The projects listed below are merely a sampling of the many research opportunities at The Wertheim UF Scripps Institute for PREP participants. All research labs at The Wertheim UF Scripps Institute are open to participants in this program.
Laura Bohn, Ph.D. (Receptor pharmacology, structure-function relationships)
The Bohn lab is interested in how ligands bind to seven transmembrane-spanning receptors (G protein-coupled receptors), and how the nature of this binding influences the subsequent engagement of intracellular and membrane-associated effectors to culminate in biological signals. These questions are studied in a collaborative environment, benefiting from medicinal chemists who develop ligands, and apply receptor pharmacological theory and experimental approaches to capture how ligands affect receptors. They also work with X-ray crystallographers to understand the binding poses that the receptors assume, and use mouse models to understand these ligands’ impact on physiological and behavioral measures.
Undergraduate interns and trainees have contributed to studies of how ligand-directed signaling downstream of G-protein coupled receptors (GPCRs), such as the mu opioid receptor, can be used to turn on one signaling pathway, while preventing engagement of another. Research trainees also help the Bohn lab learn fundamentally new aspects of how GPCRs refine their functional output in diverse cellular populations, as well as within a cell type.
Previous intern Nilay Shaw generated point mutations in the mu opioid receptor to help to understand how the agonists bind to the receptor and how that effected functional signaling. His poster won the first price at ASCB in 2018. Erika Serrano Diaz, who came with a chemistry background, determined optimal routes of ligand administration gaining an appreciation for compound metabolism. Diaz and Shaw are still undergraduate students.
James Burke, Ph.D. (RNA biology, immunology, virology)
The Burke lab investigates how mammalian cells respond to viral infection and how viruses circumvent these cellular responses. We use genetics, molecular biology, and advanced microscopy techniques (i.e., live-cell imaging and single-molecule RNA visualization) to study the RNA biology of the innate immune response to pathogenic viruses – dengue virus, SARS-CoV-2, and influenza A virus. Understanding these processes is important since their dysregulation can lead to viral-associated pathologies, such as cytokine storms, chronic inflammation, and autoimmune disorders.
One pathway that we study is the OAS/RNase L pathway. Our recent studies demonstrate that RNase L rapidly degrades nearly all basal mRNAs in cells in response to viral infection and double-stranded RNA (Burke et al., Mol. Cell. 2019; Burke et al., Sci. Adv. 2021; Burke et al., RNA 2021; Burke et al., 2022 bioRxiv). However, both host antiviral mRNAs (i.e., type I interferons) and viral mRNAs and can evade RNase L-mediated decay. One project will focus on characterizing RNase L-mediated decay of host basal mRNAs and understanding how viral/antiviral mRNAs evade RNase L. In addition, the degradation of host RNAs by RNase L vastly alters cellular RNA biology – the assembly of antiviral ribonucleoprotein complexes (stress granules, p-bodies), nuclear mRNA export, and RNA-binding protein localization. Several projects will focus on characterizing these processes and their function during the innate immune response to viral infection.
Matthew Disney, Ph.D. (Chemical Biology of RNA)
The number of RNAs known to play critical regulatory and functional roles in biology has grown explosively. A powerful approach to interrogate RNA biology is the development of small molecules that perturb RNA functions. Indeed, many mechanisms of ribosome function were uncovered using small-molecule antibiotics, and these remain go-to tools to study gene translation.
The Disney lab has established transformative technologies that allow for the design of chemical RNA probes from sequence. Specifically, they developed novel selection-based strategies that allow the identification of RNA motifs that bind to small molecules with high affinity and selectivity. In conjunction with bioinformatics approaches, these data are used to identify cellular RNAs that harbor the desired small molecule-binding motifs. Over the course of the last decade, undergraduate interns and trainees with a wide variety of interests have been involved in this work, and the project can be customized to a trainee’s interest and capabilities. For example, trainees interested in organic synthesis and chemical biology can use their talents to develop novel lead molecules or explore their mechanism of action. Two recent undergraduate students, Christy LaFlamme and Mary DeFeo, contributed to publications that are in preparation. DeFeo, who won an NSF predoctoral fellowship and is now in graduate school at Stanford, developed a new approach to cleave RNA targets with bleomycin conjugates and has been able to demonstrate in cellular models that these compounds specifically cleave the desired RNA target and not DNA. LaFlamme developed a new approach to cleave RNA targets by recruiting a nuclease. She has demonstrated that these compounds act substoichiometrically and turn over RNA in cells: one compound molecule cleaves 26 target RNAs.
These computational approaches are also ideal for trainees with physical disabilities, and one recent wheelchair-bound undergraduate student, Audrey Winkelsas, was included as a co-author on a recent publication (Disney MD, Winkelsas AM, Velagapudi SP, Southern M, Fallahi M, Childs-Disney JL., (2016) ACS Chemical Biology). She also wrote a successful NSF-fellowship application to attend the NIH-Oxford graduate program. Her work was featured in a segment of the PBS series “Gene.”
Katrin Karbstein, Ph.D. (Biochemistry, Yeast Genetics)
The Karbstein lab studies how ribosomes are assembled, how this process is quality controlled, and how misassembled ribosomes lead to disease in humans. In addition, they have started to define physiological situations where alternate ribosomes accumulate and are investigating the mechanisms by which they arise. They largely address these questions using yeast as a model organism and employ a wide variety of techniques from biochemical assays with recombinant proteins, over biophysical techniques, to yeast genetics and yeast biochemical experiments. Many of these are well-suited for undergraduate projects, and most of the lab’s previous undergraduate students and both previous post-bac trainees have ended up as co-authors on manuscripts and entered graduate school at top institutions.
Several ongoing projects in the lab indicate that Rps29-deficient ribosomes arise commonly in cancer cells and are linked to decreased survival. Moreover, the current post-bac student has shown that poikiloderma, a skin-disease, is caused by misassembled ribosomes that lack Rps29, and data in the literature indicate a role for Rps29-deficient ribosomes in aging. The Karbstein lab has previously described roles for Rps26-deficient ribosomes (Ferretti, MB, Ghalei, H, Ward, EA, Potts, EL, and Karbstein, K* (2017). Nat Struct Mol Biol. 24(9):700-707; Ferretti, MB, Barre, JL & Karbstein, K (2018) Cell Chem. Biol. 25(11)1372-79), work that has included two summer undergraduates and a year-long high school student. They are now interested in better understanding how Rps29-deficient ribosomes differ from fully-assembled ribosomes, and how these differences might lead to the above-described diseases.
A second possible project revolves around mechanisms of RNA folding during ribosome assembly. They have recently used a combination of structural, biochemical and genomic analyses to dissect the role of assembly factors in ensuring the correct order in which secondary structures are adopted (Huang, K & Karbstein, K. (2022) Proc.Natl. Acad. Scie. Jun 22;118(25):e2101164118). Moreover, they have also analyzed the structure of a misfolded intermediate and discovered mechanisms by which the assembly machinery avoids misfolding (Liu et al., BioRxivs). These have led the Karbstein lab to consider additional roles for assembly factors in guiding the folding landscape of nascent ribosomes, work that will build students skills in genetic, genomic and biochemical analyses.
Laura Solt, Ph.D. (Nuclear receptor function)
Nuclear receptors are highly conserved, ligand-regulated transcription factors that sense the metabolic state of the cell by sampling their respective ligands and adjusting gene transcription accordingly. This is particularly important for cells lining the intestinal tract, including immune cells, which are constantly exposed to environmental insults. The Solt lab uses a combination of genetic, molecular biology, immunology, and chemical biology approaches to understand how nuclear receptors function, using the immune system as a model. Much of their work has focused on the biology of REV-ERBa/b in Th17 cell development.
Previous undergraduate intern Laura Chopp determined the kinetics of REV-ERBa/b expression during Th17 cell development and showed that loss of REV-ERBa led to increased IL-17A expression. Her results helped the lab establish that the REV-ERBs were key negative regulators of Th17 cell development and laid the foundation for current work exploring the relationship between the REV-ERBs (Amir M, Chaudhari S, Wang R, Campbell S, Mosure SA, Chopp LB, Lu Q, Shang J, Pelletier OB, He Y, Doebelin C, Cameron MD, Kojetin DJ, Kamenecka TM, Solt LA. (2018) Cell Rep. 25(13):3733-3749). Chopp, now a graduate student at the University of Pennsylvania, won an NSF fellowship based on these studies.
Another previous undergraduate student, Ashley Nichols, further explored how ligand regulation affects co-factor recruitment to various nuclear receptors, including REV-ERBa and PPARg. (Shang J, Mosure SA, Zheng J, Brust R, Bass J, Nichols A, Solt LA, Griffin PR, and Kojetin DJ. A molecular switch regulating transcriptional repression and activation of PPARg. Nat Commun. 2020 Feb 19;11(1):956). Nichols is currently a graduate student at the Gerstner Sloan Kettering Graduate School of Biomedical Sciences and also won a NSF fellowship based on her studies in the Solt lab.
Finally, Molly Bassette, another previous undergraduate student who is currently a PhD candidate at the University of California, San Francisco, worked on another family of nuclear receptors, the RORs, which are positive regulators of transcription and Th17 cell development (Wang R, Campbell S, Amir M, Mosure SA, Bassette MA, Eliason A, Sundrud MS, Kamenecka TM, Solt LA. Genetic and pharmacological inhibition of the nuclear receptor RORa regulates TH17 driven inflammatory disorders. Nat Commun. 2021 Jan 4;12(1):76).