Some environments hinder microbes from gaining the nutrients they need for a chemical reaction. Species in this circumstance collaborate to produce energy by clearing toxic substrates or providing nutrients. This is mutualism, where two species benefit from each other’s fitness. Previously, Desulfovibrio vulgaris and Methanococcus maripaludis were forced to rely on mutualism for survival over time. D. vulgaris catabolizes lactate, producing hydrogen as a byproduct. Hydrogen is inhibiting at high concentrations. M. maripuladis consumes this hydrogen and uses it for energy, producing methane as a byproduct. To learn how these microbes adapted to mutualism over time, 22 communities were propagated for 5000 generations. Each species was also propagated alone in environments that were as similar as possible to the mutualistic evolution environment. The whole genomes of the entire population of D. vulgaris or M. maripaludis were sequenced for the first 1000 generations using Illumina sequencing. This was done from 8 mutualism-evolved and 8 solitary-evolved cultures. Our research goal is to identify alleles that were substituted because they had a beneficial effect on mutualism fitness instead of other generic features of the environment. We can rule out generic adaptations in the population data by comparing mutations in community versus solitary-evolved clones. Clones were collected from each population that had been stored at –80 °C by streaking them on plates. Plates were also used to test if the clones could grow on sulfate. The DNA of these clones will be sequenced to confirm the presence and determine linkage of alleles that are beneficial to mutualism. It is anticipated that the mutation frequency of catabolic proteins will increase within D. vulgaris solitary clones in comparison with community D. vulgaris clones. This experiment has greater implications for mutualistic interactions between microbes, specifically in terms of the methane greenhouse gas that microbes produce.

Every second, living human cells are executing complex functions to initiate or influence various processes such as energy production, growth, metabolism and reproduction. This also means that intracellular structures are rapidly moving and morphing as the various proteins and organelles interact. Two important goals for scientists attempting to understand and manipulate intracellular processes are to visualize structural movements within live cells and to use specifically generated proteins to adjust interactions within the cell. Recently, members of the University of Washington’s Gao Lab have made a breakthrough by creating a cholesterol tag that allows delivery of small proteins across the cell membrane. I have explored the use of this method for delivery of immunological agents such as sdAbs (single domain antibodies) and synthesized Fab fragments for fluorescent visualization of structural cellular elements. These types of proteins have very specific target-binding which makes them optimal for imaging and therapeutics. Each of the tested immunological agents was delivered into the live cells successfully and the imaging proteins provided clear pictures of cell structures. Better understanding the functions and limits of this cytosolic-delivery method will increase the accessibility of live-cell fluorescent imaging, while the success of delivery opens up new possibilities in the field of intracellular protein therapies.

The process of development in mammals is always accompanied with high amounts of brain plasticity. Similarly, the maturation of sex organs during puberty in mammals serves as the foundation for important cognitive development. Researching centers of high plasticity could lead to key discoveries for the molecular basis of behavior. The medial preoptic area (MPOA) is an example of a very active site in pubertal gene expression. Estrogen-receptor 1, or Esr1, is a gene that is essential for hormone binding. We aim to gain insights on the neural basis of behavior by analyzing Esr1’s level of control in the MPOA, and on sexual behavior. This was completed by identifying single-cell types, determining the full transcriptome of the MPOA, and then observing any changes in gene expression after selectively knocking out the Esr1 gene. We utilized scRNA sequencing to identify single-cell types. The mating behaviors of mice was also studied to pinpoint any phenotypic differences. We have found that the deletion of Esr1 leads to severe hinderances in maturation, as well as sexual behavior, due to reduced function of Cis-regulatory elements. This provides important clarity regarding the key effectors of brain development in the pubertal stages. We aim to apply this approach to uncover the transcriptional dynamics of other areas in the brain, eventually forming a full, detailed representation of the brain. This would be a massive step forward in understanding the neural foundation governing sexual behavior.

Humans are incapable of regenerating a majority of their major tissues following traumatic injury. Xenopus tadpoles have the ability to regenerate a variety of complex tissues quickly following tail amputation, but lose this regenerative competency during metamorphosis. Though tadpoles have been extensively used to study regeneration, we do not yet understand the roles that stress signals from injury play in directing regenerative gene expression. We have found that inhibition of the stress responsive transcription factor Hypoxia Inducible Factor 1α (Hif1α) with the Hif1α inhibitors 2-methoxyestradiol (2ME) and Echinomycin (Ech) prevents regeneration. In particular, inhibition of Hif1α decreases Wnt mediated gene expression. Wnt is known to be one of the primary signaling processes necessary for proper Xenopus regeneration, specifically tail regeneration. While we have shown that Hif1α and Wnt regulate expression of similar gene programs in regeneration, we predicted that there are unique processes that Hif1α regulates to facilitate growth. To investigate how Hif1α and Wnt regulate regeneration, we utilized the two Hif1α antagonists, as well as the Wnt antagonist, IWR-1 (IWR) which I have previously shown inhibits regeneration. To determine Hif1α and Wnt regulated genes, we performed RNA-sequencing 24 hours post amputation. I then identified genes downregulated in Hif1α inhibited tadpoles, which are likely Hif1α dependent. I then removed genes downregulated by Wnt in order to isolate genes uniquely regulated by Hif1α. With the 250 genes uniquely regulated by Hif1α, I used PANTHER to perform gene set enrichment based on Gene Ontology terms. The main biological process of interest found to be regulated by Hif1α during regeneration was DNA replication. By determining how Hif1α uniquely regulates DNA replication during regeneration, we will continue to enhance our understanding of the roles that stress signals play in directing how regenerating cells meet increased proliferative demands post traumatic injury.

Osteoporosis is an orthopedic disease in which old bone begins to dissolve, but is not replaced by new bone. This reduces overall bone density and increases a patient’s risk for fractures. Our lab studies genes that contribute risk to osteoporosis. One human gene associated with osteoporosis is WNT16, which is also expressed in zebrafish. Previously, we mutated the wnt16 gene at four different sites to produce a range of skeletal phenotypes. These studies show that wnt16 mutant fish have skeletal defects. The aim of my project was to find how wnt16 influenced spine segmentation in mutant zebrafish. Through GNOMAD, a Genome Aggregation Database, I collected data on the types of mutations that naturally occur in human Wnt genes. Across all human Wnts, there was no strong bias for stop-gained mutations in the terminal coding exon; however, in WNT16, stop-gained mutations were underrepresented in the terminal coding exon, which was of interest. Using CRISPR-based gene editing, we used a guide RNA to mutate the terminal coding exon in zebrafish wnt16 and found a range of severe skeletal abnormalities along the spine. Segmentation of the wnt16 mutant zebrafish was disrupted compared to the controls. I determined how wnt16 influenced segmentation of the spine by quantifying centrum fusions and other morphological abnormalities in these mutant zebrafish. Our results implicate the wnt16 gene as essential for spine morphology and contributes to understanding human phenotypes of osteoporosis.