Current Scholars and Projects

Following the loss of a loved one, people often seek reminders of them such as photos, favorite places, or familiar activities. This behavior, known as partner-cue seeking, is also seen in prairie voles, a socially monogamous species, after separation from a bonded partner. The purpose of it, however, remains unclear. This project aims to identify whether exposure to partner cues during the loss state modifies behavioral response in a stress test and biological markers of autonomic stress response activation.

Prairie voles will first be paired with an opposite-sex genetically compatible partner for one week, and bonding will be confirmed through a partner preference test. After separation for one week, voles will be exposed either to partner-scented bedding or unscented bedding.

Behavioral stress will be assessed using the elevated plus maze to evaluate sympathetic nervous system activation and determine behavioral stress responses. Biological measures of the sympathetic nervous system will be evaluated in two ways. Blood samples will be analyzed for hormone and peptide levels, including oxytocin, vasopressin, and corticotropin-releasing factor (CRF). Brain tissue will be sectioned, and immunohistochemistry (IHC) will be used to examine the paraventricular nucleus of the hypothalamus for levels of vasopressin, CRF, and oxytocin. Special attention will be given to the anterior cingulate cortex (ACC) and the insular cortex (IC) given that these regions are known to regulate both social attachment and the autonomic stress response, suggesting these processes may be interlinked.

There are two potential outcomes to this project. One possibility is that partner cues may soothe, reducing anxiety and biological stress markers in bonded animals. Another possibility is that these cues cause distress, heightening the stress response. Both explanations may be accurate depending on factors such as the time since separation, the strength of the bond, and the context in which the cues appear. Nevertheless, either outcome would provide valuable insight into the loss model and a deeper understanding of how the experience of chronic loss stress affects the brain's response to acute stressors.

Flower symmetry is important for reproductive success in fruiting plants. Symmetry allows appropriate pollinators to recognize rewarding flowers and facilitates reproduction through the symbiotic relationship of pollination. Mimulus lewisii is a model species for studying flower symmetry. In M. lewisii two genes, CYCLOIDEA 2A (CYC2A) and CYCLOIDEA 2B (CYC2B), encode transcription factors important to forming bilateral symmetry in developing flowers.
My goal is to demonstrate CRISPR knockout of CYC2A and CYC2B in M. lewisii to characterize their contributions individually and in combination to overall flower symmetry. Using Agrobacterium mediated stable transformation techniques I will introduce a CRISPR construct into wildtype M. lewisii. The construct will produce functional CRISPR/Cas9 complexes when transcribed and translated. The complexes will cleave the DNA on both sides of a conserved region, the TCP domain, of the M. lewisii CYC genes. Cell machinery will repair the DNA with a fragment of the TCP domain deleted, presumably rendering the CYC genes nonfunctional. With CYC gene loss of function, CYC2A and CYC2B proteins will not be able to carry out their roles as transcription factors, and the effect on flower development can be carefully determined.
To screen for plants affected by the treatment, the CRISPR construct includes a resistance to Basta herbicide. Seeds from self-pollinated, infiltrated flower buds will be grown and treated with Basta. Surviving seedlings likely have the CRISPR/Cas9 machinery integrated into their genome. The DNA from surviving seedlings will be extracted and tested for mutations in CYC2A or CYC2B (depending on CRISPR/Cas9 construct and gene targeted by gRNAs) via PCR and DNA sequencing. CRISPR lines should be heterozygous for the TCP mutation. If PCR and/or DNA sequencing show mutations, it will indicate success, and the plants will be further propagated by self-fertilization for additional flower trait characterization.
Through CRISPR knockout of CYC2A and CYC2B, the effects of these transcription factors on M. lewisii flower symmetry can be carefully determined. Analysis of CYC2A and CYC2B function will improve understanding of both the model system and many related organisms.

Chromobacterium subtsugae is a non-pathogenic soil-based Proteobacteria that is used as a model to study bacterial interactions. We previously discovered that C. subtsugae uses antibiotic resistance to defend against other bacteria during competition. One mechanism of antibiotic resistance is with the CdeAB-OprM efflux pump, which can pump antibiotics and other substrates from within the cell to the environment. This pump allows the bacteria to defend against competing antibiotic-producing bacteria.
My lab also previously found that the CdeAB-OprM pump is regulated by antibiotics. Regulation is through a transcription regulator, CdeR, that represses the cdeAB-oprM genes. CdeR is a member of the TetR family of gene repressors, which often bind directly to the promoter of their target gene to repress transcription. A potential binding site of CdeR was identified as the cdeA promoter based on conservation with that of other closely related TetR-family proteins. A mutation at this site in the cdeA promoter was found to abolish repression of cdeA transcription by CdeR. This strongly suggests that CdeR directly interacts with the cdeA promoter to regulate the efflux pump genes.
To further this research, I will test whether CdeR directly interacts with the efflux pump gene promoter. I will complete this by first purifying CdeR by expressing a His-tag CdeR in E. coli and filtering through a nickel column; second, testing for binding of purified CdeR to the cdeA promoter using an electrophoretic mobility shift assay, and third, mutating the conserved binding site in the cdeA promoter and testing for reduced binding interactions with CdeR compared with the wild-type cdeA promoter.
Completing these experiments will allow my lab and I to further our understanding of how C. subtsugae uses CdeR to regulate the CdeAB-OprM efflux pump in response to antibiotics. These results will also add to our understanding of how bacteria integrate cues from the environment to optimize strategies to compete with one another.