The inner ear consists of hair cells which are responsible for hearing and balance. Over time, these hair cells die and get damaged, resulting in hearing loss. Curiously, chickens and other related species are capable of regenerating their lost hair cells via supporting cell differentiation, while mammals can only regenerate hair cells in the balance organ. To better understand the mammal regeneration process we are studying Cre-lox mice that allow us to change the gene expression in supporting cells which can differentiate into hair cells. These mice allow us to track supporting cells during regeneration, which types of hair cells they turn into, how they turn into hair cells, and to delete or misexpress genes in supporting cells, in order to test the role of various genes in hair cell regeneration. My research is examining three lines of Cre-lox mice, id2, glast, and sox9, to assess their usefulness for these studies. I studied the images of the mouse utricle, one balance organ, and determined which types of supporting cells, and how many, can be monitored or manipulated in each line of mice. Through the results of this project, we hope to better understand the specific process of mammalian hair cell regeneration, as well as prove this method as viable for analyzing specific gene expression in other cells.

Plants’ ability to sense ambient light conditions is critical for their fitness. Phytochromes (phy) are photoreceptors that translocate to the nucleus when activated by red light. One consequence of this translocation is rapid phosphorylation and turnover of basic helix-loop-helix (bHLH) transcription factors called Phytochrome-interacting factors (PIF). PhyB has been shown to recruit and inactivate PIF5 in seedlings and inhibit elongation of the stem (hypocotyl). Phytochrome mutants in Arabidopsis thaliana exhibit reduced CO₂ uptake, abnormal accumulation of sugars and metabolites during the day, and shade avoidance-like phenotypes. We aim to compare findings in A. thaliana with phenotypes of light signaling mutants in Brassica rapa, a related crop species. We have isolated phyB, and pif5 mutants and are growing them in ambient and high CO₂ conditions. Measurements of photosynthetic rate as well as levels of chlorophyll, sugars, total protein and starch will provide insights into the role of Phytochrome signaling on metabolic processes. Preliminary results show that phyB mutant seedlings exhibit longer hypocotyls in comparison to wild-type plants at ambient CO₂ but have a reduced growth response to elevated CO₂ compared to wild-type. Adult plants have a reduced photosynthetic rate compared to wild-type. In contrast, pif5 mutant seedlings have short stems and reduced root growth in comparison to wild type. Our results to date are consistent with previous studies in A. thaliana. Examining the role phytochromes play in resource allocation, specifically in response to Carbon availability will aid in identifying key phytochrome-mediated developmental pathways. This research has implications in engineering more efficient crops and in improving understanding of plant metabolism as a whole.

This research examines dancers with dance-related injuries due to hypermobility and hyperextension. Initial research has been conducted through interviews and a survey. The author interviewed students and dance instructors who have personal experience or are familiar with hypermobility and hyperextended joints. The author then conducted a survey on the topic titled, “Hypermobility in Dancers” which was distributed to the University of Washington Dance Program faculty and students (18 or older), as well as the dance community in Seattle. The final step of this initial research will consist of a movement laboratory, whereby the author will ask dancers to perform a series of set movements in order to analyze how dancers problem solve differently depending on the presence or absence of hypermobility/hyperextension. The results of this research aims to show that dancers with this structural phenomenon are more prone to specific types of injuries than dancers without.

Many court cases in the United States are resolved through plea bargaining. A plea bargain is an agreement between the prosecutor and the defense for a reduced sentence in exchange for a guilty plea from the defendant. This study hypothesized that a) strength of evidence, b) client acknowledgment of their substance use problem, and c) substance use rehabilitative history would predict attorney recommendations for taking a plea bargain. Participants were recruited through the Washington State Bar Association and included 491 active and retired attorneys. Each received an informed consent and one of eight counterbalanced vignettes. In this 2x2x2 between subject’s design, a potential client was described and the possibility of conviction was manipulated based on strength of evidence, defendant’s acknowledgement of a substance use problem, and prior history of substance abuse treatment. 91 participants had completed surveys and identified as Public Defender (51%), Private Defender (34%) and both (15%). This study found a significant interaction η2 = .08, F(1,85) = 8.12, p < .01 and three significant main effects that indicate defense attorneys consider all of the aforementioned variables when making recommendations that clients accept a plea of diversion to treatment. They placed greatest importance on clients’ rehabilitative histories η2 =.48, F(1,85) = 77.47, p = .00, followed by acknowledgements of substance use problems η2 = .26, F(1,85) = 30.24, p = .00, and then strength of evidence η2 = .19, F(1,85) = 19.52, p = .00. A possible limitation, though anonymous, the self-report nature may have been impacted by participant’s social desirability. This study had a strong counterbalance design, which eliminated the potentially confounding impact of order effects. Future research would benefit from investigating differences factors that may be significant to prosecutors offering a plea and moderating or mediating factors involved in this decision-making process.

Prior research has demonstrated that there is a relationship between sleep and academic performance, yet few studies have assessed the role of stress on these variables. It was hypothesized that stress would act as a mediator between sleep and academic performance. Seventy-six undergraduate students made up the preliminary sample from a public university in Washington. Participants completed an online survey which asked demographic questions, including self-reported GPA. Additionally, students completed the Perceived Stress Scale and the Pittsburgh Sleep Quality Index. The preliminary regression analysis indicated a nonsignificant effect of sleep on GPA, R² = .03, F(1, 74) = 2.13, p = .15. The effect of stress on this relationship was also a nonsignificant effect, R² = .01, F(1, 73) = .84, p = .36. However, a power analysis indicated a minimum of 107 participants is necessary to reach beta of .95, alpha = .05, with an effect size of F² =.15, the number of predictors at 1 for level A and 2 for level B. Correlations show predicted, but nonsignificant, relationships between GPA and sleep (r = -.17, p = .07) and GPA and stress (r = .01, p = .45), with a moderate correlation between stress and sleep (r = .47, p < .01). The current sample is underpowered, making it difficult to confidently draw conclusions. Participants will continue to be recruited but if power is met and results continue to be nonsignificant, it is probable that this is due to GPA failing to have a normal distribution for each level of the independent variable. The lack of heterogeneity in GPA would then raise concern. If the necessary sample size is met and results are significant, this is likely due to poor sleep increasing student’s susceptibility to high levels of stress, which consequently leads to a decline in GPA. 

Sorsby Fundus Dystrophy (SFD) is a rare autosomal-dominant macular dystrophy that causes central vision loss in early adulthood due to mutations in the TIMP3 gene, yet its pathogenesis remains unclear. SFD is clinically similar to age-related macular degeneration (AMD), the leading cause of vision loss in older persons in the United States. Preliminary data revealed structural and functional differences between retinal pigment epithelial (RPE) cells differentiated from SFD patient-derived induced pluripotent stem cells (iPSC) and control iPSC-derived RPE cells. In contrast to control iPSC-derived RPE, SFD RPE does not accumulate any detectable extracellular matrix (ECM) and demonstrates a 40-fold increase in the level of intracellular hydroxyproline, a collagen degradation product whose increased expression adversely impacts SFD RPE metabolism and increases its susceptibility to oxidative damage. We wanted to determine whether differential protein expression between SFD and control RPE cells could elucidate the mechanisms behind these changes. A shotgun proteomics approach using isobaric Tag for Relative and Absolute Quantification (iTRAQ) identified peptide sequences present in these cells, and proteins were identified based on these sequences. Proteins with a twofold increase or decrease between cell types were noted, which included glutathione-s-transferases, gamma-glutamyl transferase 5, and chitinase-3-like 1. I am confirming these results through Western Blot and will analyze them by protein network analysis to determine their relevance to the disease model based on previously obtained metabolic data. The results from this experiment will help reveal the mechanisms behind SFD pathogenesis and potentially identify targets for disease treatment.

Myocardial infarctions (MI) are ischemic events which cause heart tissue to die and scar tissue (fibrosis) forms to maintain integrity of the walls of the organ. Fibrosis is crucial in the initial healing process but continued formation can compromise the elasticity of the tissue and lead to heart failure. Fibrosis is formed by myofibroblasts. These cells differentiate from fibroblasts after MI. Currently, the differentiation process is not well understood. The RNA binding protein muscleblind like (MBNL1) is known to be critical in this process, however. This project aims to use fluorescent imaging to track the localization of mRNA bound by MBNL1, particularly serum response factor (SRF) which has also been shown to play a role in myofibroblast differentiation. We aim to use regularly interspaced short palindromic repeats (CRISPR) and nuclease-null, RNA-targeting CRISPR associated protein 9 (RCas9) to specifically bind to the desired sequence of SRF mRNA. Enhanced green fluorescent protein (EGFP) is bound to the RCas9 in order to be able to visualize the RNA. Two target sequences on SRF mRNA were identified and protospacer adjacent motif presenting oligonucleotides (PAMmer) and single-guide RNA (sgRNA) were designed to bind to these sequences. PAMmer and sgRNA allow Cas9 to bind to the target sequence. This system was transfected into mouse embryonic fibroblast (NIH 3T3) cells and differentiation induced using transforming growth factor beta. During differentiation, live cell imaging was used to visualize RNA dynamics and localization. Results are expected to show colocalization with MBNL1 in the cytoplasm. This data will allow for the correlation of RNA trafficking and cell differentiation. It will also allow for a better understanding of myofibroblast differentiation and fibrosis formation after a myocardial infarction.

The plant hormone auxin controls nearly every aspect of plant growth and development. Proteins involved in the perception of auxin are closely studied in an effort to elucidate drivers of plant morphology. Potential applications of such research lie in engineering of plant architecture and ultimately maximizing crop yield. Auxin triggers binding between two proteins: auxin receptors (Auxin-signaling F-Boxes or AFBs) and transcriptional repressors (Aux/IAAs). This interaction leads to the ligation of ubiquitin to Aux/IAAs, triggering their degradation, and allowing transcription of auxin-induced genes. In the model plant Arabidopsis thaliana, the AFB family has six members (TIR1 and AFB1- AFB5). While the AFBs are highly similar in sequence to one another, individual AFBs may play specialized roles. In our preliminary studies, we have found that a reduction in AFB2 activity in the afb2-3 mutant produces a leaf phenotype not observed in TIR1 mutants. To confirm that AFB2 is responsible for this phenotype, we are engineering transgenic afb2 mutants that express a wild-type copy of AFB2. We expect that leaves of these transgenic plants will resemble those of wild-type. We are also analyzing the leaf phenotypes of other AFB mutants. To test whether small differences in AFB2 function can change leaf phenotype, we are currently transforming natural hypomorphic and hypermorphic AFB2 variants into afb2-3 mutants. By quantifying leaf morphology of these transgenic plants, we plan to map the relationship between AFB2 function and leaf phenotype.

Climate change is threatening global food security. There is a pressing need for better crops that can prevent severe losses. We hope to apply our current understanding of plant developmental mechanisms that are involved in responses to harsh external stimuli to engineer more resistant crop varieties. The plant hormone auxin plays an important role in organogenesis, the process of new organ formation. Auxin controls organogenesis by accumulating in specific tissues at specific developmental times. These accumulations are due to the polar flow of auxin through the plant. These flows are in turn reinforced by auxin through a feedback mechanism called canalization. PIN1 is a an auxin efflux transporter that is required for polar auxin transport. Loss of PIN1 results in several developmental defects. These phenotypes and other analyses have led to mathematical models of organogenesis. We used these models to predict plant phenotype in response to increased canalization. We have engineered transgenic plants with a library of synthetic promoters with varying auxin sensitivity driving expression of PIN1. We are characterizing the shoot architectures of these plants, specifically the number of branches and the pattern of lateral organ initiation. We are also studying how the increased canalization affects the distribution of auxin in our plant lines using fluorescent reporters. We hope that our results will demonstrate how plant synthetic biology and predictive models can be used to rationally design plant developmental phenotypes.

Brassinosteroids and auxin are plant hormones that are crucial in plant growth development. Although it is known that brassinosteroids and auxin signaling interact, it is not known exactly how. We are focusing our attention on the question of how auxin and brassinosteroids interact during the formation of lateral roots. Lateral roots are roots that extend horizontally from the primary root and the developmental tradeoff between the primary and lateral root formation helps the plant optimize its water and nutrient uptake. Previous studies have demonstrated that auxin promotes specification of founder cells, the first step in making a new root. To test if brassinosteroids are also involved in this process, I am quantifying the density of founder cells in seedlings treated with brassinosteroids alone, auxin alone or both hormones together. In this study, I will be able to distinguish whether the hormones act additively or synergistically (a much greater effect than a simply addition of the effect of each hormone individually). Our study will deepen our understanding of root formation which will have agricultural significance as strong roots lead to effective nutrient and water intake and stability for the plant under varying weather conditions.

Crop production will have to double by 2050 to meet projected global food demands. Insect herbivory currently accounts for nearly 15% of global crop losses; thus, plants better able to repel insect attack should have a significant impact on food security. One successful strategy for engineering plants is the constitutive expression of insecticidal toxins such as Bacillus thuringiensis endotoxin (BT). However, the constant production of these toxins decreases crop yield and increases the development of BT-resistant pests. When plants are damaged by insects, they produce the phytohormone jasmonate. We propose a new plant engineering strategy that links the expression of BT to jasmonate signaling. To create this link we have built a synthetic transcription factor called a Jasmonate Degradable CRISPR Transcription Factor (JDCTF). This transcription factor is easily retargeted to any gene of interest by changing the dcas9-associated guide RNA. The JDCTF responds to jasmonate via a jasmonate sensitive degradation domain. Using the plant model Arabidopsis thaliana, we’ve generated transgenic plant lines expressing a JDCTF that targets a Venus-Luciferase reporter. We have shown the system works using a bioluminescence time course assay on seedlings treated with the jasmonate analog coronatine. After induction with coronatine, we observed an increase in bioluminescence in the treated seedlings and no increase in biolumescence for the control. Currently we are characterizing the dynamic response to both exogenous jasmonate, as well as simulated and real insect herbivory on mature leaves. Future work will focus on regulating BT expression with the JDCTF system. Our system has the potential to increase yields, increase consumer safety, and slow down the development of insecticide resistance in pests.

Both content knowledge and problem-solving strategies are important components for academic success in biology. All problem-solving strategies begin with the ability to understand the problem being presented. In the context of biology, this means identifying the core concept being asked in complex questions. Identifying keywords, words that are important or significant, can help students identify these core concepts. The purpose of this study was to explore how well students can identify keywords and if this ability is correlated to exam performance. Students in an Introductory Biology course took weekly practice exams (PE), after each PE they either graded their own or a peer’s PE. In addition, students were asked to “identify the keywords in the question that should have caused you to provide the complete and correct answer given in the key”. This experimental design also allowed us to explore how grading an anonymous PE affects quality of students’ keyword selections. Students’ keyword selections were compared to a keyword rubric created by the research team. Data was coded by two researchers to 90% inter-rater reliability. On average, students only correctly identified 39% of keywords each week. Multiple regression models were used to explore correlations between correct identification of keywords and performance on both PEs and course exams. Interestingly, students who self-graded were more consistently engaged with the keyword activity as compared to peer-graders. In both cases, percent keywords correct could predict overall exam performance. These results indicate that if instructors want to help students improve their problem-solving skills of complex biological problems, helping students recognize keywords may be one solution. Furthermore, identifying which students have trouble simplifying complex exam problems and helping these students improve their ability to identify keywords may be beneficial in addressing learning disparities within large introductory classes.

Student-graded practice exams (PEs) have been used to facilitate learning in large classrooms. In a large introductory biology class, we investigated the impact of self vs peer-grading of PEs. We hypothesized that students who graded their own PEs would grade more accurately than peer graders because they are more motivated and reflective in their grading. Over time this more accurate grading would lead to increased performance on course exams. One question from each week’s PE was graded by a team of experts. We calculated PE grading accuracy by comparing the expert grade to the grade assigned by the student. We analyzed the correlation of PE grading accuracy with exam performance. For each metric we investigated the impact of treatment, incoming GPA, and student demographics (e.g., gender, socioeconomic status, first generation, race/ethnicity) using general linear models. There was no difference in average expert PE grade between the two treatments. Incoming GPA was the only significant predictor of expert PE grade with GPA positively correlated with expert PE grade. There was a significant interaction between incoming GPA and treatment on grading accuracy. Students with high GPAs who graded peers consistently underestimated their peers’ PE performance. Students with lower GPAs who graded peers either graded similarly to experts or slightly overestimated their peers’ PE performance. Students who graded their own PEs more accurately predicted their PE performance regardless of incoming GPA. There was an interaction between GPA and treatment on course exam performance, as high GPA self-graders earned more course exam points than high GPA peer-graders. Average GPA students earned the same amount of course exam points regardless of treatment. Given these findings and the logistical complications of peer-grading in large classes, instructors can use the more easily administered self-grading method of providing feedback on low-stakes practice exams.

 Many children have lasting pain problems after surgery that can greatly impact their recovery and health-related quality of life. Emerging research has identified psychosocial factors as important predictors of children’s postoperative outcomes, suggesting that an intervention addressing these factors could positively impact both short-term and long-term recovery from pain and quality of life. However, there are few data from children, families, or healthcare providers assessing psychosocial needs and receptiveness to a behavioral pain management intervention in the perioperative period. Involving healthcare provider stakeholders in the development of an intervention is essential to ensuring feasibility, acceptability, and successful implementation of the intervention. Seventeen semi-structured interviews were conducted with pain clinicians, nurses, surgeons, physical therapists, and child life specialists who provide perioperative care at a children’s hospital. Interviews probed the following areas: 1) current experience with perioperative preparation and care, 2) recommendations on content and format of an ideal psychosocial intervention, and 3) potential barriers to implementation and participation in a perioperative program. After transcription, we coded and analyzed interviews using inductive thematic analysis to identify consistent and relevant themes across provider groups. Independent members of the research team reviewed codes at each stage for credibility and representativeness. Providers said that a pain management program would address current gaps in surgery preparation, and that children and their parents need to learn effective coping strategies to positively impact their recovery. Providers also suggested necessary content, delivery methods, and ways to enhance program effectiveness. Provider themes, as well as themes from patient and family interviews, are being used to develop a family-based psychosocial intervention targeting children at risk for poor postsurgical outcomes. Next steps for this research include design, implementation, and evaluation of the perioperative intervention aimed at reducing the likelihood that children will develop lasting pain problems after surgery.

Prolonged activation of cardiac fibroblasts after acute wound healing post myocardial infarction results in excessive extracellular matrix deposition in the myocardium and formation of fibrotic scar at the site of injury. This adverse fibrotic remodeling disrupts physiological function in the heart and leads to increased risk for heart failure which is detrimental for patient outcomes. The reasons why activated fibroblasts, known as myofibroblasts, persist after wound stabilization are poorly understood due to the lack of a myofibroblast reporter system capable of direct longitudinal study. Current reporting modalities are invasive, resource and time intensive, wasteful, and indirect. The present project aims to design and develop an in vivo, repeatable, and real time reporter system for myofibroblast cell fate as a surrogate for cardiac fibrosis. To achieve this, we have designed a chimeric promoter to drive firefly luciferase vector expression specifically in myofibroblasts. We have built 9x tandem repeats of serum response factor (SRF) and nucleated factor of activated T-cells (NFAT) binding sequence since they are key mediators in myofibroblast activation. These repeats were ligated onto smooth muscle alpha actin (ACTA2) and collagen type 1 alpha 2 (COL1A2) minimal promoters, and these constructs were cloned into pGL3 luciferase reporter vectors. The specificity of the promoter was evaluated and optimized through in vitro luciferase assays with NIH 3T3 primary mouse fibroblasts.  With the successful creation of this reporter, wasteful use of animal models will be mitigated and direct mechanisms in the pathobiology of chronic cardiac fibrosis can be elucidated. Furthermore, efficacy of novel therapies can be evaluated in vivo.

We investigated how grading treatment, self-evaluation versus peer-evaluation, on weekly practice exams (PE) in an Introductory Biology course affects the advice students give to improve academic performance. We hypothesize that students who suggest more resources and study strategies perform better on course exams because they are aware of how improvements need to be made and what resources to use. Students in the class (approximately 550) take a PE on Thursday and then grade the PE responses on Friday. The grading groups were split up into two treatments: half of the class self-evaluated whereas the other half evaluated their peers’ responses. In addition, they were prompted to answer two questions: 1) Pick a single thing that you/this student should work on. 2) What resource should they consult before the next exam? We used this information to explore how study strategies and resources that students suggest relate to their test scores. To analyze the responses, we created specific codes corresponding to student answers. Results of the codes indicate that the students’ responses fall into four categories for question 1 and two sets of categories for question 2 (with three and two codes in each set). When asked to provide ways to improve PE performance, the four specific codes were being descriptive, increasing knowledge of content, interpreting the question, and time management. When providing resources, the two sets of categories were active versus passive resources and expert, peer, and self resources. Over the course of ten weeks, students who suggested more strategies and resources tended to perform better on exams. As well, both self-evaluators and women in the course suggested more strategies and resources than peer-evaluators and men respectively. Given this study, instructors can better understand what resources students are utilizing but also which study strategies tend to be more successful for students.