Chimeric antigen receptor (CAR) T-cell therapy is a revolutionary cancer treatment that utilizes the body’s immune system to recognize and fight malignancies. It describes the process of extracting T cells from a patient and genetically engineering them ex vivo to express CARs that direct T cells to kill cancer cells. Multiple FDA-approved CAR T-cell products have shown promising clinical efficacy in treating cancers such as relapsed/refractory CD19+ B-cell leukemia and lymphoma. However, these products still have limitations in targeting other hematological and solid cancers. Solid tumors are difficult to treat due to their heterogeneity and ability to down-regulate or mutate antigen expression in response to treatment. Also, T-cell exhaustion and treatment associated toxicity result from the inability to precisely control CAR T-cell activity with these current treatments. Biological intermediates such as antibodies may be used to address these barriers, but they are expensive and can elicit an immune response. We aim to develop a universal CAR T-cell system that uses synthetic, high-affinity biomaterials to bind receptors expressed on cancer cells for targeted T-cell killing. For this project, we engineered CD8+ T cells, a cytotoxic subset of T cells, with lentivirus to extracellularly express universal CARs. Using flow cytometry, we confirmed strong expression of CARs on the T-cell surface and demonstrated their ability to interact with a synthetic cancer-targeting intermediate. Lastly, we show that CD8+ CAR T cells pre-armed with the targeting intermediate were capable of selectively lysing cancer cells in vitro that express the receptors of interest. For future studies, I hypothesize that we can expand this universal CAR system by utilizing other synthetic intermediates, such as a heterobifunctional small molecule peptide intermediate, for the treatment of solid tumors.

Archaeal cellular envelopes are quite simple compared to bacterial surfaces. Unlike bacterial cells, S-layers are the dominant component of most archaeal surfaces. Archaeal S-layers are composed of proteins or glycoproteins symmetrically arranged in an almost crystalline lattice. S-layers are in direct contact with the surrounding environment and facilitate important processes including protection, cell adhesion, molecular sieving, ion trapping, and nutrient adsorption. It is clear that S-layer proteins play an important role in an organism's ability to survive, especially within extreme environments. However, it remains unclear if S-layer proteins change in response to environmental stressors such as increasing temperature. This project is aimed at quantifying variation in bulk protein concentrations within microbial cultures containing a hyperthermophilic deep-sea methanogen (Methanocaldococcus sp. FS406-22) grown at 65, 73, and 85°C. Total protein concentrations were gathered via spectrophotometric analysis after growth. Optimal growth temperature for Methanocaldococcus sp. FS406-22 is 65C but can grow at temperatures up to 92°C, the highest known temperature limit for biological nitrogen fixation. I hypothesize that changes to Methanocaldococcus sp. FS406-22 S-layer proteins will be observed in order for the organism to acclimate to more extreme growth conditions. These data give important preliminary information regarding Methanocaldococcus sp. FS406-22 ability to physically adapt to changing environmental conditions. This project ultimately provides context for future work to be directed at exploring not just if S-layer proteins change but also how.

The weathering—or breaking apart by physical and chemical means—of common silicate (silicon dioxide based) minerals is a major mechanism of the freeing and distribution of nutrients into the environment at large, as well as a major component of the planetary carbon cycle. Effective extraction of nutrients by weathering is particularly important to organisms living along bedrock surfaces, such as the bacteria found in the basal ice of the Greenland ice sheet. Microbial surfaces have reactive sites which bind to ions and minerals, and may be a mechanism for internalization of nutrition in the incredibly harsh Greenland ice sheet basal water pores. These reactions may encourage the further dissolution of minerals as their constituent ions are pulled out of solution. Therefore, my research seeks to understand the effects that bacterial surface binding has on the dissolution of gneissic bedrock from the Greenland ice sheet’s base. I hypothesize that the presence of endospores will increase the rate of silica dissolution above control sample rates. To test this hypothesis, two experiments were performed. Ground gneiss obtained from the base of the Greenland ice sheet was placed into dialysis pouches. One dialysis pouch was placed in water containing Bacillus subtilis endospores and the other dialysis pouch was placed in pure water. The dissolution products were monitored over time. Initial results suggest that increased mineral dissolution occurs in samples with endospores present, implying that endospore surface reactivity may have important implications in chemical weathering processes. Better understanding of weathering processes, particularly as they are used to extract nutrients and control climatic conditions, allows us to better understand habitability in our present, past, and changing future.

Cancer is often a life-changing diagnosis that can be extremely difficult to navigate. Parents diagnosed with cancer face not only the burden of their disease, but also the burden of the disease on their children. In addition, previous research has shown a difference in experience for parents diagnosed with terminal cancer compared to those diagnosed with non-terminal cancer, with or without children. These families need support in communicating with their children. However, there is insufficient research about interventions that support parents' engagement in meaningful conversations about the illness. To begin providing these families with the tools they need to endure a cancer diagnosis, one must first discuss what concerns need to be addressed. The purpose of this study is to examine and contrast parent-reported concerns about the children's response to the parent's cancer for parents with early-stage cancer (stage I-III) compared to late-stage cancer (stage IV). Thirty-six mothers from a recently published clinical trial comprised the sample of parents with early-stage cancer. Parents were eligible if they had one or more dependent children five to twelve years of age and were recently diagnosed with stage I-III cancer. Parents' responses to the question "What questions do you have about what your child is thinking or feeling about the cancer?" were transcribed and inductively coded by these authors using content analysis methods adapted from grounded theory. Peer debriefing and an audit trail were established to protect the trustworthiness of results. Four of the resulting domains from parents with early-stage cancer mirrored those of parents with late-stage cancer and there were two major differences found between the two study samples. Contrasting these study results adds to our understanding of the unique or common needs for parents with early-stage versus late-stage cancer and informs future supportive interventions. 

Prostate cancer is the second most prevalent cancer among men in the United States. Characteristic biochemical markers include abundant Androgen Receptor (AR) and Prostate Specific Antigen (PSA), a downstream marker for AR activity. A rare neuroendocrine prostate cancer (NEPC), however, is characterized by low AR and PSA activity and high Synaptophysin (SYP; NEPC marker) expression. AR pathway inhibitors (ARPIs), the first-line treatment for prostate cancer, have minimal therapeutic effects on NEPC. This suggests that suppressed AR activity inhibits ARPI effect, and restoring AR could induce sensitivity to ARPIs, such as Enzalutamide. This study investigates the impact of overexpressing a tRNA, Arg-TCT-1-1, in NEPC. tRNA-sequencing of NEPC cell lines identified reduced expression of the Arg-TCT-1-1 tRNA isodecoder (Arg-TCT). Stable cell lines were generated for adenocarcinoma (AD) and neuroendocrine (NE) phenotypes, with overexpression of Arg-TCT or mutant TCT (mut-TCT). Reverse Transcriptase Quantitative Polymerase Chain Reaction confirmed Arg-TCT and mut-TCT overexpression. Cell lysate was immunoblotted for AR, PSA, and SYP; cell growth assays were then performed with Enzalutamide in DMSO to assess cell proliferation and sensitivity. Enzalutamide inhibited AR activity in LNCaP (1uM) and C4-2B AD (20 uM). Immunoblot suggests that Arg-TCT-1-1 overexpression rescues AR activity in NE cells, while mut-TCT does not affect AR activity. Cell growth assays reveal Arg-TCT-1-1 upregulation induces increased cell proliferation and enzalutamide sensitivity in NE cells. These novel findings demonstrate that upregulated tRNA promotes ARPI sensitivity in NEPC cells. Further research on how Arg-TCT-1-1 regulates AR activity and its transferability to other cancers is needed. These results suggest a promising therapeutic vulnerability if confirmed in murine models.

Treatment of individuals with HIV using antiretroviral therapy (ART) is highly effective, but effective clinical management depends on maintaining therapeutic drug concentrations. Antiretroviral (ARV) drug concentrations in patients with HIV can vary due to differences in drug metabolism, medication adherence, or interactions between multiple drugs. These individuals may have subtherapeutic or supratherapeutic drug concentrations, putting them at risk of treatment failure, acquisition of drug resistance, and risk of hospitalization or death. Current measurement of ARV concentration is done through liquid chromatography tandem mass spectrometry, which requires expensive equipment and requires a labor-intensive protocol. This restricts accessibility to specialized laboratories, making it difficult for persons with HIV to have routine measurements of ARV drug concentrations. The goal of the project is to develop an assay that is simple to perform and uses standard equipment to increase access to routine clinic-based drug level monitoring to improve HIV care. We designed an assay using a 2-step process of DNA strand transfer and quantitative polymerase chain reaction (qPCR) to quantify integrase strand transfer inhibitors (INSTIs). We tested for dolutegravir (DTG) and cabotegravir (CAB) in both buffer and plasma -- the latter to simulate patient blood samples. We were able to demonstrate that the assay could quantify clinically relevant drug concentrations of DTG and CAB. By developing an assay that can be readily integrated into most clinical laboratories, we will contribute to increasing access to routine HIV drug level monitoring to improve clinical HIV care and maintaining viral suppression in persons with HIV.

Nature uses only four nucleobases to store genetic information in DNA. However, additional synthetic bases which use Watson-Crick pairing have been developed and are known as non-standard bases (NSBs). NSBs P, Z, B and S incorporated alongside standard bases A, G, C and T compose DNA strands using a new genetic alphabet. Nanopores offer the potential capability for direct single-molecule sequencing of DNA containing non-standard bases (NSBs). Using a voltage gradient, DNA strands were directed through a nanopore, the biological membrane protein MspA, while we measured the ion current through the pore over time. In studying the effect of NSBs on the ion current through the pore, we observe current measurements corresponding to the Z base have a different noise profile compared to other bases. We hypothesize this noise may be associated with pH-dependent protonation of the base. To test this hypothesis, we conducted experiments with identical sequences in buffers of pH 8 and pH 7, as Z is known to have a pKa of 7.8. I analyzed the noise from the ion current signals to look for signs of protonation. I found increased current noise values associated with the Z NSB in pH 7 compared to pH 8, while the canonical A base had no change in noise values from pH 7 and pH 8, supporting the hypothesis that the increased current noise is due to protonation of the Z base. In addition to indicating potential sensing abilities of nanopores for probing protonation kinetics of DNA, this research contributes to a better understanding of the fundamental mechanisms that control the currents in nanopore sequencing of DNA.