Asthma is one of the most common chronic pediatric illnesses, affecting nearly 10% of school-aged children in the United States and disproportionally impacting low-income populations and people of color. More than 50% of childhood asthma cases in the United States are uncontrolled, characterized by frequent, severe symptoms that contribute to missed school days, sleep disturbance, and hospitalization. Outcomes can be improved by rigorous daily management. However, due to both individual and system level factors, adherence to treatment regimens is low. Several studies have suggested a relationship between family functioning and asthma morbidity, making it a promising target to improve asthma outcomes. The purpose of this study was to further elucidate the relationship between family functioning and asthma control in school-aged children. This study involved a cross-sectional, secondary analysis of baseline data collected as part of a larger intervention trial. Thirty-three children aged 6-11 years old and their caregiver completed the Childhood Asthma Control Test. Caregivers completed the Family Assessment Device to assess family functioning. Analyses compared family functioning in children with controlled versus uncontrolled asthma. Fifty-one percent of participants had asthma that was uncontrolled. Asthma control was positively correlated with family functioning, r(32)=.41, p=.02, such that those with better asthma control had better family functioning. However, there was no significant difference in family functioning between children with controlled versus uncontrolled asthma, nor was asthma control predictive of family functioning. Although there appears to be an association between asthma control and family functioning, the nature of that relationship is still unclear. It is possible that our sample size was too small to detect a predictive relationship. Future studies could continue to explore this relationship with a larger sample size and consider additional potentially modifiable variables that may predict asthma control.

Asthma is a very common chronic childhood illness, affecting approximately 10% of US children. It can lead to significant morbidity and decreased quality of life (QOL) in pediatric patients and their caregivers, on whom the burden of daily asthma management may take a significant toll. However, existing research does not sufficiently describe the relationship between asthma control and parent- and child-reported QOL. The purpose of this study, therefore, is to describe asthma-related QOL in school-aged children and their caregivers and to determine whether asthma control is predictive of QOL. Children aged 6-11 and one caregiver participated in this cross-sectional study. Child-caregiver dyads completed the Childhood Asthma Control Test, Paediatric Asthma Quality of Life Questionnaire, and the Paediatric Asthma Caregiver’s Quality of Life Questionnaire. Descriptive statistics were used to describe the sample demographics and asthma control. Pearson correlations and linear regressions were computed to assess the relationship between asthma control and parent- and child-reported QOL. The study included 33 child-caregiver dyads (child mean age 8.4 years). The children were predominantly male (52.9%), non-Hispanic (73.5%) and white (70.6%). 51.5% had asthma that was not well-controlled. Asthma control is associated with child-reported QOL (r(33)=.49, p=.004) and the parent-reported activity QOL (r(33)=.44, p=.011). While there was not a significant difference in QOL between children with controlled versus uncontrolled asthma, there was a significant difference in parent QOL (t(33)=-2.3, p=.02). A regression analysis found that child asthma control was a significant predictor of child QOL (F(1,31)=9.90, p=.004, R2=.24) but not parent QOL. Parent and child QOL and child asthma control appear to be related, though additional research is needed to clarify this relationship. Our preliminary findings suggest that clinical efforts to improve asthma control might not only decrease risk for asthma-associated morbidity, but also positively impact how patients feel and function in day-to-day activities.

The health of marine ecosystems is often determined by water chemistry balance and the levels of nutrients. Nutrients, such as nitrates, are essential to primary productivity. When they are present in excess, nutrients can lead to harmful algal blooms (HABS), eutrophication, and hypoxic conditions at depth due to phytoplankton decomposition. With decreasing dissolved oxygen, respiring plants and animals are at risk. Because of the potential negative impacts to the ecosystem, it is crucial to monitor these factors. We investigated the health of Possession Sound through levels of nitrate, dissolved oxygen (DO), and phytoplankton densities to investigate changes over time and indications of eutrophication. We hypothesized that the plankton populations would be less dense in areas with low DO levels and denser in areas with high nitrates. We used long-term water quality monitoring data collected by Ocean Research College Academy (ORCA) from 2014-2019 from five different sampling locations. Overall, trends from 2014 - 2019 showed that there was no linear correlation between nitrates, DO, and phytoplankton densities. It appeared that plankton density generally alternated every other year between highs and lows. Seasonally, DO was higher in the spring and summer, and Nitrates were higher in the fall and winter. Phytoplankton appeared to be greatest in the spring, with 2014 and 2018 having the largest blooms. Over time, nutrient levels changing can greatly affect the marine ecosystem in Possession Sound by creating an environment for excess plankton and algae blooms to occur and potentially harming fish populations. By observing a smaller sample of oceanic environments, we can see what could potentially occur in the open ocean if human influence goes unchanged.

Lateral roots are branches originating from the main branch of a primary root. They provide stability to the plant, and assist in acquisition of nutrients and water, similar to the primary root. It is well understood from studies in Arabidopsis thaliana that lateral root development is regulated by the plant hormone auxin. A few undifferentiated cells respond to a  pulsatile auxin signal to become ‘specified’ lateral root stem cells, retaining potential to proliferate and ability to differentiate into a lateral root. These specified lateral root stem cells are arrested in G2 phase of cell cycle, respond to auxin signaling and undergo rounds of cell division marking the onset of lateral root development. The focus of my study is to better understand how cell cycle control influences lateral root developmental transitions from undifferentiated to specification to initiation. In particular, I am interested in addressing the question of whether cell cycle arrest in G2 phase crucial for lateral root development, and if so at which step of cellular transitions get altered specification or initiation. An important tool necessary to address this question is the ability to control the length of G2 cell cycle arrest for lateral root stem cells, and we have recently generated transgenic lines for this purpose. I am inducing lateral roots in a number of transgenic plant lines and using fluorescence microscopy to observe the early stage of lateral root development. These experiments allow viewing of both the staging of development and cell cycle stage associated with development over time. The understanding gained from these experiments will help build a framework for how cell cycle contributes to lateral root development, allowing for future genetic modifications to improve root structure in crop plants.

Plants respond to environmental changes by changing their growth patterns. For plants to continue to grow throughout their lives, they are constantly undergoing cell fate determination—how the plant determines the specific fate of new cells they are generating. One example of this is the development of roots that emerge from the primary root, called lateral roots. The number and spacing of lateral roots determine the overall root structure, which determines how well the plant takes advantage of resources in its environment. Auxin is a plant hormone involved in many aspects of growth and development, including the regulation of lateral root production. Using data from an experiment where mRNAs were sequenced from single cells isolated from roots, the Nemhauser Lab identified specific genes that may be active during lateral root development. My research question is: are these genes targets of auxin signaling? To test this, I will use quantitative RT-PCR to measure expression of the candidate genes in wild type and in plants that are deficient in the auxin response pathway. Genes that are targets of auxin should show lower expression levels in the mutants relative to wild type. The more we understand about how cell fate determination occurs in lateral roots, the more we can understand the underlying mechanisms by which plants arrive at their final root structure. Our understanding can then guide engineering or breeding projects, allowing optimal root growth that could drastically increase plant survivability and yield.

Plant hormones are critical to their growth, development, and overall function. Gibberellins (GAs) are a class of plant hormones that are crucial for cell elongation and growth. The GA pathway has been genetically manipulated in many crops to enhance agricultural yields. Through my research, I hope to test whether modulation of GA signaling pathways can make crops that are more productive and better adapted to climate change. These types of interventions are needed because climate change is occurring more quickly than plants are able to adapt or evolve. Rewiring of the GA pathway is a potentially significant solution to this problem because we can modify plants in a manner that is likely transferable across many species. By using a novel genetic tool called a GA-sensitive Hormone Activated Cas9-based Repressor (GA HACR), we can modulate targeted genes in the GA hormone response pathway to turn down their transcriptional activity. The GA HACR targets specific genes through guide RNAs to repress the gene with complementary DNA sequence. The GA HACR is degraded in the presence of GA, which allows them to have a natural response to the activating hormone signal. We hypothesize that by targeting the HACRs to genes involved in GA biosynthesis (GA20 oxidase) and GA response (GID1 genes), we can modulate root growth of these plants. Once we identify plants with modulated growth behaviors, I will grow them in a CO2-enriched growth chamber to simulate future climate conditions. In this way, I will determine whether genetic intervention targeting the GA is a feasible strategy.

Tracing the lineage of individual cells within a multicellular organism has been one of the key struggles of modern developmental biology. The ability to trace the differentiation of individual cells over various timescales would give extensive insight into many fields of biology. Techniques using large scale genomics based on natural DNA mutations have been used in the past. However, in recent years, novel techniques using CRISPR-Cas9, and more recently recombinase, have been developed to study cell lineage in a more precise and dynamic manner. Each of these methods have different specifications in their readout methods, time-dependent resolution, spatial integrity, and accuracy. I have constructed a review summarizing these methods, and will present their impact on dynamic cell lineage tracing. While most of this research has been done in animals, I will also propose a design for cell lineage tracing in plants based on these reviewed methods.

CRISPR is a powerful gene-editing tool with several advantages over state-of-the-art viral vectors used for gene therapy. However current methods to deliver CRISPR to human cells requires electroporation, which is cytotoxic and not viable for in vivo delivery. The Adair lab previously demonstrated gold nanoparticles (AuNPs) to passively deliver CRISPR gene editing into blood stem/progenitor cells, with Cas12a (Cpf1) nuclease resulting in high levels of homology-directed repair (HDR) compared to Cas9 when co-delivered with a single-stranded, homology-directed DNA template (HDT). Here we tested this AuNP-mediated delivery system in primary human T cells. We hypothesized that AuNP delivering Cpf1+HDT would also mediate high levels of HDR in T cells, compared to Cas9+HDT. To facilitate HDR readouts, the HDT encoded an 8 bp NotI restriction enzyme site. Both guide RNAs targeted the same C-C chemokine receptor type 5 (CCR5) gene locus. A mutation at this locus confers resistance most human immunodeficiency virus (HIV) strains. Briefly, CD3+ T cells from 8 different human donors (n=8 biological replicates) were either cultured overnight and exposed to AuNP-CRISPRs, or activated with CD3/CD28 beads prior to AuNP-CRISPR treatment. Test conditions for each donor included Mock-treated cells (negative control), naked AuNP, AuNP-CRISPR/Cpf1, AuNP-CRISPR/Cpf1+HDT, AuNP-CRISPR/Cas9, or AuNP-CRISPR/Cas9+HDT at identical cell and AUNP concentrations for 48 hours. Cell viability and counting by trypan blue dye exclusion assay demonstrated >70% viability and yield across all conditions tested. Gene editing analysis by tracking of indels by decomposition (TIDE), demonstrated the highest levels of gene editing with AuNP-CRISPR/Cpf1+HDT, with >90% of all editing being successful HDR. In non-activated T cells, mean gene editing levels were 19.7% across all donors (n=4). Final results for activated T cells are currently pending. These data demonstrate AuNP-mediated CRISPR/Cpf1 delivery as an efficient method for HDR in primary human T cells and suggest utility in human clinical applications.

Nitrate, silicate, and phosphate are essential nutrients in diatom based food webs. Chlorophyll and nutrients are good indicators of phytoplankton abundance and diversity. Phytoplankton, being integral to the Possession Sound ecosystem, can be indicators of greater change in an ecosystem. By studying phytoplankton abundance and diversity along with chlorophyll and nutrient levels spatially and temporally, correlation can be determined and used to help understand the health of Possession Sound. It was predicted that nutrients and chlorophyll abundance are inversely proportional, while chlorophyll and phytoplankton abundance and diversity are directly related. Thus, higher nutrient levels indicate less chlorophyll and fewer nutrients indicate higher chlorophyll and plankton abundance and diversity. Increased levels of nutrients in the fall and winter were expected, with greater chlorophyll and plankton levels in the spring and summer. The chlorophyll and plankton abundance and diversity were anticipated to have gone down over the past four years, while nutrient levels will have gone up slightly. Students at the Ocean Research College Academy (ORCA) collect monthly samples as part of the longitudinal study: State of Possession Sound (SOPS). Results from three locations were utilized from 2016 to 2019. Chlorophyll is measured by a YSI EXO2 Sonde, while nutrient samples are taken using the Niskin bottle and sent to the University of Washington Marine Chemistry lab to test for results. By evaluating seasonal data, temporal trends of chlorophyll, nutrients, and plankton abundance and diversity were discovered. Changes in data can be linked to environmental and anthropogenic variations. It would be compelling to analyze ecosystem changes by exploring dissolved oxygen and pH levels.

Phytoplankton, one of the primary sources of dissolved oxygen in marine ecosystems, are dependent upon nutrients for growth. However, there is evidence that eutrophication, the overabundance of nutrients, can lead to hypoxia in marine ecosystems. Because they are a primary source of dissolved oxygen and are dependent upon nutrients, phytoplankton density can indicate how nutrients are affecting dissolved oxygen at depth. It was hypothesized that an increase in phosphates, nitrates, and nitrites would correlate with an increase in phytoplankton density. Furthermore, it was predicted that with greater phytoplankton density there would be a greater difference in dissolved oxygen at the halocline versus dissolved oxygen 30 meters below the halocline. This study used data collected from 2016 to 2019 by students at the Ocean Research College Academy (ORCA) at two sampling stations in Possession Sound, WA. Water was collected and sent to the University of Washington Marine Chemistry Lab for nutrient analysis. Phytoplankton density was calculated using samples collected during 3-minute horizontal tows at the halocline. Dissolved oxygen data was collected using a YSI Exo2 Sonde at different depths. Preliminary results suggest that greater levels of phosphates, nitrates, and nitrites may show a steeper oxycline due to an increase in density of phytoplankton from the nutrients. The potential for hypoxia is increasing because of anthropogenic nutrients, so understanding the influence humans have over nutrients in marine environments is critical. This study will help us to understand how humans are influencing Possession Sound and marine ecosystems as a whole as a result of the impact of nutrients on phytoplankton and dissolved oxygen.

Makerspaces are an emerging tool in the engineering education field. Compared to the current standard of formal, class-based education, makerspaces provide a multitude of resources meant to support students through more informal, project-based learning. This study is part of a larger project exploring supporting design learning through design decision making. Here we investigate how students make material decisions when pursuing projects in a university makerspace. What kinds of questions, options, and criteria do they explore, and what rationale do they use to make their final choice? How does this change in time, across different projects, and across different students? In previous work, 7 undergraduate students completed a self-driven project while documenting their process and anything else they felt was relevant. During this study, 6 researchers analyzed written traces of the students’ project progress. Material and tool decisions were identified, and coded to present questions, options, and criteria over time using the Design Space Analysis framework. Trends were identified across students, time, and different materials or tools. Through our analysis, we discovered the following results. Though students are pursuing different projects, they all deal with similar decisions around material and tool choice during their processes. For most decisions, students consider very few options, although there are some where more are contemplated. Regarding criteria, students consider cost, aesthetics, and availability, but often not specific functionality requirements. Students naturally provide design rationale as part of their process, but it is not very well developed. The results from this study will allow us to gain greater understanding on what students tend to consider, and develop methodology to make a greater number of potential options more visible to students during their project processes.