorescent. Our NMR comparison between the CF3, the purified conjugate, and the OMA drug also gave us interesting results which we will see in the further slides.

This slide shows a comparison of the absorbance and emission data graphs that we collected for the conjugate to help us understand the composition of our molecule. As stated in the legend at the bottom of the slide, we can see that the CF3 is shown in red, the OMA is shown in blue, and the conjugate is shown in green. And if we look at the graphs, we can see that the red and green lines were very similar while the blue line was not as high. Because the red and green lines are so similar, we came to question if the dye may be overpowering the drug.

To see a more detailed and more accurate composition of the molecules we had worked with we conducted NMRs for each of the molecules. The purpose of an NMR is to analyze the magnetic properties of atomic nuclei to study the structure, the dynamics and interactions of the molecule. The area outlined here by black lines is where we will zoom in for the next slide.

Enlarging that small area shows us these multiple peaks that appear between the three samples. The yellow highlight indicates peaks that belong to the OMA drug. The blue highlighted peaks indicate the CF3 molecule, and the darker blue highlighted peaks indicate that we may still have some starting material remaining in our conjugate.

Based on the results of our NMR and the other test that we conducted we plan to move forward by trying to figure out where the CF3 is attaching on the OMA drug and how we can predict an NMR for it. Because of the dark blue highlight peaks that were present in our NMR on the previous slide, we decided to re-crystallize the CF3 coumarin to purify it further before we make another conjugate, and to try and get rid of those extra peaks. Finally, we plan to expand our range of molecules that can be conjugated with the OMA drug to see which one will be most efficient. We did a quick experiment in vials using small samples of different types of small molecules which you can see in this bottom image here and you can see were able to fluoresce. We plan to go forward with molecules numbers 2, 5, and 8, and study them further to see if they will be able to conjugate with the OMA drug.

Lastly, I’d like to acknowledge and thank Dr.Ozlem Dilek, Eva-Maria Rudler, and the rest of the Dilek team for their support and guidance throughout this project along with the GMU Department of Chemistry and Biochemistry. Additionally, I would like to express my gratitude to Dr.Karen Lee and the OSCAR team for giving me this unique research opportunity. Thank you for listening to my presentation.

College of Science OSCAR

A Non-Invasive Buffer Based Alternative to DNA Extraction from Teeth

Author(s): Fatima Durrani

Mentor(s): Joseph DiZinno, Forensics

Abstract

In forensic and anthropological contexts, DNA extraction from teeth is critical in cases where individuals must be identified from mass disaster sites, crime scenes, or ancient skeletal remains. Traditionally, the standard method adopted by the FBI and forensic laboratories is an invasive one, involving the drilling, crushing, or creating a powder of the tooth to access DNA within the dentin. This approach is impractical, since the tooth is a piece of evidence and can be important for analysis in the future. My study aims to provide a non-destructive alternative. I followed a protocol proposed by Monalisa Heng and Éadaoin Harney, using a guanidine-based buffer to extract DNA without damaging the tooth. Cleaned third molars were placed in the buffer, leeching DNA out of the cementum. This buffer proved successful, extracting DNA suitable for analysis, demonstrating that non-invasive extraction is a feasible alternative to destructive techniques. Additional research should be done in the future, including DNA sequencing to assess the quality and completeness of the genetic material. In addition, sodium or potassium acetate buffers could also be tested for comparative studies of non-invasive DNA extraction

Audio Transcript

Hello everyone, my name is Fatima Durani. I am a biology major and a forensic science minor. Or rather I have a minor in forensic science I conducted my research under the guidance of Dr. Madden in the biology department. I am a predental student as you can probably tell from my research so let’s get straight into it. My research focus is on a non-invasive buffer-based method of DNA extraction from teeth you’re probably asking Fatima why the heck are you doing research on teeth so some up when it comes to ancient remains or mass disasters where corpses are decay beyond identification, forensic scientist Take the teeth of these corpses extract DNA from these teeth and identify individuals, however All these scenarios have one thing in common can be used as evidence fortunately, the standard protocol to extracting DNA from these teeth is to destroy the truth. Now this visual I included actually is a minimally invasive method where the tooth is drilled and the DNA is extracted that way once it’s placed in the buffer however, in many cases, the entire tooth is destroyed and into a powder. And DNA extracted that way this is impractical when you’re looking as teeth as a source of evidence for future analysis So a solution to this issue is a buffer-based extraction protocol, which is what my research focuses on I used a buffer proposed by Mona, Lisa, Hank and Dr. Harney So this buffer includes guineas HCL which help DNA extract from the tooth while preserving the pH in the integrity of the DNA so as you can see here, I included a visual that shows you that the pulp in the Denton this pink flesh inside the tooth contain most of the DNA in the in invasive protocol which includes crushing the tooth aims to extract DNA from this area however, DNA is also included in the cement in the enamel, which is the outer portion of the tooth The invasive method that I will use that the invasive method that I will use using the buffer plants to extract DNA from this cement in the enamel, especially around the root right here, so the invasive protocol includes powder rising. The tooth placing get a protein is buffer and then quantify it using PCR. The noninvasive method includes emerging in a buffer created right here and then purifying the DNA quantify it using PCR and then and then both results or both DNA extractions from both methods will be analyze and analyze using gel electrophoresis This is just me. I gonna be so loud because teeth can contain diseases so I worked under a hood. This is the invasive method that I used crushing the juice to extract the DNA. This is the buffer and the that was placed in the buffer and this was me just cleaning the truth with. To remove any debris or blood so ask for the results in the conclusions the gel for the noninvasive method actually showed a successful amount of DNA these bands represent DNA at the 450 base pair Portion Well, one well one was the negative control which was just a buffer and two was the positive control which was a swab of my cheek and then the remaining well not including the ladder were the third molars that were placed in the buffer and DNA was extracted that way Invasive method actually was supposed to be a control method that had a positive result however, as you can see deep, something went wrong. There is no bands like the invasive method and there are two but they’re very faint so the likely reasoning behind this was. When I placed the powder in the button the Chex buffer I placed too much tooth powder, which let you contamination. Some future improvements to the study. Include adding DNA sequencing to check for contamination. Using degraded samples to allow my buffer to be implemented in anthropological settings and testing other buffers such as sodium and potassium acetate, which are stronger and more likely to extract more DNA from the acknowledgments. I’d like to know who is my mentor Doctor Who ordered all of materials necessary for this project and Dr. Madden for providing the lab and the resources I needed to conduct my research. As for the work protocol, buffer were inspired by Mona Lisa Hayes study on the development of non-invasive DNA extraction method. Definitely check her study and research out. It’s very interesting. She worked on dog teeth thank you guys so much for listening to my little research. Blurb have a nice day.

College of Humanities and Social Science College of Science OSCAR

Pharmacological Characterization of Chimeric NMDA Receptors in Xenopus

Author(s): Diborah Gutema

Mentor(s): Theodore Dumas, Department of Psychology, Interdisciplinary Program in Neuroscience

Abstract

NMDA receptors (NMDARs) play a critical role in synaptic plasticity and cognitive function by mediating both ionotropic and non-ionotropic signaling. During postnatal development, NMDA receptors undergo a subunit shift from GluN2B to GluN2A, a transition associated with synaptic maturation and the emergence of mature cognitive function. While previous research has explored the physiological and behavioral consequences of this transition, the specific mechanisms driving these changes remain unclear. In particular, the relative contributions of ionotropic and non-ionotropic signaling have not been fully defined. Ionotropic signaling involves calcium influx through the receptor channel upon ligand binding, while non-ionotropic signaling refers to intracellular signaling cascades initiated by conformational changes in the receptor that occur independently of ion flow. This study aims to dissect these signaling pathways by utilizing chimeric GluN2 subunits engineered to separate ionotropic and non-ionotropic functions. Chimeric constructs are currently being subcloned into Xenopus laevis–compatible vectors for functional expression in oocytes. This work has involved preparation of the pGEMHE-membrane-EGFP backbone through bacterial culture and plasmid isolation via alkaline lysis mini prep, followed by gel electrophoresis and spectrophotometric analysis to assess plasmid integrity and purity. Restriction digests were performed to linearize the plasmid and confirm backbone identity. In parallel, GluN2A, GluN2B, and chimeric constructs have been cultured from glycerol stocks and are currently undergoing PCR amplification with construct-specific primers. Following amplification, the inserts will be digested, purified, and ligated into the vector. Preliminary results confirm successful plasmid preparation, and insert amplification and ligation are ongoing. Once subcloning is complete, the constructs will be injected into Xenopus laevis oocytes, followed by two-electrode voltage clamp (TEVC) recordings to measure receptor responses to varying concentrations of glutamate and glycine, as well as pharmacological modulators of NMDA receptors. Statistical analyses using two-way ANOVA will compare current amplitudes across receptor type (GluN2A, GluN2B, ABc, BAc) and treatment condition (agonist/modulator concentration), with the goal of identifying distinct electrophysiological profiles associated with each subunit composition. This study will characterize how differences in GluN2 subunit composition and intracellular domain identity affect NMDA receptor-mediated signaling in an isolated system.

Audio Transcript

Hello all. I am Diborah Gutema and this is my video presentation for my project, Pharmacological Characterization of Chimeric NMDA Receptors in Xenopus laevis Oocytes.

NMDA receptors are ion channels located on neurons that allow calcium ions to enter the cell when activated by the neurotransmitter glutamate. This calcium signaling, known as ionotropic signaling, is critical for synaptic plasticity, learning, and memory. NMDA receptors can also engage in non-ionotropic signaling, where conformational changes in the receptor trigger internal signaling pathways without ion movement. Each receptor is composed of two GluN1 subunits and two GluN2 subunits. A developmental shift occurs where GluN2B subunits are gradually replaced by GluN2A, a transition essential for synapse maturation. Understanding how these subunits contribute to ion flow and conformational signaling is the focus of our project.

To investigate how different regions of NMDA receptor subunits contribute to signaling, we are working with chimeric GluN2 constructs developed by Dr. Dumas’s lab. These chimeras are engineered by swapping specific intracellular domains between the GluN2A and GluN2B subunits. In doing so, we can separate the functional contributions of individual regions, such as the intracellular tail, to ion flow and to non-ionotropic signaling. By studying receptors with these controlled domain swaps, we aim to determine which portions of the subunit structure are responsible for differences in calcium permeability, activation properties, and downstream signaling. This semester, we focused on preparing the DNA constructs necessary for expressing these receptors in Xenopus laevis oocytes for future functional testing.

The overall goal of this project is to express wild-type and chimeric NMDA receptors in Xenopus laevis oocytes and compare their ionotropic signaling properties using two-electrode voltage clamp recordings. By analyzing how domain swaps between GluN2A and GluN2B affect receptor function, we aim to better understand the molecular basis of NMDA receptor signaling. This semester, we focused on preparing high-quality plasmid DNA, optimizing restriction digests, and initiating PCR amplification of the GluN2 receptor inserts to prepare for future subcloning and expression studies.

First, upon receiving the plasmid pGEMHE-membrane-mEGFP, we transferred a sample from the backstab into a 3 mL bacterial culture, which was incubated overnight at 37 degrees Celsius for 16 to 24 hours. The plasmid includes a Xenopus laevis promoter sequence, which enables later expression in oocytes. Following incubation, we isolated and purified the plasmid DNA from the bacterial culture using a alkaline lysis mini prep protocol. To ensure the integrity and purity of the plasmid, we assessed DNA quality using agarose gel electrophoresis to check for intact plasmid structure and spectrophotometry to measure the 260/280 absorbance ratio.

Next, we performed restriction digests to prepare the plasmid for future subcloning. We used the enzyme NheI to linearize the plasmid and carried out diagnostic digests to prepare for the later excision of the GFP segment originally present in the vector.

In parallel, we grew bacterial cultures containing the DNA for GluN2A, GluN2B, ABc, and BAc constructs. Using these templates, we initiated PCR amplification with construct-specific primers to selectively amplify the inserts. PCR amplification is currently ongoing. Once complete, we will purify the amplified products and verify insert size by gel electrophoresis before moving on to the next phase of subcloning.

After the inserts are fully amplified and purified, we will digest them with restriction enzymes to create compatible ends with the plasmid vector. We will then use a DNA ligase enzyme to join the inserts and vector together, creating new plasmids that carry either the wild-type or chimeric NMDA receptor sequences. Some ligation reactions will be performed in-house, while others may be sent for commercial cloning depending on efficiency. Sequence verification will follow to confirm successful ligation.

Following sequence confirmation, we will synthesize capped RNA transcripts from the recombinant plasmids using in vitro transcription. These RNA molecules will then be injected into individual Xenopus laevis oocytes, allowing the cells to produce functional NMDA receptors for electrophysiological testing.

Two to three days after RNA injection, we will perform two-electrode voltage clamp recordings, a technique that holds the membrane potential constant while measuring ionic currents. By applying glutamate and glycine, we will evaluate receptor function based on current amplitudes, activation and deactivation kinetics, and dose-response characteristics. Comparing wild-type and chimeric receptors will help us determine how specific subunit regions influence NMDA receptor ionotropic signaling.

This semester, we focused on growing bacterial cultures, isolating and purifying plasmid DNA, troubleshooting purification and digestion protocols, and beginning PCR amplification of the NMDA receptor inserts. These steps are critical for setting up RNA synthesis, oocyte injection, and functional testing. Moving forward, we aim to complete subcloning, synthesize RNA, and characterize receptor function using TEVC recordings.

I’d like to take a moment to thank those who have been instrumental in this project.

Dr. Herin who has been an invaluable mentor in electrophysiology and molecular biology.

Dr. Dumas who has provided expert guidance on receptor signaling and chimeric constructs.

Hannah Zikria-Hagemeier who was essential in training me on plasmid preparation.

Finally, I’d like to thank the rest of the PBNJ Lab for their collective support through guidance and resources, which has been key to my growth as a researcher.

Thank you all for your help and support!

College of Humanities and Social Science College of Science OSCAR

Secretory Mitophagy Exports p53: A new pro-tumor survival mechanism.

Author(s): Layla Hasanzadah

Mentor(s): Purva Gade, Center for Applied Proteomics & Molecular Medicine

Abstract

Mitochondrial dysfunction is associated with many life-threatening illnesses, from Parkinson’s disease to malignant cancers. Cells remove damaged, aged, or stressed mitochondria through a process called mitophagy. Our team is investigating a potential pro-tumor survival mechanism cancer cells enact with the help of mitophagy initiation and subsequent export. Mitophagy initiation is sensed by the molecule PINK1, triggering the isolation and packaging of the damaged mitochondrial segment for degradation through the lysosome. Cancer cell mitophagy is triggered by elevated oxidative stress and mitochondrial DNA damage caused by hypoxia, chemotherapy, radiotherapy, and immunotherapy. In our recent study, we discovered a secretory form of mitophagy in which damaged mitochondrial segments are packaged and exported outside of the cell within PINK1+ extracellular vesicles (EV). Additionally, we found that these PINK1+ EVs export tumor suppressors (TS) phospho-P53 and Merlin along with mitochondrial segments outside the tumor cell. It has been discovered that p53 is phosphorylated by PINK1, ultimately enhancing mitophagy. We also found that Merlin, an unappreciated TS associated with neurofibromatosis, interacts with p53 through the MDM2 mediator. We hypothesize that secretory mitophagy export of TS and other mitophagy-related proteins is an adaptive mechanism of cancer cells to withstand oxidative stress and avoid apoptosis. The export of TS p53 and Merlin were analyzed in 4T1 & IOMM-Lee cells. We isolated EVs via differential ultracentrifugation followed by immunoprecipitation of purified EV samples with PINK1 and phospho-p53 to identify protein-protein interactions. Western blotting and mass spectrometry were performed to validate immunoprecipitation results and PINK1 and p53 interaction. Understanding the role of EV-mediated export of TS in secretory mitophagy can reveal novel mechanisms aiding cancer cells’ survival under oxidative stress caused by chemotherapy or other treatments, offering potential therapeutic targets for various cancers.

Audio Transcript

Mitochondrial dysfunction is associated with many life-threatening illnesses, from Parkinson’s disease to malignant cancers. Cells remove damaged, aged, or stressed mitochondria through a process called mitophagy. Mitochondria are known as the powerhouse of the cell for a reason: they help produce the majority of the energy needed to power our cells. In Parkinson’s disease, scientists have found that the process of mitophagy is impaired, leading to an accumulation of dysfunctional mitochondria within cells. On the other hand, in cancer cells, mitophagy plays a more complex role and may even be enhanced. As evidenced by my research, our lab at the Center for Applied Proteomics and Molecular Medicine found that the process may serve as a kind of survival mechanism used by cancer cells in the face of oxidative stress caused by chemotherapy or radiation.
Mitophagy initiation is sensed by the molecule PINK-1, triggering the isolation and packaging of the damaged mitochondrial segment for degradation through the lysosome. Our team discovered a secretory form of mitophagy in which mitochondrial segments are packaged and exported outside of the cell within extracellular vesicles (EVs) derived from the interstitial fluid of breast cancer tumors. Moreover, it has been discovered that the tumor suppressor molecule p53 interacts and becomes phosphorylated by PINK-1, ultimately enhancing mitophagy and carcinogenesis. Pancreatic cancer (PC) p53 mutations are associated with tumor aggressiveness. Decreased levels of intercellular p53 leads to increased genetic instability, higher tumor growth rate, and survival. Pancreatic cancer is the 3rd most fatal cancer in the U.S. due to high rates of metastasis and late diagnoses. Understanding and targeting this imbalance may result in new forms of personalized medicine for PC patients.
In terms of materials and methods, I cultured PANC-1 and BXPC-3 cell lines and incubated them for 5 days, then applied a drug called CCCP to each flask. CCCP induces oxidative stress to mimic the conditions experienced by cancer cells undergoing treatment like chemotherapy or radiation. Afterwards, I confirmed that I had protein in my sample, and used various stages of ultracentrifugation to isolate the extracellular vesicles produced by these cells. The vesicles came in 3 sizes: 2K (the largest), 10K, and 100K (the smallest). To analyze the proteins within my samples, I used Western Blotting and mass spectrometry, and used the ExoView R200 to examine and categorize the EVs used in the experiment.

Here you can see some images of me working in the lab: doing cell culture, running Western Blots, and observing my pancreatic cancer cells.

My project produced some very interesting results. I compared the relative concentrations of p53, the tumor suppressor protein, and PINK-1, the mitophagy-associated signalling molecule, and found that there is a very high and positive correlation between the export of PINK-1 p-p53 via EVs when oxidative stress is induced, indicating that p53 is degraded and exported alongside PINK-1 in EVs.Exported p53 may aid tumor progression and constitute a novel diagnostic method of non-invasively determining the mitochondrial health and p53 status within PC. PC EVs positive for phospho-p53 represent a novel diagnostic biomarker indicative of tumor stress. Targeting EV pathways in combination with oxidative stress could be a novel method of treating PC. Our lab is currently investigating if secretory mitophagy & EV export of tumor suppressors is common among other kinds of cancer, as well.

We recently published a paper on the topic of secretory mitophagy, but again, we hope to connect secretory mitophagy to the export of other tumor suppressors in future studies.

I wanted to thank my mentors and colleagues at the Center for Applied Proteomics and Molecular Medicine for their continued guidance and support, including the following people: Purva Gade, my direct mentor, Dr. Lance Liotta, Dr. Marissa Howard, Sofie Strompf, Angela Rojas, and Thomas Philipson.

I would also like to thank the GMU OSCAR URSP program and Dr. Karen Lee, as I received funding and guidance from OSCAR throughout the past semester.

Thank you very much for listening to my presentation!

College of Engineering and Computing College of Science OSCAR

THE DIRTY CONSEQUENCES OF POOR SLEEP: MODELING GLYMPHATIC EFFICIENCY ACROSS DIVERSE SLEEP-WAKE CYCLES QUALITY.

Author(s): Alvaro Olmo Jimenez

Mentor(s): John Robert Cressman, Department of Physics and Astronomy, Krasnow Institute for Advanced Studies

Abstract

The glymphatic system plays a vital role in maintaining brain health by facilitating the clearance of metabolic waste, a process most active during sleep. This clearance is facilitated by changes in extracellular space due to glial and neuronal shrinkage, enabling enhanced flow of interstitial and cerebrospinal fluid. The relation between the change in brain volume and the effectiveness of the glymphatic system has already been described. Despite the evidence linking sleep to brain clearance, the relationship between the quality of the sleep-wake cycle and glymphatic system efficiency remains unexplored. Thus impeding the understanding of how disrupted sleep may increase vulnerability to neurodegenerative diseases by impairing brain waste clearance. This study investigates the relationship between sleep-wake cycle quality and glymphatic system effectiveness by utilizing an existing computational model of neural dynamics. We calibrated the model to replicate real brain activity – matching frequencies and activity with data collected through EEG– during healthy NREM and REM sleep. These cycles were modeled and their response in brain volume change examined to assess the performance of the glymphatic system. Then, parameters – such as ionic conductance or vascular volume– were modified to simulate poor or high-quality sleep-wake cycles and the glymphatic system’s response examined. Early findings suggest that high-quality sleep cycles induce higher volume changes and therefore better glymphatic performance. Nevertheless, further analysis is required to more fully assess the system’s behavior across all sleep conditions.

Audio Transcript

Hello everyone. My name is Álvaro Olmo Jiménez, and today I’ll be presenting my research on THE DIRTY CONSEQUENCES OF POOR SLEEP: MODELING GLYMPHATIC EFFICIENCY ACROSS DIVERSE SLEEP-WAKE CYCLES QUALITY.
First, we will start by explaining why we are doing this research. Basically,we know that there are established links between sleep and brain clearance. The glymphatic system acts as the brain’s cleaning system and during sleep changes in glial and neuronal cell volume expand the extracellular space, which promotes convective fluid flow and waste clearance. Nevertheless, the specific impact of sleep quality on the glymphatic functions remains unexplored. This knowledge gap limits our understanding on how disrupted sleep may contribute to neurodegenerative disease risk
Thus, this study aims to explain how the quality of the sleep-wake cycles affect the glymphatic system during sleep.
To do so, we first established what was going to be our indicator for sleep quality → Brain volume change. This is because variations in extracellular and intracellular volumes during sleep enhance the glymphatic performance. Also, because the release of sleep-promoting molecules like prostaglandin induces blood vessel dilation and further volume changes.
At this point, we could state that our main focus was to study how the volume change is affected by varying the sleep-quality.
Once we had our indicator for good sleep, we used an existing model of neural dynamics implemented with glial dynamics whose behavior is determined by the concentration of ions.
This model was calibrated to replicate real brain activity – matching frequencies and activity with data collected through EEG. For example, a frequency of 2.8Hz was used to simulate NREM and 5.6Hz to simulate REM.
Moreover, we used an electrical and a volume stimulation as parameters to determine the sleep quality. The higher these parameters, the higher the simulated sleep quality. Therefore, from a bigger volume stimulation, a bigger volume change and thus the better glymphatic performance.
In order to replicate regular sleep, we did numerous simulations. However, just the most significant ones are going to be shown.
In this figure we can see 3 different simulations. In the three of them, the same electrical stimulation is used. The difference between the high and low volume stimulation is that the stimulation effort is halved. We can observe that over 20 cycles, there is not a significant brain volume change if we don’t stimulate the volume and that there is some difference in the final volume depending on the stimulation.
Now, we can see the transmembrane potential change for the high volume stimulation. One can see that the voltage deeply decreases with the volume stimulation. This makes sense because while the volumes vary, ion concentration varies too. Thus, we can state that the alterations of pump dynamics and diffusion result in a decrease in the transmembrane voltage.
In this figure, which again outputs high volume stimulation over the last cycle of the simulation, we can clearly appreciate the change in frequency from NREM to REM with the change in volume. Also, for further visualization, the right has been done and the change in frequency revealed.
From these figures, which show the change of concentration of intracellular sodium and extracellular potassium over the last cycle between electrical and non-electrical simulation outputs, we can see how electrical stimulation is fundamental for the correct simulation of sleep dynamics. Although it does not seem that important for volume change, we can see that in the simulation with electrical stimulation there is a balance between the intracellular and extracellular potassium and sodium. While in the non electrical-stimulated run, there is no apparent difference. This happens because the ATP-pump is shut-off due to the low extracellular potassium and thus cannot transport these two ions correctly. Although these ionic effects may not seem that important, they can be highly significant, as they can alter the signalling properties of the neuron.

This figure shows how the overall volume change varies if the sleep quality is disrupted. It is important to remark that in the microarousals simulation, 3 random intervals ranging from 1 and 5 seconds for each cycle were done and volume stimulation was stopped. Something similar was done for the less quality sleep simulation. In it 3 random intervals ranging from 5 and 15 seconds for each cycle were done and volume stimulation force was halved.

We can see the final values for each volume in this next figure.

Although it seems that the volume decrease is higher in the simulation with microarousals – suggesting that it has better glymphatic performance than varying sleep quality simulation – it is not. This is because the microarousals last less than the low stimulation stages. Thus, the simulation (with microarousals) would have less volume decrease if both periods– microarousals and low stimulation stages– lasted the same.

Now, from this data we can conclude that as sleep quality decreases, we observe a reduction in both overall volume changes and thus in glymphatic efficiency. This is consistent with previous findings that link slow-wave activity and stable sleep patterns with enhanced interstitial fluid movement and metabolic waste clearance.

Moreover, while volume stimulation contributes to mechanical shifts in brain tissue, electrical stimulation proves essential for preserving ionic balance. Without it, ATP-dependent pumps like the sodium-potassium pump become ineffective, leading to disrupted ion gradients and impaired homeostasis.

This underscores the critical role of electrical activity in maintaining proper cellular function, beyond just facilitating volume changes. The breakdown of ionic regulation in the absence of electrical stimulation highlights the interdependence of mechanical and electrophysiological processes in sleep. Together, these findings reinforce the complexity of accurately simulating sleep.

Ultimately, further research is needed in order to flawlessly replicate sleep, accounting not only for volumetric shifts and electrical rhythms, but also for how these elements dynamically interact over time. Accounting for the metabolic rate of the pumps.

College of Science OSCAR

Validations of Estrogen Assays in Baleen of North Atlantic Right Whales (Eubalaena glacialis)

Author(s): Sarah Fenstermacher

Mentor(s): Kathleen Hunt, George Mason University Department of Biology & Smithsonian-Mason School of Conservation

Abstract

Whale baleen has proven to be an accurate method in the retrospective longitudinal analysis of hormones. Baleen plates, the filter-feeding apparatus attached at the upper jaw in mysticete whales, continuously grow and represent a multi-year endocrine record that remains stable without undergoing post-mortem decomposition. While previous studies have quantified steroid and thyroid hormone concentrations in baleen from multiple species to evaluate different life-history events, the role of estrogens remains relatively understudied. Understanding reproduction in the critically endangered North Atlantic right whale (NARW), for example, is vital for accurate population estimate models.Therefore, archived baleen samples from 2 female NARW baleen plates were drilled every 4 centimeters using a dremel, and pulverized into a fine powder. Hormones were extracted from the baleen powder and Arbor Assays enzyme immunoassays (EIA) were used to quantify hormone concentrations. Three estrogen hormones: estrone (E1), estradiol (E2), and estriol (E3), were all validated for NARW baleen through parallelism tests using a pooled sample from non-pregnant females. This demonstrated a sample curve that was parallel to the standard curve, both of which were serially diluted samples: estrone (F1,8 = 0.09058, P = 0.771, r2 = 0.99), estradiol (F1,8 = 4.482, P = 0.0671, r2 = 0.98), estriol (F1,8 = 0.9084, P = 0.3685, r2 = 0.99). These hormones were quantified and compared to previously collected progesterone, stable isotopes, and confirmed calf-sightings to determine the behavior of these hormones during pregnancy, lactation, and resting periods. The data from these two females showed a spike in E2 at the end of pregnancy (after the progesterone (P4) spike) and was stable before pregnancy, which was the expected result. These estrogens appear to provide valuable insight in the study of reproduction (including gestation length and inter-calving intervals) in baleen whales.

Audio Transcript

Hi everyone! My name is Sarah, and I will be presenting my project on Validations of Estrogen Assays for Baleen of North Atlantic Right Whales. The samples used for this research came from the two whales pictured here…
Their names are Stumpy and Staccato, and they’re females who both died from vessel strikes in 2004. Ship strikes and entanglement are the two top-killers of NARW, and they are critically endangered with only 370 individuals remaining. Of those, only 70 are reproductively active females, meaning that the rate of population growth is limited to how often these females can have calves. Before these two were killed, they were a part of the breeding population; so they had documented pregnancies from regular calf sightings, and Stumpy also died with a full-term fetus.
Previous research on their baleen also confirmed that certain pregnancy hormones were elevated at the same time as these two were assumed pregnant, and subsequently seen with calves.
So what is baleen?
Baleen is keratin (the same structure as your fingernails and hair), and it is what they use to filter feed. It’s arranged in vertical strips that hang from the upper jaw as shown in these photos.
My mentor, Dr Hunt, was on the team that first determined that these baleen plates contain stable steroid and thyroid hormones, and repeated sampling along the length of a baleen plate can represent an endocrine record that spans multiple years of a whale’s life.
Because there is still debate in the large whale research community regarding length of gestation and exactly what happens during pregnancy, I was interested in re-examining these two females, this time, focusing on three estrogen hormones: estrone, estradiol, and estriol.

One of these hormones has been measured in NARW before (estradiol), but the other two (estrone and estriol) have never been measured in baleen whales before. We assumed that hormone extraction methods previously used would also work with these hormones, so we followed the protocol that Dr. Hunt developed.
Briefly, we measured the length of the baleen plate and used a dremel to generate powder every 4 cm along the length of the plate and then weighed the powder to 20mg. Hormones are then extracted from the powder using a MeOH-based protocol, followed by resuspension in assay buffer. Next, we performed enzyme immunoassays for each target hormone. This test allows us to calculate the target hormone concentration in each sample.

Because only one of these hormones has been previously validated for use in NARW baleen, my first objective was to ensure all three estrogen hormones could be reliably used in these samples. Specifically, I ran a parallelism test in each estrogen, and these are my results for that. On the x-axis of each graph, you see the log of the relative dose, and on the y-axis of each graph, you will see the percent of bound antibody. The goal for parallelism is for the standard curve to match the sample curve- both of which are made with serially diluted samples. I used a pooled dilution of non-pregnant samples from the two females (Stumpy and Staccato), and all three estrogens passed for parallelism. This meant that the sample curve was not significantly different from the standard curve (that means they were parallel to one another). We can see that the sample curve for E3 (estriol) only has 3 points; we did test other samples, but it appears a dilution greater than 1:4 did not have high enough concentration of the hormone to be detectable (but a 1:1 to 1:4 is detectable).

This project will continue into the summer, but I wanted to provide preliminary results of what we have seen so far. Because estradiol is typically a major pregnancy hormone, we wanted to assess it along the length of each baleen plate, providing longitudinal information during pregnancy, lactation, and non-pregnant (or resting) periods. We are working on continuing these assays along the length of the plate, so you will see some missing points, but we do have the results from one full pregnancy (in Staccato). Just to orient you on this graph, the x-axis provides the distance from the base (in cm), which really means time, and time moves forward from left to right (the very right side of the graph represents when the baleen plate was collected, meaning when she died).
On each of these graphs, the left y-axis and in the color blue, we can see the concentration of estradiol, while on the right y-axis and in the color green, is the previously published progesterone longitudinal profiles for each female. Stumpy on the left graph (a), has roughly the second half of a pregnancy shown on the left side of her graph (earlier in time), while Staccato (graph b) has an entire pregnancy and beginning of lactation shown. Though we are still working to fill in gaps, the results so far match what we expected. The hormone estradiol (E2) was relatively stable before pregnancy, but rose and peaked toward the end of pregnancy. Progesterone starts to elevate at the start of pregnancy, and maintains higher levels to the majority of a pregnancy.

So to summarize, assay parallelism validations were successful for E1, E2, and E3, which means that I will be able to analyze all three hormones along the length of both Stumpy and Staccato’s baleen plates. This furthers our understanding of the relationship between progesterone and the estrogens before, during, and after pregnancy. Once this is established, we may find similar patterns in other baleen whales, which will be interesting upon further study. This type of research will contribute to our understanding of large whale reproductive cycles, which is generally unknown, and will hopefully aid in population models and conservation efforts for this endangered species.

This project was funded by the OSCAR Undergraduate Research Student Program at George Mason. I’d also like to give a special thanks to my mentors Dr Hunt and Ms. Jelincic, for providing me with the guidance needed to complete this project. I also would like to acknowledge the Woods Hole Oceanographic Institute, for letting us borrow these archived baleen plates.
Thank you so much for listening and I hope you enjoyed learning about these incredible females, Stumpy and Staccato.

College of Science OSCAR

Behavior of Estuarine Crab Hosts as Affected by Parasite Infection

Author(s): Kiersten Jewell

Mentor(s): Amy Fowler, Environmental Science and Policy

Abstract

Parasites are an understudied portion of ecosystems, considering the impacts they have on their host species. Marine invertebrates such as crabs serve as both primary and intermediate hosts for several different parasite species. In the Chesapeake Bay region, the white-fingered mud crab (Rhithropanopeus harrissi) has been shown to host entoniscid isopods (Cancrion and Cryptocancrion spp.), a rhizocephalan barnacle (Loxothylacus panopaei). Given previous studies that parasites can change host population densities, alter predator-prey dynamics, and impact food web function, we sought to determine how parasite infection affects crab host behavior in the presence of a predator. These parasites are not trophically transmitted; if the host dies, they do too. Therefore, we hypothesize that infected crabs will spend more time hiding and resting compared to uninfected crabs. To test predator response, crabs were placed into an aquaria with open space and shelter habitat available. Their habitat use and behavior were videoed and calculated before and after the addition of a blue crab predator scent cue. Preliminary results show that uninfected crabs spend less of their time moving and more hiding and resting, as compared to their infected counterparts. This project will continue in the fall of 2026, expanding the sample size of crab hosts across all infection statuses.

Audio Transcript

Hello my name is Kirsten and I am an undergraduate researcher in Dr Fowler’s aquatic biology lab here at the Potomac Science Center, and my project for Oscar for this semester is looking at parasite infections in crab hosts and how it affects the behavior at- specifically the white finger mud crab or Rhithropanopeus harrisiiand two parasites that are found in it. One is Loxothylacus panopaei which is a parasitic barnacle that is actually invasive to the Chesapeake bay and it is a barnacle that creates an externa on the outside of the crab’s reproductive organs and it has a lot of morphological changing properties. It feminizes the male crabs and it completely castrates all crabs. I’m also looking at a species of Cryptocancrion which is an entoniscid isopod. The thing about both these parasite species is they are not tropically transmitted. That means thatif the host dies the parasite dies. So this led me to hypothesize that an infected crab is going to spend less time doing bold activities. That means it’s going to have an increase in hiding and resting and a significant decrease in moving around- especially in the presence of a predator. So what does it look like for us to test this? We have an aquaria setup where we are simulating conditions of both presence of predator and absence of predator. We use scent cues which are frozen ice cubes- the predator cue ice cube has frozen water that a blue crab was marinating in and the control ice cube has plain water with no predator scent. The aquaria has water that the crabs are acclimated too, a base layer of substrate along the bottom, a shelter that is comprised of PVC and tiles, as well as a red light because red is the go-to for crab behavioral studies, and a hammock that is saran wrap where the ice cube can rest.
For each crab each experiment starts with an acclamation period where they’re allowed to be in the tank for 20 minutes before they are videoed. Then we start the video and we record a control period – so this has no scent ice cube- It is just the crab in the tank. And then we add either the control ice cube or the predator ice cube with the scent cube and we record again. Then afterwards I analyze this with for an ethogram. So I have a whole suite of behavior options and a suite of location options. At 30 second intervals I record what the crab is doing and where it is, So for our preliminary results we simplified these behaviors into three categories: resting, moving, and hiding. This graph is showing theproportion of times that crabs are spending in these different activity levels per their infection status. And as you can see the uninfected crabs are actually showing less time moving and more time resting and hiding. This graph is again showing proportion of time I these different activity bins but it is showing these in the different cue presences. So we have the control, the predator, and the no cue. And we would expect there not to be a big difference between the no cue and the control cue because there’s no scent on that control cue. But we are noticing here on this percent change graph that there is a
difference which is indicating that maybe it is the ice cube itself that is impacting the crab’s behavior not so much the scent cube I am continuing this project in the fall as an independent research project where I will be increasing the amount of replicates we have across all infection statuses. So hopefully this will allow us to draw some cool conclusions about how parasite level is affecting crab behavior and itwill culminate in a publishable unit. I want to thank you guys for listening and thank you Oscar for funding this project for the spring of 2025.

College of Science OSCAR

Assessing symbiont diversity in restored and wild coral populations in Honduras

Author(s): Karina Cabrera

Mentor(s): Jennifer Salerno, Environmental Science and Policy Department

Abstract

Elkhorn (Acropora palmata) and staghorn (A. cervicornis) corals are important reef builders on Honduran reefs, and their coverage has declined by >90% since the 1970s due to disease and bleaching. These corals form obligate symbioses with photosynthetic dinoflagellate endosymbionts, and different symbiont taxa provide the host coral with benefits that aid coral resilience, such as thermotolerance or disease resistance. Ongoing coral restoration projects in Honduras have not yet identified the symbiont taxa in their corals, which is helpful to ensure effective restoration. Here, we used restriction fragment length polymorphism analysis to screen and identify symbionts from 266 wild and restored corals across different reef sites. This information will be given to the restoration programs, enabling them to assess the genetic and symbiotic diversity of their restored corals and improve their approach to slowing the population decline of these important corals.

Audio Transcript

Hello, everyone. My name is Karina Cabrera, and I am a Junior here at GMU pursuing a BS in Geology and a minor in oceanography. Today, I will be talking about the work I have done this semester to develop a protocol for identifying coral symbionts in elkhorn and staghorn corals.

Corals are important ecosystem engineers that build up coral reefs and provide habitat for extremely diverse organisms to live in, supporting as many as 1/3 of marine species. They also benefit human communities near the coast by supporting ecotourism and reducing coastal erosion. In the Caribbean, staghorn and elkhorn corals were historically dominant reef-builders but have experienced over 90% decline in the past 4 decades due to bleaching and disease.

This unfortunate decrease not only puts reef ecosystems at risk but also threatens the organisms that depend on reefs for survival, including humans. One way to combat this decline is through coral restoration, and specifically a method called coral gardening, in which samples are taken from wild corals and then grown in controlled conditions so that the coral population for staghorn and elkhorn corals are restored. Despite this collection method being an easy and fast way to restore corals additionally helping increase population numbers, because this process relies strictly on asexual reproduction, it also means that the coral host and symbiont diversity decreases over time.

These photosynthetic dinoflagellate symbionts form obligate symbiotic relationships with the corals, and different symbiont taxa provide the host coral with benefits that aid coral resilience, such as thermotolerance or disease resistance. Because of this, understanding the phylogenetic diversity of these symbionts will help improve the effectiveness of coral restoration efforts. I am working with four coral restoration programs in the Bay Islands of Honduras, seen on this map, but these restoration programs do not currently have the necessary molecular facilities or financial resources to perform molecular symbiont identification. To address this need, my URSP project focuses on developing a relatively cheap and efficient assay to identify the coral symbionts.

Samples were collected from wild and restored populations of the two coral species being restored in Honduras, staghorn and elkhorn corals. 100 wild corals were collected from sites all around the island of Roatan, and 166 restored corals were collected from the four different restoration programs on Roatan and Utila. To identify the symbionts in these samples, I developed a protocol based on polymerase chain reaction (or PCR) and restriction fragment length polymorphisms (RFLP), originally developed by Rowan and Powers. This protocol amplifies the 18S rRNA gene in the symbiont and then cuts up the DNA. These different length fragments from different DNA sequences are what cause different banding patterns. These different patterns then correlate to the taxonomic clades that the symbionts belong to. As you can see these are the banding patterns for clade a, b, c, and d. Getting into my results, I first optimized the PCR step. Based on the original protocol, which incorporated lower-quality DNA extractions, I was not getting good amplification of the target gene from most of the samples as shown in this PCR blank gel. This is due to the DNA being too short for the banding to show up. Because this gene is very long, I switched the protocol to use higher quality DNA instead and received much better results. In this optimized gel there are clear bandings due to the DNA being of higher quality and longer. I am now working to optimize the RFLP portion of the protocol. The restriction appears to be working from the gel there is some banding appearing at 30 minutes and there is some double banding patterns present, which is expected for these symbionts, but was not separated enough so I let the gel run for an hour and saw that it had become blurry. Because of this, my next steps are to try optimizing the time in which the gel is run since an hour seems too long, but 30 minutes is not enough for the bands to become clear, so hopefully reducing the time will give us better and more clear results. Once I have optimized this portion of the protocol, I will screen all the wild and restored corals and share my results and the protocol itself with the four restoration programs in Honduras. This will help them design out planting schemes that maximize genetic diversity and ensure that the restored populations mimic the diversity found in the wild. This will help improve the effectiveness of restoration efforts in Honduras and help to build future reef resilience against ongoing climate change.

This research would not have been possible without the OSCAR URSP Program and the environmental science and policy department here at mason. Thank you to Teagen Corpening, Jennifer Keck, and all of the RIMS interns who helped to collect samples and made this research possible. Finally, I acknowledge all the funders who supported this project. Thank you for your attention!

College of Science OSCAR

Navigating the Healthcare System: Barriers and Resources for Individuals from Low-Income and Immigrant Backgrounds.

Author(s): Kabir Toor

Mentor(s): Blake Silver, Department of Sociology and Anthropology

Abstract

This study investigates the barriers individuals from low-income and immigrant backgrounds face when navigating the U.S. healthcare system. While much existing research focuses on health outcomes, this project centers on the process of accessing care, including how individuals identify needs, seek services, and confront structural and cultural obstacles. The project originally involved an anonymous online survey featuring multiple choice and open-ended questions distributed through community organizations, hospitals, and doctors’ offices. Due to time limitations, primary data collection was minimal, and peer-reviewed scholarly sources were analyzed to identify trends aligned with the study’s goals. The literature revealed consistent barriers such as healthcare costs, limited insurance coverage, communication difficulties, transportation challenges, and fears related to immigration status. Facilitators of access, such as community health centers, family support, and bilingual social workers, were also commonly cited. Findings emphasize that even insured individuals often struggle to access care, illustrating a gap between insurance coverage and actual service use. These findings suggest a need for reforms that address not just insurance coverage but also cultural, logistical, and systemic obstacles. Underscoring the importance of community-informed research and policy interventions that reflect the complex experiences of low-income and immigrant individuals across the healthcare landscape.

Audio Transcript

Hello, my name is Kabir Toor, and I’m a student in the Department of Biology at George Mason University. Today, I’ll be presenting my research project titled “Navigating the Healthcare System: Barriers and Resources for Individuals from Low-Income and Immigrant Backgrounds.”

Accessing healthcare in the U.S. is challenging for many, but especially for individuals from low-income and immigrant backgrounds. My research asks: How do individuals from these communities navigate the healthcare system, and what barriers and resources shape their experiences? While much existing research has focused on health outcomes, this project focuses on the process of accessing care itself—how individuals recognize needs, seek services, and confront obstacles along the way.

Existing studies show that access to care is shaped by insurance status, financial barriers, language differences, and trust in healthcare institutions. For example, the author DeVoe et al. (2007) found that having insurance doesn’t always guarantee actual access to services. Further, Ngondwe et al. (2024) emphasized that immigrant communities often face additional bureaucratic and cultural hurdles. Given limited primary data collection, I analyzed trends across multiple major scholarly sources to anticipate key themes my survey was designed to capture.

The original study design involved creating an anonymous online survey distributed through community centers, hospitals, and doctors’ offices. The survey included multiple-choice and open-ended questions aimed at individuals identifying as low-income and/or immigrants. Participants were asked about their experiences navigating healthcare, including barriers encountered and resources utilized. Although direct survey responses were limited this semester, the survey framework was developed and approved for community distribution.

Using peer-reviewed studies as reference, several consistent themes were identified. For Barriers: High healthcare costs, insurance gaps, communication difficulties, and transportation challenges were identified. For Facilitators: Access to community health centers, family support systems, and bilingual healthcare providers were identified.  It is important to note that even individuals with insurance often struggled with actual access to needed services, showing that coverage alone is not enough.

Due to timing constraints, comprehensive primary data could not be collected during the allotted time. The current findings are based on anticipated trends and literature synthesis rather than direct participant responses. This limitation highlights the need for continued participant outreach to fully validate the study’s themes.

Moving forward, I plan to continue gathering survey responses through additional outreach at community centers and clinics. Once a robust sample is collected, I will perform a qualitative analysis using open codebook methods. This process will allow for the identification of emergent patterns directly from participants’ narratives, strengthening the study’s contributions to healthcare policy and access research.

So what are the implications, well, the findings suggest that reforms must go beyond expanding insurance access to address cultural, logistical, and systemic barriers. Community-driven solutions and culturally competent healthcare systems are critical to bridging gaps in access. This project reinforces the importance of centering underserved voices in future healthcare policy discussions.

I would like to thank Dr. Silver, my mentor, for his ongoing support and guidance. I would also like to thank the OSCAR URSP for funding this research, and I would like to thank the Department of social science at George Mason University. That concludes my presentation. Thank you for your time and attention.

College of Science OSCAR

Digitizing colors of soils in mesocosm wetlands using Nix sensor

Author(s): Seung Han

Mentor(s): Changwoo Ahn, Environmental Science and Policy

Abstract

Forty mesocosms located at the Ahn Mesocosm Compound at George Mason University have been a part of legacy studies for 12 growing seasons since 2012. In a previous study, each mesocosm wetland was planted with different amounts of species richness. After pre-COVID mesocosm studies halted, vegetation communities gradually changed. The Nix color sensor scanned mesocosm soils that were collected to produce 15 unique color variables for each mesocosm. Data was grouped by mesocosms that shared the original number of species planted. Preliminary results show that there are two out of five groups that have similar color variable values. Further analysis will continue to see if any differences arise and whether the soil conditions have been altered.

Audio Transcript

Hello! My name is Seung Han, and my project is “Digitizing color of soils in mesocosm wetlands using Nix sensor.”
When we look at soil, the color tells us about soil health and properties such as the presence of organic matter, mineral contents, and changes from fluctuating water levels between the different layers of soil. Here we can see the differences between the soil colors with the very grey wetland soil on the right versus the dark, fertile soil on the left. The standard method of analyzing soil color has been performed by using the munsell soil color chart. This method requires training, is costly, and analysis can be varied from person to person.
A new digital method of analyzing soil color is the Nix color sensor. Each Nix scan produces values for 15 color variables from 5 different color spaces. The color variables are l-a-b, c-h, r-g-b, x-y-z, c-m-y and k.
The study occurred at the Ahn Wetland Mesocosm Compound located in George Mason University’s West Campus. Back in 2012, forty mesocosms, or experimental ecosystems, were set up to study the effects of planting different amounts of species in each tub, ranging from no plants to 4 different wetland plants. The mesocosms were maintained by weeding out unwanted species and keeping the water levels above 5 cm. After pre-COVID studies were completed, the maintenance of these mesocosms stopped in 2019. Now for the big question. After studies and maintenance stopped, are the mesocosm wetland soils still wetland soils?
So now we begin. Each mesocosm had soil cores extracted from each of these five sections. The soil core was cut along the top to create a flat surface to scan, transferred on top of a white sheet, and the Nix was placed on top of a flat, smooth section of the soil for a scan to be performed. This was repeated at least 3 times for each core. Each scan data was saved in the Nix app on a smartphone, and the scans were exported into a csv file for analysis.
Data was organized by grouping mesocosms together with their original amount of planted species. Preliminary results analysis show that mesocosms originally planted with two and four species seem like they both have similar amounts in ten different color variables. A, b, and c, h in these graphs, and z, g, b, c, m, and y in these graphs.
Data analysis continues to see if the differences can be explained by differences in vegetation communities by identifying, counting species, and calculating the amount of percent cover.
I would like to thank my mentor, Dr. Changwoo Ahn, for guidance, encouragement, and believing in me. Thank you to Dr. Stephanie Schmidt for feedback and support this semester. Thanks to Rylee for coming out on hot days to help with some cores and note taking. Special thanks to Trinity Lavenhouse for being a great lab mate, giving support, and being a positive presence. And thanks to OSCAR for funding this URSP project. Take care!

College of Humanities and Social Science College of Science OSCAR

Use of a Novel In-Vivo Acetylcholine Sensor, GRABACh3.0, to Quantify the Temporal Dynamics of Acetylcholine (ACh) Release in the Cornu Ammonis 1 (CA1) Hippocampus Sub-Region

Author(s): Muhammad Shah

Mentor(s): Holger Dannenberg, Interdisciplinary Program in Neuroscience

Warning Video Contain Graphic Images

Abstract

Ongoing research aims to uncover how the brain processes spatial information, with the
long-term goal of informing therapies for spatial memory dysfunctions seen in
dementias. One key neuronal substrate is the place cell—neurons located in the Cornu
Ammonis 1 (CA1) subregion of the hippocampus that exhibit spatially tuned firing.
Recent studies suggest that acetylcholine (ACh), released into CA1 from the medial
septum, modulates place cell activity. Investigating how ACh modulates hippocampal
circuits during real-time behavior is essential to understanding its role in spatial
encoding.

The recent development of the fluorescent ACh sensor GRABACh3.0 enables real-time
measurement of cortical ACh activity in animal models. Using this sensor, we aim to
quantify the temporal dynamics of ACh release in the CA1 region during free-roaming
behavior in mice. To accomplish this, we will perform a stereotaxic injection of an
adeno-associated virus (rAAV) encoding GRABACh3.0 into CA1, followed by
implantation of an optic fiber above the injection site to permit fluorescence-based
recording via fiber photometry.

After allowing two weeks for sensor expression, a fiber photometry system will be used
to deliver constant excitation light specific to the sensor and record resulting
fluorescence during 15-minute free-roaming trials in a novel boxed environment.
Simultaneously, mouse velocity will be estimated using DeepLabCut, a markerless AI-
based pose estimation tool. Fluorescence signals will be synchronized with velocity data
to assess their temporal relationship.

Preliminary data revealed a positive correlation (r = 0.60) between ACh sensor
fluorescence and mouse velocity during free-roaming trials—a relationship consistent
with prior literature, supporting the validity of our recorded ACh signal. Next, we aim to
replicate this model and examine ACh release in CA1 during behavioral assays of
novelty and familiarity, to further investigate the neuromodulatory role of ACh in spatial
encoding.

Audio Transcript

Hello

My name is Muhammad, and my project is “Using a Novel In-Vivo Acetylcholine Sensor, GRABACh3.0, to Quantify the Temporal Dynamics of Acetylcholine (ACh) release in the Cornu Ammonis 1 (CA1) Hippocampus Sub-Region.”

In 1971, John O’Keeffe, a Nobel Prize winning neuroscientist, probed the electrical activity of hippocampal CA1 pyramidal neurons with electrodes in mouse models. He noticed that as the mouse traversed the environment, certain populations of neurons would increase firing rates in select regions of the box, which is now known as spatially tuned firing.

Discovery of these cells offered a new insight into how individual neurons encode space, and these CA1 neuron types would cleverly be named place cells! With this discovery came renewed interest in understanding the place cells within the CA1 region, with the goal of further uncovering the mechanisms of spatial cognition to inform future treatments for spatial amnesia in patients with, for example, Alzheimer’s disease.

Recently, it was discovered through retrograde tracing that Acetylcholine or ACh is released into CA1 region from distant cholinergic afferents from the medial septum. It is believed that these cholinergic afferents play a role in either directly stimulating place cells or stimulating other surrounding interneurons to inhibit place cells in the CA1 region.

Based on the multimodal modulation of place cells by ACh, understanding the release dynamics of ACh in real-time within the CA1 during behavior is of great interest.

To accomplish this, we used a novel ACh sensor called GRABACh3.0. Its major feature of interest is its binary nature, where it is capable of emission when ACh is bound but is incapable of emission when ACh is not bound. Thus, by using this sensor, the real-time neurodynamics of ACh within neuronal circuits can be observed.

To express the ACh sensor in mouse models, it is first packaged into a viral vector and then injected into the CA1 region, as seen here. We were able to confirm the expression of the sensor through immunohistology, shown here.

A fiber optic receiver is then implanted into the same location within the CA1 as the virus injection to measure the expressed ACh sensor. Then, a fiber photometry system is used to stimulate the sensor. Fiber photometry is a powerful technique that allows both excitation light, specific to the sensor, and control light to be transmitted through the same wire into the mouse fiber optic. It also allows, from the same input wire, to receive and record any resulting emission from either excitation or control light source, respectively, thus separating their influence on fluorescence within the sample. The fluorescence data is then converted into an electric signal using a phototransducer within the system.

Here is the system on the left where you can see both of the excitation cords that go into the sample and the emission cord coming back from the sample. We record fiber data for 15 minutes in a boxed environment. We also sync the fiber system with a bird’s eye video recording of the mouse to see how ACh levels correlate with the velocity of the mouse during the free roaming trials.

This correlation is found between ACh levels and velocity data to confirm whether we truly see an ACh signal from the mouse, as prior literature has established this positive correlation. After subtracting the control signal from the ACh signal and finding the velocity data using an AI markerless pose estimator, DeepLabCut, we obtained these preliminary results.

The graphs on the left show the isolated ACh signal, the velocity data, and their superimposition. Zooming in, we can see at specific time points when the animal increases its running speed, as marked by the arrows, that ACh activity tends to increase along with it. Furthermore, the overall correlation between the two variables was 0.60, which is very unlikely to be due to chance, especially over almost 45000 data points.

These findings were very exciting, but we face the next challenge of getting replicable results. The current findings come from our most recent and successful mouse, which resulted from a series of trial and error from previous mouse models using the sensor, as it is our lab’s first time using this specific sensor within the CA1. We are currently waiting on a new cohort of mice to see if we can consistently get results similar to those seen recently.

If we achieve consistency, we hope to use our standardized procedure and combine it with other techniques, such as optogenetics or spatial behavioral assays, in the future.

I want to give a big thank you to URSP for funding, Dr. Holger Dannenberg for his mentorship, and the Spatial Cognition Lab team for their support—this project would not have been possible without them.

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Warning Video Contain Graphic Images