This project focuses on analyzing Raman spectroscopy mapping data of a density-wave phase in graphene. Raman spectroscopy is a spectroscopic technique that allows us to determine vibrational modes of molecules and solids by their unique response to light. By implementing fundamental principles of solid-state physics in the analysis of Raman data, we can expand our knowledge about the electronic properties of quantum materials and how these properties can be tuned or manipulated. Utilizing a graphene sample grown on a copper substrate, our goal is to understand how this new phase of graphene affects graphene’s vibrational modes. Due to defects in the substrate some areas of the sample do not display this distorted lattice. Using Raman spectroscopy allows us to directly visualize this density wave in graphene emerging from the underlying substrate. For future experiments, comprehending the effects of substrate used is crucial to being able to control this type of distortion.

Mutated epidermal growth factor receptor (EGFR) is found in approximately 15-20% of non-small lung cancer (NSCLC) patients, where it drives tumor proliferation and metastasis. Despite clinical success of conventional EGFR tyrosine kinase inhibitors (TKIs) against primary EGFR mutant NSCLC, these TKIs fail to demonstrate efficacy in brain-metastatic NSCLC tumors. This failure can be attributed to the inability of these conventional TKIs to reach the necessary pharmacological levels to inhibit oncogenic EGFR in brain metastases due to the need to cross the blood-brain barrier (BBB). We recently developed a novel EGFR TKI, ERAS-801, which demonstrates the ability to penetrate the BBB, inhibit EGFR signaling in non-metastatic NSCLC and GBM, and minimize off target effects; therefore, we postulate that these properties make ERAS-801 a potential candidate for the treatment of EGFR-driven brain metastases. Thus, we focused on examining the efficacy of ERAS-801 against EGFR-driven brain metastatic NSCLC harboring the L858R mutation. To test this hypothesis, we compared ERAS-801 to the current standard of treatment for NSCLC, osimertinib, and other conventional EGFR TKIs, lapatinib and erlotinib. Utilizing 〖IC〗_(50 ) assays and western blotting, we concluded that ERAS-801 has higher potency and selectivity against EGFR compared to the other tested EGFR TKIs including osimertinib. This promising in vitro data supports the in vivo comparison of ERAS-801 against osimertinib in L858R EGFR mutant xenograft brain tumors. Together, this data suggests ERAS-801 may have efficacy in EGFR-driven NSCLC brain metastases and provides the foundation for further inquiries into the compound’s effectiveness in EGFR-driven brain metastatic cancers.

Sharing exosomes between cells provides a mechanism for cancer cells to communicate during metabolic stress. Exosome contents aid in tumor metastasis by enabling the invasion and colonization of organs to occur along with reprogramming recipient cell metabolic rates to facilitate disease progression. Additionally, mitochondria are central organelles of metabolism that enable cellular respiration and the production of energy and metabolites. We hypothesize that mutations in the mitochondrial genome (mtDNA) that frequently occur in cancer cells can affect intercellular signaling by altering exosome activity, potentially causing an upregulation of growth signals. To examine this hypothesis, I will measure functional metabolic changes using cell mass accumulation, proliferation, and growth assays and observe the corresponding impact on exosome expression. First, I generated cancer cell lines with mutated mtDNAs acquired from patient donor fibroblasts using the MitoPunch technique. I quantified tumor exosome production and the surface protein content of isolated exosomes characterized from these engineered mtDNA mutant cancer cells. Preliminary data obtained from Seahorse respirometry assays of cells containing either a large mtDNA deletion (Kearns Sayre Syndrome/KSS patient fibroblast donors) or an electron transport chain Complex I point mutation (Leber Hereditary Optic Neuropathy/LHON patient fibroblast donors) showed reduced glycolytic activity when compared to wildtype mtDNA cells. These results form the beginning of an interesting investigation and manipulation on the role mtDNA mutations and copy number in intercellular signaling via exosomes in cancer.

Small nucleolar RNAs (snoRNAs) are a class of noncoding RNA that guide post-transcriptional modifications mainly on ribosomal RNAs. In humans, snoRNAs are predominantly intronically encoded, making splicing an important process in the generation of mature snoRNAs. When splicing is interrupted, snoRNAs encoded within introns are left in precursor mRNA transcripts, leading to the production of hybrid mRNA-snoRNA (hmsnoRNA). HmsnoRNAs were recently characterized and consist of unspliced mRNAs that underwent exonucleolytic processing up to the mature 3’ end of the intronic snoRNA. Previous work showed that hmsnoRNAs are exported to the cytoplasm and associated with ribosomal complexes. Following these results, here we aimed to determine whether hmsnoRNAs are capable of translation into proteins. To test this hypothesis both classic and Gibson cloning methods were used to create two plasmid constructs containing a synthetic IMD4 gene with its intron and intron-encoded snR54 snoRNA. Both constructs were identical aside from one containing a hammerhead ribozyme sequence, which ensured that hmsnoRNA was the only transcript capable of producing the targeted protein. Using these constructs, we were unable to show via Western blot that IMD4-snR54 hmsnoRNAs are translated into proteins, despite the accumulation of the IMD4 hmsnoRNA that we confirmed by Northern blot. This is in opposition to what was preliminarily observed for another hmsnoRNA. These results suggest possible differences between individual hmsnoRNAs and provide insight into alternative translation methods.

Tauopathies, a subclass of neurodegenerative diseases characterized by the aggregation of hyperphosphorylated tau protein, progress along specific disease-dependent pathways. The progression of these diseases occurs through the templated misfolding of monomeric tau by tau aggregates, referred to as tau seeding. Though the exact mechanism of this seeding process is not well understood, previous studies indicate that brain-derived exosomes play an essential role by providing a vehicle for misfolded proteins to reach recipient cells from parental cells possessing tau inclusions. We hypothesize that the extent of tau seeding increases in samples derived from disease-affected cell types and brain regions relative to those untouched by the disease. Exosomes were isolated from brain tissue samples from the hippocampus, occipital cortex, and cerebellum via differential ultracentrifugation and immunoprecipitation using neuronal and oligodendroglial markers. Tau seeding activity then was determined using a FRET-based flow cytometry biosensor assay. While these experiments are still ongoing, we predict that in Alzheimer’s (AD), AD with cerebral amyloid angiopathy (CAA), and frontotemporal lobar degeneration (FTLD) brains neuron-derived exosomes from the hippocampus will show the highest amount of activity. Additionally, in progressive supranuclear palsy (PSP) brains, we anticipate that both neuronal and oligodendroglial samples from the cerebellum will possess significant activity. The results of this study will further our understanding of the contribution of exosomes to neurodegenerative disease progression and may enable the development of diagnostic testing, progression markers, and treatment options for these disorders.