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!

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!