Glutathione S-transferase (GST) is an enzyme that is present in most organisms. Its primary function is the detoxification of xenobiotic compounds. In mammals, some GST isoforms have a role in modulating the MAPK signaling pathway. The GST protein from Schistosoma japonicum (26 kDa) is also an important tool used in biochemistry labs as a tag for the isolation of proteins via affinity chromatography. Reduced glutathione (GSH) is a substrate for GST that binds to its G-site with a high affinity, and this interaction can be exploited to purify GST-fusion proteins on agarose beads containing a GSH moiety. In the research presented, GST was expressed in E. coli, purified, and dialyzed to remove GSH ligand completely. The protein concentration of the purified, dialyzed GST sample was quantified using Qubit™ fluorescence spectroscopy. Isothermal titration calorimetry (ITC) was utilized to measure the dissociation constant and thermodynamic properties of the binding of GSH to GST. ITC is a highly sensitive analytical method that monitors changes in heat produced from noncovalent chemical associations. In order to obtain accurate ITC measurements, various concentration ratios of GSH/GST were explored. Quantifying the binding affinity and thermodynamic properties of GSH binding with GST provides useful information for investigators who use GST as a fusion tag or who are interested in reactions between the GSH-GST complex and its substrates.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder consisting of altered social and communication difficulties and restrictive and repetitive behaviors. In the United States, 1 in 68 children are diagnosed with ASD, however, the underlying causes of ASD are unknown. Neuroscientists commonly use magnetic resonance imaging (MRI) to noninvasively study the brains of children with ASD. However, many MRI studies suffer from a small sample size. To address this issue, 17 institutions came together to share anatomical and functional brain images and formed the Autism Brain Imaging Data Exchange (ABIDE). Although ABIDE tackles sample size issues, new pipelines introduced by the human connectome project (HCP) require reassessment of data quality. For our study, an ABIDE dataset went through a rigorous, yet teachable quality control assessment protocol created by the Developmental Cognition and Neuroimaging Lab (DCAN) at Oregon Health & Science University. The scans were graded as usable, probable, and unusable. Grades were determined by precise delineation of gray and white matter brain segmentations in anatomical scans, signal dropout using blood oxygen level detection (BOLD) in functional scans, and proper atlas registration of functional and anatomical data. Preliminarily, 116 subjects were analyzed and numerous issues were found. Anatomical scan segmentation showed signs of poor delineation, and functional brain images had notable loss of signal. Thus far, the analysis yielded a 35% acceptance rate. Early results of this analysis indicate that the ABIDE dataset would benefit from an improved quality assessment protocol along with updates to processing pipelines to increase data quality.

With a society that consumes pharmaceuticals daily, the need to develop more efficient syntheses of bioactive molecules is vital. Organocatalysis is a field in chemistry that has been looked upon to have immense potential to succeed in this area. A type of organocatalyst that has been gaining notoriety is the bisphosphorylimide, which is a stronger acid and more sterically hindered than chiral BINOL-derived phosphoric acids. This upsurge can be attributed to the simple preparation and functionalization of bisphosphorylimides. In this investigation, several bisphosphorylimide organocatalysts are prepared and their performance in a Friedel-Crafts reaction between sesamol and benzylidenecarbamates will be presented. Ultimately, the enantioselective products synthesized from this research may serve as bioactive components in the preparation of pharmaceuticals such as trabectedin, an anti-tumor drug.

During the formation of the early embryonic zebrafish body, bipotential neuromesodermal progenitor cells (NMPs) that are located at the posterior end begin to differentiate via regulation of the Wnt pathway. Relatively higher Wnt-signaling activates the expression of the tbx16 gene, which in turn inhibits the neural fate and commits cells to a mesodermal (muscle) fate. The aim of this study is to understand the downstream effects of tbx16 on the rate and mechanism of anterior migratory transitions of mesodermal cells as they leave the progenitor region and travel to the somite-forming mesoderm region. We have previously shown that successful anterior migration and differentiation of mesodermal cells is dependent upon tbx16 downregulating the expression level of two Arhgaps, a Rho GTPase-activating protein. Hence, we heat shock hsp70: Arhgap29 and hsp70: Arhgap35 zebrafish transgenic lines at early somite stages in order to compare and contrast the effects of sustained Arhgap activation during the critical period of mesodermal cell anterior migration. At 24 hours post-heat shock, embryos are sorted by wild-type and transgenic, with transgenics having severe defects in their posterior somite formation. Followed by immunocytochemistry for a muscle antibody, confocal imaging on whole-mount embryos captures the shape and number of somites from each transgenic line. My results show that while Arhgap29 affects both the number and morphology of somites, Arhgap35 only affects their morphology. This suggests that these two Arhgaps do not have overlapping roles in the control of mesodermal cell migration. I am currently performing a separate in-situ hybridization experiment to help me understand the role of these Arhgaps on the essential genes involved in muscle cell differentiation. This study contributes to understanding early vertebrate development as a model for human embryogenesis.

Heterotopia are organized structures consisting of mixed elements of neurons, glia and neuronal progenitors arranged in an architectural pattern inappropriate to the considered tissue. Heterotopia are observed in pathological preparations of the cerebral cortex and the cerebellum as a feature of many neurological disorders yet the mechanisms driving their development remain unclear. We have analyzed 28 Dandy Walker Malformation (DWM) human fetal cerebella and found that more than 40% of cases contain heterotopia. DWM is the most common structural birth defect of the human cerebellum and is characterized by hypoplasia and foliation defects. A subset of cases are caused by loss of FOXC1, a transcription factor expressed in the mesenchyme adjacent to the developing cerebellum. We previously reported that in mice, loss of FOXC1 causes loss of the mesenchyme-secreted factor SDF1-alpha. Further, loss of SDF1-alpha is sufficient to cause cerebellar heterotopia, highlighting the importance of mesenchymal signaling in the maintenance of the highly stereotypical laminar architecture of the mature cerebellum. We hypothesized that granule neuronal progenitors (GCPs) adjacent to the mesenchyme are the primary cellular target of SDF1-alpha mesenchymal signaling and thus, the primary cell type driving heterotopia formation. To test our hypothesis, we specifically deleted the SDF1-alpha receptor from just GCPs using a Cre-loxP genetic scheme in mice. We determined that loss of SDF1-alpha in just this one cell type caused their premature migration into the developing cerebellar anlage and secondarily caused other cerebellar cell types to form structured heterotopia. We also observed defects in cerebellar foliation in the treatment group. Our data further highlights the importance of SDF1-alpha dependent mesenchymal signaling in cerebellar development and identifies heterotopia as a new phenotype in DWM.

STUDIO, a near-peer STEM (science, technology, engineering, and mathematics) mentoring program, fosters growth for both the mentors and mentees. In partnership with Neighborhood House, a local multiservice community-based organization, UW mentors support low-income, immigrant and refugee youth to develop motivation, interest, and identification with STEM education and careers. Mentors do this by creating units that provide youth opportunities to learn at the intersection of their stated interests and mentor expertise. This cultivates a community of learners that values youth and mentor agency. STUDIO focuses on both mentees and mentors, but our initial research and evaluation supported by National Science Foundation has focused primarily on mentees. In this study, we investigate mentor learning and growth. We are currently lead undergraduate mentors in STUDIO and are therefore positioned to reflect on our own and other mentors’ learning. Mentors are given the opportunity to advance their leadership and mentoring skills by documenting occurrences in program time, writing weekly reflections, creating and leading curricula, and participating in weekly seminars to share experiences and address issues important to increasing diversity in STEM. In this study, documentations, reflections, and interviews are analyzed to identify shared themes and unique personal stories that illuminate mentor learning, commitment, and collaboration in the STUDIO learning environment. In early thematic analyses, we have identified several emergent themes such as the powerful role of collaborating, relationship building, and mentors’ development through leadership that support their overall academic and personal connection with STEM. As students of color remain underrepresented in the STEM field, mentors empower youth to develop their identification with STEM, despite challenges they face due to persistent stereotypes and limited resources. This research will help us better understand mentor growth and learning so that we can continue to strengthen the STUDIO mentoring team.

The three essential structures of life are RNA, protein and a lipid bilayer membrane. We hypothesize that these three structures formed co-dependently in an aqueous environment, as opposed to forming independently in diverse environments. In our scheme, a vesicle (membrane) formed and bound nucleobases and amino acids, the building blocks of RNA and proteins, respectively. Then, the resulting confinement and conformational constraints imposed by the membrane on the nucleobases and amino acids facilitated the formation of RNA and proteins, leading to the origin of life. The work presented here addresses one part of the hypothesis: “Do amino acids increase vesicle formation?” An increase in vesicle formation due to the addition of amino acids would support the hypothesis that membranes and proteins evolved co-dependently. It would suggest that amino acids stabilize vesicles, increasing the likelihood that fatty acid membranes and amino acids co-localized. Our experiments used decanoic acid, a prebiotic fatty acid that spontaneously forms membranes when dissolved in water. In the first part of the research, we added aqueous solutions of amino acids to a solution of decanoic acid at pH 6.83 and determined the extent of vesicle formation by measuring light scattering with a spectrophotometer. In the second part, we added amino acids to a decanoic acid solution above the pH at which vesicles form, and then slowly acidified the solution until vesicle formation began; again, we used light scattering as a measure of the extent of vesicle formation. We found that specific amino acids do in fact increase vesicle formation and also have an effect on the pH at which vesicles form. The next step in this research will be the addition of multiple types of amino acids and peptides additions in order to search for combinations that increase vesicle formation the most. The results contribute to the the possible driving mechanism behind the emergence of protocells.

The Marshall Islands are often recognized as the victims of US nuclear weapon testing, leaving a lot unsaid about who they are as a people. This dark past often times overshadows the resilience and ingenuity within the Marshallese culture. This research project is aimed to gain a better understanding of Marshallese culture through their canoes and navigation techniques. The way that they navigate and construct their vessels plays a large role in their lives; their voyages, their developed culture, language and livelihood. Our methods include interviewing Marshallese community members within Seattle, analyzing navigation articles, wave charts and using the Burke Museum collection database for information on Marshallese canoes. We anticipate that this project will result in the general public walking away knowing the strengths of the Marshallese and a better grasp of one of the cultures in Oceania that is often underrepresented. It is important to look into other cultures, especially those we do not know a lot about, because you can learn so much from them. We also anticipate making relationships with those we interview and by understanding the views and values of their culture. By the end we hope to have opened the door for future research in this realm to encourage people to explore this on their own and participate in clubs and organizations.

Eelgrass (Zostera marina) is a vitally important organism to the Puget Sound that exists along the shallow, muddy seabed. Many different living creatures of all trophic levels rely on the eelgrass beds to survive and absorbed contaminants could be a real threat. Toxics metals in the eelgrass, sediment and water column could have critical implications to the ecosystem. The heavy metal concentrations in the eelgrass, sediment and the water column of two locations in the Possession Sound (Mukilteo and Buoy) were analyzed in order to determine the specific levels of metals and contaminants that travel up through the trophic pyramid. The study endeavors to determine the metal absorbance ability of eelgrass and check if factors including temperature of the water column, seasonality or time of day influenced heavy metal uptake by eelgrass. A sediment grab was utilized to collect the sediment and eelgrass samples. Sample analyses were conducted at the Everett Environmental Laboratory and in partnership with a University of Washington trace metals laboratory to find the concentration of the metals arsenic, copper, lead, zinc and cadmium in the surrounding water column and the eelgrass. It was predicted that greater concentrations of copper and zinc would be found near Buoy because they are legacy chemicals and due to anthropogenic influences near the Everett Port of Everett. It was also hypothesized that a higher concentration of metals would be found in the eelgrass during the spring and summer as more direct sunlight leads to greater primary productivity and uptake of chemical constituents. Determining these types of correlations and catalysts is significant to the entire Possession Sound ecosystem as it would lead to possible strategies on how to prevent further contamination of eelgrass. Furthering our understanding of the connected network of interactions here can only benefit the future inquiries.

The incidence of many human diseases, such as cancer, Alzheimer's and heart disease, increases with age. Studying the aging process could provide insight into new treatments and therapies for these aging-related diseases. Our research uses the yeast species, Saccharomyces cerevisiae, as a model organism to explore the effects of specific genetic and environmental factors on aging. Aging in yeast can be measured in terms of replicative lifespan, which is the number of progeny (daughter) cells produced by a parental (mother) cell. In order to measure the replicative lifespan, we utilize a microdissection microscope outfitted with a fiber optic needle that is used to separate individual mother cells from their daughter cells in order to quantify the number of daughter cells produced. The total number of daughters produced by a mother cell is the replicative lifespan of that cell. To determine which genes play an instrumental role in yeast replicative lifespan, our lab has studied the lifespan of 4,698 yeast mutants containing single gene deletions. Many mutants were identified that increase the lifespan of S. cerevisiae. In this study, we have re-evaluated our data to identify potential false-negative results from our initial work, in order to identify additional genes that may influence lifespan. Identifying the false-negative results will be beneficial in finding genes that may play a role in the aging process and had been previously overlooked. This will allow future research to continue in developing treatments that target the prevention of age-associated disease in humans, potentially allowing people to live healthier lives and decreasing the prevalence of cardiovascular disease and cancers. 

The vaginal microbiome has a mutualistic relationship with the immune system and can have a major impact on health and immunity to diseases, specifically its role in prevention against STI’s and Bacterial Vaginosis (BV). BV has been found to be associated with the prevalence and incidence of multiple STI and HIV. We hypothesized that changes in bacterial composition would correspond to changes in inflammatory cell frequencies in the vagina and examined this using rhesus macaques (RM) treated with various antibiotic regimens.12 rhesus macaques were administered paromomycin (n=3), enroflaxin (n=3), clindamycin (n=3), or cephalexin (n=3). Vaginal biopsy samples were taken twice before antibiotics (ABX) and at 5 timepoints post-ABX. DNA was extracted according to standard protocol and bacterial 16s rDNA was amplified and quantified by PCR and sequenced using Illumina kits and the Illumina MiSeq platform to determine abundances of relative bacteria. Flow cytometry was performed on cells isolated from matched biopsies to assess inflammatory cell frequencies. For animals treated with cephalexin or paromcyin, we found that CD4+ T cells and CD8+ T cells spiked after treatment around day 14 and declined to normal levels by day 63. Additionally, we found that neutrophil abundances spiked between days 28-63. Animals treated with clindamycin increased CD4+ T cells and CD8+ T cell counts around day 28 through necropsy while neutrophil levels stayed constant. Those given enroflaxin spiked in CD4+ T cell and CD8+ T cell counts at day 14 then returned to baseline, while neutrophil levels spiked between day 14-63. We observed alterations in bacterial composition that was dependent on the ABX regimen given and observed associations between these changes and the immune cell changes we observed. Taken together, these data show that altering the vaginal microbiome results in changes in inflammatory cell frequencies that could have implications for STI risk.

Cystic fibrosis-related diabetes (CFRD) complicates up to 50% of adult cases with CF. However, its pathogenesis is poorly understood. One thing that has been shown, however, is that amyloid deposition occurs in the islets of 60% of subjects with CFRD, which is known, from data in other forms of diabetes, to be toxic to insulin-producing islet β-cells. Currently existing animal models do not reproduce this feature of human CFRD, since islet amyloid deposition requires the expression of an amyloidogenic form of the islet peptide islet amyloid polypeptide (IAPP). To address this gap in the field, our group is generating a CF mouse model that expresses the human amyloidogenic form of islet amyloid polypeptide (hIAPP). To do this, we have generated a double transgenic mouse from a male homozygous for the CF mutation and a female hemizygote for the hIAPP transgene. Mice will be followed for up to 12 months, or until hyperglycemia develops. During this time, body weights and non-fasted blood glucose levels will be measured monthly. When hyperglycemia is identified, animals will be euthanized in order to determine if islet amyloid is present in the pancreas. We are expecting to find that mice bearing the CF mutation and expressing the hIAPP transgene will develop increased and/or accelerated amyloid deposition in their islets as compared to the control mice generated by the cross. In addition to this, we expect that these mice will also show increased glucose levels (hyperglycemia) and decreased β-cell mass. There are currently 16 mice on study, and data collection is ongoing, with additional animals being enrolled approximately every two weeks. The goal of this study is to generate a new model of CFRD, which better reflects the human disease state, and can be used to develop and test improved therapies for human CFRD.

The way adolescents describe their emotions shapes how they view themselves, peers, and the world around them. Observational and experimental studies have shown feelings of gratitude in particular to be linked to an increased sense of well-being, academic performance, and prosocial behavior. Previous studies have defined “gratitude” as an appreciation of what is valuable or a state of appreciation. This study explored 1) how gratitude varies across different peer interaction scenarios and ethnic groups, and 2) how adolescents explain the emotion gratitude. Studies have yet to examine how ethnically diverse students interpret the emotion. Our sample size consisted of 300 students from ages 12-18, evenly divided between African, European, Mexican, and Native Americans across Washington state. Students provided explanations of times where they were an actor and recipient in four different scenarios: calming, amplifying, avenging, and reconciling a peer. In each scenario students rated how grateful they felt and why. Quantitative analyses demonstrated that students’ levels of gratitude varied by role and scenario, but did not vary by ethnic group (all ps < 0.001). This suggested that adolescents’ definitions of gratitude could vary in different roles as well. To understand influences on gratitude in adolescents, we coded students’ explanations for their actions across all four scenarios to see if different definitions of gratitude were being used in each role. We also present results of demographic analyses relating to gratitude levels (gender, ethnicity, grade level). The results of this study contribute to research on adolescent mental health and peer relationships in school contexts, and could also help school practitioners work towards learning environments that promote student well-being and more stable peer interactions.

Flowering plants have coevolved with insect pollinators in a mutualistic relationship; plants provide nectar to insects and insects in turn cross pollinate flowers. Plants have employed a range of colors, scents and shapes to attract their insect pollinators. Vision, olfaction, and mechanosensation all have roles in the identification and interactions with flowers. While the roles of vision and odor sensing have dominated the literature, recent work has shown that mechanosensation plays as much a role as vision in ensuring that moths stay engaged with a moving flower. Moths use their straw-like proboscis to probe a flower, using the overall shape of the flower as well as tiny grooves in the petals to guide the insect’s mouthparts toward the nectary. Using the feeding behavior of the nocturnal hawkmoth, Manduca sexta, we ask how moths use mechanosensory information to find the nectary opening in flowers. We combine 3D printing technology--to generate different 3D flower curvatures, with micro-sensing technology--that allows us to detect the proboscis tip inside nectary, and computer vision techniques--to detect the motion of the hovering moth, to test the relationship between flower shape and efficiency in locating the nectary. We find that flower exploration depends on its 3D curvature. Moreover, over repeated trials, moths take less time to find the nectary across all curvatures suggesting that moths use tactile exploration to learn flower shape. Mechanosensation is one of many inputs used by moths in the wild to interact with their environment. In future studies we wish to explore the integration of mechanosensation with other sensory information, and the salience of each modality.

The Kaeberlein laboratory utilizes the budding yeast Saccharomyces cerevisiae to study aging, a progression characterized by the gradual deterioration of cellular components and bodily functions. Aging is a progression every organism eventually undergoes, and by better understanding the molecular mechanisms behind it, we can determine how to delay its progression and increase our healthspan, the portion of our lifespan in which we are healthy and free from serious disease. S. cerevisiae functions as an effective model organism because it contains many conserved biological processes, making lifespan simple and fast to measure. The replicative lifespan assay (RLS) is used to quantify the lifespan of the yeast. Individual yeast cells undergo asymmetric mitotic division producing a daughter cell that is distinctively smaller than the original mother cell. Yeast are incubated on growth promoting plates, and the yeast produce daughter cells that are microdissected, counted, and moved away. The cycle repeats until the original mother cell can no longer produce a daughter. The total number of daughter cells produced by individual mother cells is then used to understand the effect of the gene on the lifespan of the yeast. This technique aids in developing models that can be used to understand the conserved biological pathways that influence longevity. Aging is an underlying risk factor for many non-communicable diseases such as Alzheimer’s Disease, Cancer, and heart disease. Our laboratory has performed thousands of gene knockouts on S. cerevisiae and has identified several genes that modulate replicative lifespan. This research is focused on identifying the genes that play a role in reducing the rate of aging to delay the onset of age associated diseases. By understanding how specific genes play a role in aging, we can apply this research to other organisms, with the ultimate intent of increasing the length of a healthier human life.

The ability to regulate emotion does not develop completely until young adulthood, meaning teenagers are at risk for disorders associated with difficulties in regulating emotions. Identifying one’s emotions is essential for taking active steps towards regulating them; thus emotion differentiation is an essential skill that facilitates psychological well being. Although there have been multiple questionnaires developed to assess emotion differentiation, there are limitations of self-report. Behavioral tasks provide an opportunity to observe an individual’s ability to identify emotions. The Photo Emotion Differentiation (PED) task is a computer task designed to assess emotional differentiation, validated predominantly in adult populations. Participants are shown 20 standardized emotional images and are asked to report the intensity with which they experience each of the 19 listed emotions. The limitation to this method is that it has only been used once in child populations. We aim to validate the PED for use in adolescents by comparing it to a self-report questionnaire [the Levels of Emotional Awareness Scale for Children (LEAS-C)] that assesses emotion differentiation. The instructions for the LEAS-C ask youth to respond to 12 scenarios by indicating how they might feel and how the other person in the scenario might feel. A validated scoring system is used applying specific criteria to the emotion words used in the response. This is an ongoing study and we have currently recruited 14 participants from ages 13 to 16 who will come for a lab visit and complete both the PED and the LEAS-C. A positive correlation between the scores from the PED and LEAS-C will show that the PED is measuring the construct of emotion differentiation and can be used to obtain valid assessment in youth in subsequent studies. With this information, the PED can be used to examine the relationship between pain and emotion differentiation in adolescents.

The ability to quantify rat and mice ultrasonic vocalizations (USVs) can provide ethological validity to animal models of affective disorders. Vocalization frequencies are key indicators of a rodent’s subjective state and are closely tied to their behavior. For example, 50-kHz calls in rats are emitted during positive events such as sucrose drinking, and are attributed to a positive affective state. In contrast, 22-kHz calls are ascribed to a negative affective state, and are emitted during negative events such as shock. Current methods for manually scoring vocalizations are slow and labor intensive, while automated programs lack the accuracy to produce satisfactory scoring under non-ideal recording conditions. In order to create a more precise program for detecting these USVs, we applied deep learning, which has revolutionized the field of bioacoustics through image detection and recognition of audio files. By converting soundwaves produced by mice and rats into spectrograms, we can train convolutional neural networks to distinguish between vocalizations and noise. Using these methods, we created the software DeepSqueak©, a Deep Learning Based System for Quantifying USVs and Extended Analysis Kit, which outperforms current state-of-the-art, commercially available software. Hundreds of rat calls from the 18-75kHz were manually isolated from spectrograms and used as training data for our neural network, allowing it to differentiate calls from noise. As a result, USV detections using DeepSqueak are far more rapid and accurate in comparison to existing software. The detected calls produced from the software were then reviewed and re-entered into the neural network as another training set to consolidate its precision and efficiency. With the speed and reliability of DeepSqueak, quantification of USVs can become more widespread and lead to a wealth of new knowledge relating to affective disorders.

Hydrogen Sulfide (H₂S) has potent physiological effects. In large doses, it is a deadly toxin, but in small doses, it acts as a signalling molecule with a wide range of physiological effects. The worm C. elegans is a good model organism for the study of H₂S because of its short lifespan, ease of genetic manipulation, and ease of maintenance. In C. elegans, H₂S induces a stress response pathway, coordinated by the HIF-1 and SKN-1 transcription factors, that plays a role in extending lifespan and improves resistance to various stresses. A previous screen in our lab identified several mutations that suppress the requirement of hif-1 for survival in H₂S by increasing SKN-1 activity. One SKN-1-upregulated transcript, rhy-1, was sufficient to rescue hif-1 lethality in H₂S. RHY-1 is an integral membrane protein that is localized in the hypodermis, intestine, and some head neurons of C. elegans. It has predicted acyl transferase activity, but its specific molecular function is unknown. The role of RHY-1 as a hif-1 lethality suppressor indicates that it acts in a hif-1 independent H₂S response pathway. The goal of this study is to better understand the role of RHY-1 in this hif-1 independent response pathway. In order to identify its binding partners and elucidate its role in cellular signalling, we performed immunoprecipitation of RHY-1 for use in tandem mass spectrometry. This approach allows for the identification of its binding partners under baseline and H₂S stress conditions, which may further define the role of RHY-1 in the stress response to H₂S. Defining the role of RHY-1 will enhance our understanding of how the H₂S stress response leads to increased lifespan in C. elegans. Since several members of this pathway are well conserved in humans, this work may provide a mechanism for the therapeutic use of H₂S in human medicine.

Nanoparticles have begun to see wide adoption in commercial application particularly in fields relating to medicine and electrical engineering. While their small size provides unique properties furthering advancements in these field, their effect on human health are still relatively unknown. As their use increases, their potential to cause health effects will need to be considered. This is especially true for workers who manufacture engineered nanomaterials (ENM) and who use them in commercial products. Quantum dots (QD) are an emerging application of nanoparticles using their semiconductor properties for optical imaging. To determine the effects of QD to human neural development, an in-vitro model was established using an adverse outcome pathway (AOP) framework. This methodology provided observations of QD on their ability to alter cellular morphology and developmental endpoints. H9 human neural progenitor cells were cultured and maintained in vitro for up to 21 days in 96 well plates. Normal morphology and biochemistry was characterized under both proliferating and differentiating conditions. H9 cells were then treated with two different QD, Qtracker non-targeted QD and Qdot ITK carboxyl QD, under the concentrations of 0, 2.5, 5, 10, 20, and 40 nM at culture day in vitro (DIV) 1, 6, 13, and 20. After 24 hours of exposures, cell viability was evaluated for dose-response relationships using LDH and MTT assays. H9 cultures undergoing proliferation saw resistance towards Qtracker non-targeted QDs, however cell viability declines with increasing concentrations of ITK carboxyl QDs. H9 with differentiation media under DIV 1 and 6 saw declining cell viability towards accumulative exposure to Qtracker non-targered QDs. Nonetheless, H9 cells with differentiation media showed resistance towards ITK carboxyl QDs. In the future, we will investigate the resistance of our cells to various Q dots by assessing dosimetry of QD uptake under our test conditions.

Cystic Fibrosis (CF) is a genetic disorder that is characterized by recurrent pulmonary infections and a progressive decline in lung function caused by increased inflammation. While the host immune response can help fight these infections, an overzealous inflammatory response in the lungs may be harmful. Inflammation can be induced by multimeric proteins called inflammasomes, which catalyze the maturation of pro-inflammatory cytokines and pyroptosis. Our laboratory is interested in investigating genetic variants that alter the response of the NLRP3 inflammasome in human CF patients as a means of identifying potential targets for host-directed therapies. Preliminary data from the EPIC study has identified two genetic variants in CF children that is associated with colonization by P. aeruginosa, a common bacteria in the lung that can induce deleterious inflammation. We hypothesize that genetic variants in the NLRP3 inflammasome will increase the inflammatory response to bacterial infection and will lead to worsened pulmonary outcomes in patients with CF. First, we used CRISPR/Cas9 and gene editing techniques to knock out the NLRP3 gene in human cell lines. We then inserted NLRP3 genetic variants and stimulate the NLRP3 inflammasome using Pathogen associated molecular patterns such as Nigericin and ATP. The extent of inflammation was measured using ELISA (Enzyme Linked Immunosorbent Assay), and results were compared among NLRP3 genetic variants and wild type controls. The ultimate goal of this investigation is to learn how genetic variants are associated with the clinical outcomes of CF patients. Future studies can involve harnessing host-directed therapies to target specific inflammatory pathways in order to reduce harmful inflammation in CF. Finding the relationship between the NLRP3 inflammasome and CF is critical to generating an effective treatment for CF.

Epigenetic modifications regulate chromatin structure and function, playing important roles in altering DNA transcription levels and subsequently cell fate decisions. Various next-generation sequencing (NGS) methods have been developed to detect these epigenetic changes in the genome, such as chromatin immunoprecipitation followed by sequencing (ChIP-seq). Even though ChIP-seq is extensively used to analyze DNA and histone modification levels, this method is limited to one histone marker at a time and requires significant amount of input cells, which masks the profiles of cell-to-cell variation and the complex interaction between the epigenome and gene expression. To overcome these limitations in current next-generation sequencing methods, we have been developing a multiplexed assay that could detect multiple epigenetic modifications in single cells. So far, I have developed a NGS data analysis pipeline to identify potential gene candidates that are highly differentially modified by histone markers. In the future, we hope to use these gene candidates as templates to design DNA-fluorescent in situ hybridization (DNA-FISH) probes and perform Expansion Microscopy and DNA-FISH with these probes to link histone modifications to specific gene loci at high resolution. After the assay is fully developed and validated, we plan to utilize the assay to take the epigenetic profiles of hematopoietic stem cells and study cell fate decisions in hematopoiesis.

Treatment options for kidney disease are limited to dialysis and transplantation, which are of limited efficacy and availability. Human pluripotent stem cells (hPSCs) can be used to generate immunocompatible kidney organoids, which contain the major proximal structures of the nephron, including podocytes, proximal tubules, distal tubules, and endothelial cells. They can be made on-demand to study kidney disease and regeneration. Unfortunately, these organoids lack a collecting duct system, a crucial component of the kidney. To address this need, we have developed a protocol to differentiate hPSCs into ureteric bud (UB) cells, which are the precursors of collecting duct cells. First, we identified markers for the collecting duct lineage in developing kidneys. Immunofluorescence analysis of human kidney tissue revealed co-expression of cytokeratin 8 and Dolichos biflorus agglutinin (DBA) in the collecting ducts. These markers were not expressed in the proximal tubules. Next, we investigated the ability of hPSCs to express these markers. Undifferentiated hPSCs were treated with a concentration gradient of small molecules known to induce kidney lineage differentiation, including CHIR99021 and glial cell line-derived neurotropic factor (GDNF); their effects were jointly and individually examined. Immunofluorescence was used to characterize the resulting cells, which were initially stained with DBA and Lotus tetragonolobus lectin (LTL), a proximal tubule marker. We found that CHIR99021 alone was sufficient to produce DBA positive and LTL negative cells in vitro. Notably, the DBA levels in these cells were lower than observed in kidney tissue, indicating that the structures we obtained were not yet fully mature. These studies provide the first evidence that collecting duct cells can be generated from hPSCs. Optimization of this differentiation protocol will yield kidney organoid structures that include the collecting duct system, which will make them ideal for kidney disease modeling and regenerative medicine approaches to reduce the need for kidney transplants.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor made up of cells of endothelial origin. KSHV establishes a predominantly latent infection in endothelial cells in culture and in the KS tumor. A previous study has shown that KSHV induction of the Warburg effect is required for the survival of latently infected endothelial cells. The Warburg effect, a common metabolic alteration in cancer cells, refers to an increase in glycolysis and a decrease in oxidative phosphorylation. The mechanism of Warburg induction by KSHV is currently unknown. I proposed to evaluate the role of endothelial cell specific hypoxia-induced factors (HIFs) on KSHV Warburg induction since HIFs have been implicated in Warburg induction in other types of cancer. I hypothesized that HIF2α mediates KSHV Warburg induction through expression of glycolytic genes. To test this, I constructed HIF2α knock-out cells using CRISPR/Cas9 gene editing. I then used RT-qPCR to measure glycolytic gene expression during KSHV infection of wild-type and HIF2α knock-out cells. I found that the transcript levels of certain glycolytic genes remained constant in KSHV-infected HIF2α knock-out cells as compared to KSHV-infected wild-type cells, showing that HIF2α is not responsible for glycolytic gene expression during KSHV infection. I am following up on the role of the KSHV latent gene KapA on the induction of glycolytic gene expression. KapA was previously found to interact with components of the Ras pathway. As the Ras pathway activates glycolytic genes through HIF1α, I hypothesize that exogenous expression of KapA will lead to increased glycolytic gene expression through increased expression of HIF1α. I will construct an endothelial cell line that overexpresses KapA using CRISPR/Cas9 system and then use RT-qPCR to evaluate glycolytic gene expression. These results will aid in the future efforts to develop antiviral drugs by targeting cellular metabolism.

Theodosius Dobzhansky was a prominent 20th century scientist, who proved to be an eloquent rhetor, catalyzing the interdisciplinary synthesis of modern theories of evolution by bridging the great divide between Mendelian and Darwinian schools of thought. In March 1973, The American Biology Teacher published his essay “Nothing in Biology Makes Sense Except in the Light of Evolution.” This sentence has become the memorable line from the essay, often used anecdotally against religion and in support of evolutionary theories by the scientific community. However, this use directly contradicts Dobzhansky’s goal of persuading readers that science and religion aren’t mutually exclusive, through arguing for evolution as proof of God’s greatness, attempting to unite these two disciplines. Theories of evolution have long been tied to and in confrontation with religion. This contention prevails today; in 2017 alone multiple bills were introduced into the U.S. House lobbying for equal time spent teaching both evolution and creationism in public schools. I argue that Dobzhansky’s rhetorical eloquence allows him to appeal to two audiences. Through the use of prolepsis, anecdotes, and devoutness to God he is able to address his religious audience while simultaneously giving educators persuasive tools for teaching evolution in a religious context. Going forward, we can pull from Dobzhansky’s rhetorical strategies to facilitate productive conversations between polarized schools of thought, such as seen today in the American political climate.

Abnormalities in TGF-ß signaling are thought to cause aortic aneurysms and dissection in people with inherited syndromic illnesses such as Marfan's and Loeys-Dietz Syndrome. However, the mechanisms linking changes in TGF-ß signaling to aortic disease evolution are poorly understood. We developed a mouse model in which the type II TGF-ß receptor was deleted to characterize and study the effects of this perturbation in TGF-ß signaling. My research focused specifically on characterizing any histologic changes in the aorta in 7-8 week old mice, 1 week after deleting the TGF-ß receptor. To quantify changes in aortic architecture, we employed methods such as H&E staining and fluorescence microscopy. Our results showed that the ascending aortas of the mice with the TGF-ß receptor knockout had significantly larger medial thickness and medial area, with p-values of 0.0001 and less than 0.0001, respectively. However, we did not find a significant difference in elastic breaks between the two experimental groups, although previous research has shown increased elastic breaks in aortas of mice with the TGF-ß receptor deletion when measured at a later point (14 weeks post-deletion). Together, these results suggest that there are subtle but significant aortic changes as early as one week following receptor deletion. This suggests that losing TGF-ß signaling causes early and immediate changes in the aortic smooth muscle cells and matrix proteins that are quantifiable by histology. More research is needed to understand the precise mechanisms behind why these changes occur and how they might be related to the development of aneurysms.

Many children with Autism Spectrum Disorder (ASD) suffer from decreased gut motility that manifests as debilitating chronic constipation, leading to an unhealthy aversion to food. We hypothesized that the same neurological defects that cause the hallmark cognitive impairments in ASD also negatively impact the enteric nervous system controlling gut peristalsis. Within the autism spectrum, we chose to study one particular condition called Phelan-McDermid Syndrome (PMDS), because every patient with the diagnostic chromosome 22 micro-deletion has a dramatic decrease in gut motility. Evidence suggests that the gene-of-interest in the deleted region is SHANK3 because mice with SHANK3 haploinsufficiency show decreased postsynaptic density of neurotransmitter receptors. The overriding goal of this project is to develop novel drug treatments for patients with PMDS and ASD. Finding new compounds that can increase (or bypass) postsynaptic receptor density could alleviate these patients’ gut motility problems. However, models to study the enteric nervous system either involve invasive biopsy procedures to remove enteric tissue from patients or the use of animal models that fail to replicate the pathology observed in humans. Therefore, the objective of this study is to create a human disease-in-a-dish model of enteric nervous system dysfunction by generating stem cells from PMDS patients and differentiating them into enteric neurons (ENs). We predict that molecular characterization of SHANK3-deleted enteric neurons will identify aberrant signaling pathways suitable for therapeutic intervention. Preliminary experiments from our laboratory have demonstrated the feasibility of differentiating patient-derived induced pluripotent stem cells (iPSCs) into neural crest (NC) cells, which are necessary precursors to making enteric neurons. NC plus smooth muscle co-culture experiments will then be used to drive fluorescently labeled normal and SHANK3-deficient NC cells to enteric neurons, thus enabling phenotypic characterization of the disease phenotype in vitro.

For more than a century, Drosophila melanogaster (fruit flies) have been an invaluable and versatile tool to further our understanding of cell signaling and survival mechanisms. To this day, they continue to shed light on the endogenous pathways that cancer cells can hijack in order to proliferate, metastasize, and recur following remission. The molecular conservation of these pathways invites parallels between the germline stem cells in D. melanogaster and the cancer stem-like cells in human carcinoma. In the same way that a tumor can relapse following a period of dormancy, Drosophila germline stem cells are capable of repopulating their niche after insult from Ionizing Radiation (IR). Utilizing this powerful model, we have conducted a small molecule drug screen of 512 compounds that we have narrowed down to eight candidate drugs that appear to increase cell death in Drosophila germline stem cells. Having already characterized the wild type Drosophila germline stem cell response to IR-induced DNA damage, we  probed how drug treatment and gene knockdown affected the germline stem cells' ability to recover from insult. Previous work in the Ruohola-Baker Lab has demonstrated the critical importance of the mechanistic target of rapamycin (mTOR) and the Tie receptor pathways in regulating regeneration after insult in the Drosophila germline. The Tuberous Sclerosis Complex (TSC), a heterodimer comprised of Tsc1 and Tsc2, is a known negative regulator of mTOR. Additionally, the Tie receptor is central to anti-apoptotic signal transduction in the Drosophila ovary. We have screened four candidate drugs to see if they effectively increase stem cell death in Tsc1-knockdown and Tie-null flies, in order to ascertain whether our drugs affect stem cell survival mechanisms through mTOR and/or Tie signaling. Our findings may shed light onto how to mitigate the quiescent threat of tumor relapse.

Gene amplification can be associated with genetic disorders such as cancer and varying types of autism. One such form of amplification is a neochromosome where a chromosomal segment is amplified and these two segments are joined at an inverted repeat. There are two models to explain how this event could occur: double stranded break and repair and origin dependent inverted repeat amplification (ODIRA). ODIRA hypothesizes that a replication fork error occurring near short inverted repeats could cause the leading strands to erroneously become ligated to the lagging strands and produce an extrachromosomal palindromic DNA intermediate. Because both models produce identical neochromosomes, I have worked to find which pathway produces them. To do so I utilized CRISPR/cas9 to induce breaks at commonly observed junctions, and analyzed the resulting intermediates using gel analysis of whole yeast chromosomes. If the neochromosome were to occur via the double stranded break and repair model, I expected that providing the break should increase the frequency with which these neochromosomes arise. Alternatively, if the neochromosomes were occurring due to the ODIRA model, I expected the frequency to remain unchanged. I followed this initial analysis with DNA sequencing to give further support to either outcome. In particular I verified that the CRISPR system was working by sequencing across the junctions where cas9 was cutting. If cas9 was indeed cutting, then when comparing the sequence from the cas9 survivors to that of the yeast genome, the survivors had an altered cut site. My work provides insight to the etiology to this fascinating class of chromosome rearrangements.

Small RNA molecules called microRNAs (miRNAs) are translational repressors and are involved in a variety of processes important for normal cell development and function. Previous studies in our lab showed that miRNAs are required in early (embryonic) stages of retinal development. After depletion of miRNAs in early retinal progenitor cells (RPCs), late RPCs and their progeny, such as Müller glia (MG) which are the primary glial in the retina, are not generated. Instead, only early born neurons are produced. The depletion of miRNAs can be obtained through deletion of an enzyme that generates mature miRNAs called Dicer. Since we know that miRNAs are crucial for early retinal development, this study investigated if miRNAs are also important for proper late (postnatal) retinal development. We used a Sox2 reporter mouse which allows visualizing late retinal progenitors (Sox2 is a gene expressed in RPCs) and selectively deleted Dicer in RPCs at postnatal day 2-6. The tissue was analyzed 2, 4 and 7.5 weeks after Dicer deletion and compared to normal retinas using immunofluorescent staining of retinal cross sections and confocal microscopy. We found massive disruptions in the retinal architecture such as the formation of rosette/ bubble-like structures, indicating that miRNAs are required for proper formation of the retinal layers. Since MG play a role in maintaining the retinal structure, in a second series of experiments we induced Dicer deletion specifically in MG at postnatal day 11 –14, when the retina is almost completely developed. The tissue was analyzed 8 and 20 weeks after Dicer-deletion and compared to normal tissue. We found an atypical location of MG and disruptions in the layered retinal structure, indicating that miRNAs are also required for accurate MG location in the retina, which is essential for normal retinal architecture.

Antibiotic resistance is an intractable clinical challenge that disproportionally affects much of the world’s most impoverished populations. A critical driver of resistance stems from DNA mutations. Therefore, insights into how mutations arise in the genome is critical to understanding the development of antibiotic resistance. Our lab is currently identifying factors and environmental conditions that promote mutagenesis and bacterial evolution. Utilizing mutation rate analysis, we have identified factors and conditions that promote mutagenesis in divergent bacterial species. Additionally, using short-term evolution experiments, we have found that these factors promote the development of antibiotic resistance over time. This work provides novel mechanistic insights into mutagenesis and antibiotic resistance development in bacteria.

 Polyethylenimine (PEI) is considered as the most promising alternative gene carrier to viral vectors. PEI-based carriers minimize unwanted immunogenicity and promote loading capacity. However, PEIs’ nondegradable nature determines their high cytotoxicity. To minimize the toxicity and improve gene transfection efficacy, biodegradable cross-linking agents have been screened to synthesize biocompatible PEIs. Various cross-linkers have been linked to low molecular weight PEI (MW = 800) and tested across multiple cell lines (xPEI). N,N'-Methylenebis(acrylamide) (NDA) showed the lowest toxicity and highest transfection rate among all cross-linkers. To further optimize gene transfection efficacy, xPEIs were modified with increasing amount of fluorocarbon (xPEI-FC). DNA that expresses red fluorescence protein are bound to xPEI and xPEI-FC and then incubated with cells for 48 hours. Green florescence light is later used to examine the presence of RFP. xPEI-FC has demonstrated higher biocompatibility as well as higher transfection rate in vitro with higher level of RFP. The best-performing candidates for xPEI, PEI-FC and xPEI-FC based on toxicity and transfection efficiency will be selected for nanoparticle (NP) modification. Selected candidates will be conjugated with Chitosan-poly(ethylene glycol) (PEG)-catechol copolymer (CCP) and then grafted onto iron oxide nanoparticle (IOCCP-PEI). The resulting nano-vector delivery system will be tested in vitro. Ultimately, in vivo test will be performed to evaluate its transfection efficacy in living organisms.

Many biochemical processes rely on the conversion of electrical energy into chemical energy. Tryptophan is one aromatic amino acid that facilitates electron transport through proteins by means of reduction and oxidation reactions, allowing for the energy conversion to occur. The ability for a protein to tune the reduction potential of tryptophan is critical to its function in reactions within enzymes such as cryptochromes, which are involved in regulation of circadian rhythm, and photolyases, which are involved in DNA repair. This tuning of the reduction potential is achieved by modulation of the microenvironment surrounding tryptophan residues within a protein. In order to study the relationship between protein environment and tryptophan oxidation, we have designed and synthesized a series of model peptides to engineer specific environments which have the same geometry but differing charge states and levels of solvent exposure. The reduction potential of each peptide was determined via differential pulse voltammetry over a pH range from 1-7.5 in order to determine how the pH dependence of tryptophan's reduction potential responds to changes in its microenvironment. The results indicate that the reduction potential of tryptophan at the physiologically relevant pH=7.5 increases with increasing positive charge in the surrounding microenvironment. The results across the entire pH range indicate that the pKa of oxidized tryptophan increases with decreased solvent exposure. Finally, our entire series of peptides indicates that the tryptophan oxidation reaction does not directly follow the expected Nernstian behavior for pH dependence of a single proton-coupled electron transfer reaction and thus indicates that the oxidation process may have differing degrees of reversibility in difference environments.

Breast Cancer type 1 gene (BRCA1) was first linked to a high risk for breast and ovarian cancer over two decades ago, yet much remains elusive about its functional mechanism and how inheritance of a variant of the ubiquitously-expressed gene can lead to markedly increased risk for tissue-specific cancers. A gene found in the nematode C. elegans was demonstrated to be homologous to human BRCA1 including at the protein product level. Its protein product was shown to have the same type of enzymatic activity as the human protein product of BRCA1, and to be involved in the same DNA repair pathway. Using purified protein products and both structural analysis via Nuclear Magnetic Resonance and in vitro biochemical enzyme activity assays, I have shown that the C. elegans and human BRCA1 share some structural similarities and that the enzymatic activity is conserved. These results suggest that C. elegans could be developed into a model system that would allow for experimentation to directly move between in vitro biochemical studies and in vivo genomic/genetic studies. My current work has focused on furthering the development of the C. elegans model system by using it to identify other protein-protein interactions, including the identification of new protein targets. Additionally, my work explores the effect that cancer associated variants have on the enzymatic activity of the C. elegans BRCA1. The development of C. elegans as a model system could lead to new insights into conserved functions of BRCA1 and a fuller understanding of its biological and developmental importance.

This research aims to examine the effects of nanoparticle-mediated inhibition of the phospholipid glutathione peroxidase (GPX4) pathway in mesenchymal state cells on radio-resistance in glioblastoma (GBM) therapy. GBM is a particularly deadly cancer with poor survival rates and relatively low treatment success, despite aggressive surgery and radiotherapy. Recent research has shown that biocompatible, tumor-targeted iron oxide nanoparticles (NPs) can serve to enhance radiotherapy through production of secondary electrons and subsequent reactive oxygen species (ROS) within the tumor volume. The presence of these NPs in the tumor during radiotherapy has shown to decrease damage to the healthy surrounding cells and prolong survival in mice with GBM tumors. This approach however, has not demonstrated the ability to eliminate the cancer completely, in part due to the presence of mesenchymal state cancer stem cells. The GPX4 pathway has shown to effectively kill mesenchymal state cells in sarcomas by inducing ferroptosis, an iron-dependent form of cell death. Here we plan to evaluate the efficacy of a range of GPX4 inhibitors on silencing of the GPX4 pathway and the ability to induce ferroptosis in primary human GBM cells. Inhibitors identified as effective silencers of the GPX4 pathway will be combined with a NP delivery vector to provide targeted delivery to GBM. The radioenhancement capabilities of iron oxide NPs in conjunction with targeted therapy against mesenchymal state cancer stem cells may provide a means to overcome radioresistance in GBM therapy.

Cancer, the leading cause of death worldwide, results from a combination of mutagenesis and selection for malignant phenotypes. Mutations from DNA replication errors may initiate as many as two thirds of all human cancers. Accurate DNA replication requires proofreading domains found on the major DNA polymerases as well as mismatch repair (MMR) proteins that detect and repair replication errors after DNA synthesis. Cancer-causing defects in proofreading and/or MMR lead to “mutator phenotypes”, marked by elevated mutation rates and increased cellular mutation burden. Such mutator cells occur spontaneously and drive evolution by generating mutations that enhance population survival. However, mutator cells also accumulate detrimental mutations that compromise fitness. Combined defects in polymerase proofreading and MMR cause error-induced extinction (EEX), which imposes a strong selection for cells with “antimutator” mutations that suppress the mutator phenotype. Using yeast (Saccharomyces cerevisiae), we isolated mutants that survived EEX and mapped the underlying determinants to candidate mutations (APC1, MCM5, PMS1). We are engineering these mutations into yeast strains to test whether they confer an antimutator phenotype. Understanding how mutator cells suppress elevated mutation rates will give us insights into how cancer cells survive and thrive in the face of strong mutagenesis and may suggest novel therapeutic strategies to combat this disease.

Mutator phenotypes due to mutations in genes encoding DNA polymerases or mismatch repair proteins lead to increased error rates during DNA replication that accelerate the evolution of cancer cells and contribute to chemotherapy resistance. Work in the yeast Saccharomyces cerevisiae indicates that excessive DNA replication errors can lead to error-induced extinction (EEX), where every cell within the population dies due to a random lethal mutation. Thus, a possible direction for cancer therapy may be to target antimutators, which can modulate mutation rates of mutator cells and suppress mutation rates, in order to slow the rate of tumor evolution. In a screen isolating antimutators, we identified an EEX mutation in Chromosome Transmission Fidelity 18 (ctf18-K666fs). Ctf18 directly associates with the N-terminus of DNA Polε, including part of the proofreading domain, and thus may influence mutation rates by directly affecting Polε function — a novel finding that would expand our understanding of mutator polymerases. In the current study, I determined whether Ctf18 exerts its antimutator phenotype independent of the S phase checkpoint pathway, the pathway by which all other previously isolated antimutators, such as Dun1, are known to modulate mutation rates. To accomplish this, I compared the mutation rates of double mutant strains, dun1Δ ctf18-K666fs and dun1Δ ctf18Δ, to their respective single mutants in pol2-L439V cells, where lower mutation rates would indicate additive antimutator effects from independent pathways. I also observed the degree of activation of this pathway by fluorescently tagging Sml1, a protein downstream of Dun1 in the S phase checkpoint, expecting to see decreased expression of Sml1 in cells in which the S phase checkpoint was activated. Expanding our understanding of mechanisms by which antimutators can modulate mutation rates may contribute to novel approaches for cancer treatments by targeting the mutator phenotype.

G Protein-Coupled Receptors (GPCRs) are seven-transmembrane proteins present throughout the body that can be activated upon binding of drugs, hormones, or neurotransmitters. Acting like an inbox for messages, these multi-protein complexes play a significant role in the body by regulating cell expression and signaling, making them attractive targets for drug development. One class of GPCRs, known as the adrenergic receptors (ARs) are critical in modulating the function of numerous targets, including cardiac muscle, vascular smooth muscle, and bronchial smooth muscle. There are nine AR subtypes: three α1s, three α2s, and three βs. In my research, I focus on deciphering the interactions of proteins with the α1D-AR due to a recent publication of the Hague Lab, which discovered that α1D-AR forms a multi-PDZ protein complex. To determine the participants of this complex and identify the presence of associated proteins, I first purified the proteins that potentially bind to α1D-AR. In my purification process, I first conducted Polymerase Chain Reactions (PCRs), transformed my bacteria, grew my bacteria, and finally purified my proteins of interest. With this information, my lab and I intend to identify the complexes using mass spectrometry and Octet. Determining how α1D-ARs are organized can create a deeper understanding of how drugs work in the body and assist in new drug development.

The majority of urinary tract infections are caused by E. coli bacteria. E. coli infections are strengthened due to E. coli’s ability to bind to mannosylated cells. A receptor protein on E. coli, FimH, has two domains: a regulatory pillin domain and a lectin domain (LD). In the presence of a force, the pillin domain detaches from the LD allowing the LD to go from its low to high affinity state. The naturally occuring mannose sugars on Horseradish Peroxidase (HRP) are known to bind the LD of FimH. The mannose binding pocket is suspected to open transiently, even when bound to mannose sugars. Based on the interaction of HRP with FimH, HRP may not regularly dissociate during these episodes, but we suspect that free mannose can induce the dissociation of HRP from FimH in these moments. To determine if mannose can improve HRP dissociation from LD, we are using an assay similar to a competitive Enzyme Linked Immunosorbent Assay. We expect to see a decrease in HRP bound to LD in the presence of free mannose compared to the absence. Ultimately this experiment provides an explanation of mannose monomers as an inhibitor for longer chains of mannose binding and a premise for a larger study on alternatives to E. coli antibodies that can competitively inhibit FimH from binding mannosylated cells.

In the context of the cell, molecular crowding can be described as the summation of molecular forces acting on polypeptides at the high protein concentrations that exist naturally in membrane-bound structures. This phenomenon of molecular crowding can affect protein conformations and interactions, particularly for proteins that require high flexibility to recognize and bind a large number of interactors like the chaperone Hsp90. To observe changes in protein conformations and interactions, we cross-link proteins with lysine-reactive linker molecules, then identify and quantify cross-linked sites using mass spectrometry. The linkage sites provide physical distance constraints useful for structural predictions, and the observed changes in cross-link abundance levels is informative of conformation and interaction changes. In this project, we apply chemical cross-linking to proteins under conditions that exert different levels of molecular crowding. These conditions include in vivo cross-linking of in-tact cells where high protein density causes high levels of crowding, and cross-linking of cellular lysates where protein density and molecular crowding effects are reduced. This presentation will highlight cross-linked peptides in Hsp90 and how these are being used to improve our understanding of the effects of molecular crowding on the function of this important chaperone.

The development of tubes is a conserved process observed in a wide variety of species; tubes contribute to structures such as neural tubes, digestive systems, and vascular systems. For a tube to form correctly, individual cells within a sheet of cells (epithelium) must move and change shape in very specific ways. My goal is to understand what controls this movement. Polarity proteins help to establish the directional identity of cells and tissues. This identity is important to ensure that cells coordinate their behaviors. I hypothesize that the proteins that help establish polarity also play key roles in the development of tubes, including in proper closing of the tubes and correct directional elongation. To study the involvement of polarity proteins during tube formation, I am using the dorsal appendages (DAs) on Drosophila melanogaster egg shells. Because the appendages form as a direct readout of morphology, I can see how tissues developed earlier on. These appendages, which provide the developing laid egg with oxygen, are formed from an epithelium that wraps and elongates into a tube, then fills with eggshell protein; the initial epithelium sloughs off, leaving the appendages as a visualization of the earlier tube formation. To test my hypothesis, I am using RNA interference (RNAi) to knock down the expression impact of various proteins and evaluate the impact on DA formation. The RNAi system induces transcript loss, eventually revealing the impact of protein loss. I am using RNAi in epithelial follicle cells to test 20 candidate polarity genes. This analysis will help me identify a few genes to then characterize further. These results will inform future studies and add to the knowledge of the role of polarity proteins in the patterning and subsequent growth of epithelial tubes across many species.

Osteocalcin is a protein implicated in bone growth and bone reconstruction in several vertebrates, including humans. In this project, we analyzed the decarboxylated form of osteocalcin’s first alpha helix (hOC-a1) using circular dichroism and isotherms. Circular dichroism spectroscopy uses polarized light to determine whether the peptide fragment forms alpha helices or beta sheets. Using adsorption isotherm experiments will determine the protein’s ability to bind onto hydroxyapatite, silica, and titania mineral surfaces. Osteocalcin is known to change conformation and form a helical structure in the presence of calcium ions and adsorb onto HAP. Previous studies attributed these characteristics to the gamma-carboxyglutamic acid (Gla) residues at positions 17, 21, and 24, which lie within its first of three alpha helices. However, a mutant form of osteocalcin which has its Gla residues reduced to glutamic acid has also been shown to adsorb onto HAP. It is unknown why the protein containing glutamic acid in place of Gla can adsorb onto the mineral surface. Using circular dichroism, the experiment showed that the decarboxylated hOC-a1 peptide fragment was a random coil and unable to form a helical structure with or without the presence of calcium ions. Isotherms determined the peptide fragment’s ability to non-cooperatively bind onto HAP, as well as silica and titania. This project determined that the decarboxylated peptide fragment could adsorb onto the mineral surfaces as a random coil, suggesting there are alternative mechanisms of adsorption in comparison to the native protein. The Drobny Group intends to continue studies of the peptide fragment by labeling residues and performing experiments using solid-state nuclear magnetic resonance spectroscopy (ssNMR).

The Nanoscribe is a high-resolution (500nm) Nano 3D printer that is new to the Washington Nanofabrication Facility. It was acquired through a National Science Foundation (NSF) Major Research Instrumentation (MRI) grant. The tool uses a technology called two-photon polymerization. This involves the use of photoresist, which is a light sensitive material, and an infrared laser that directs brief pulses of light through a microscope objective in different patterns to create 3D objects. The highly focused laser beam allows for dimensions of less than 500 nanometers in X and Y directions to be achieved. One major limitation of the tool is that unlike most photolithography equipment, it is not programmed to do basic alignment. Most fabrication applications require at least two layers of materials to be aligned. For example, when making electronic circuits, there is a top and bottom layer at a minimum, and a vertical layer in between, thereby requiring at least two alignments to the first layer. Our research is focused on understanding and testing test code to add pattern recognition alignment capabilities to the Nanoscribe instrument. For our test application, we applied test aligning microneedles to pre-patterned electrodes fabricated using conventional photolithography. We then 3D printed microneedles that are subsequently used to release drug therapies in test wells via an applied electrical potential (voltage). The alignment process that we developed can be used as a basic construct and capability for other applications being developed on the tool.

The reduction of drug craving and subsequent relapse is central to the recovery process for individuals with substance use disorders. One of the goals of this project is to improve pharmacological treatment interventions for substance use disorders by better understanding how the neurotransmitter dopamine is involved in relapse. We investigated the relationship between drug craving and dopamine release in the nucleus accumbens core (NAC), an area of the brain that plays an integral role in reward-based learning and addiction, in male rats that have been trained to consume cocaine or alcohol. We tested the effects of L-DOPA, a chemical precursor to dopamine and a treatment that could reduce drug consumption, on craving and relapse-like behavior. Recent evidence from our lab suggests that decreased dopamine release in the NAC correlates with increased cocaine consumption in rats, and that L-DOPA administration attenuates drug intake. Preliminary data from our current project shows a similar effect of L-DOPA on alcohol or cocaine relapse. Our characterization of dopamine release in the NAC during both periods of drug abuse and withdrawal provides new insights for future studies and our use of innovative electrode implants for monitoring dopamine neurotransmission will pilot the technology for other drug self-administration studies. This will help to determine whether L-DOPA should be considered for treatment of cocaine and alcohol addiction in humans.

Mechanistic Target of Rapamycin (mTOR) is a highly conserved serine/threonine kinase originally discovered in yeast as a central regulator of cellular growth and proliferation in response to adequate nutrients and energy levels. mTOR is activated in a complex with an essential co-activator called Raptor. Our lab recently discovered that mTOR holds an essential role in early murine B-cell development; B-cell specific disruption of Raptor inhibited mTOR activity and resulted in a complete block in B cell development at the precursor-B (pre-B) cell stage. We hypothesize that B-cell specific disruption of Raptor will inhibit B-cell transformation induced by the Myc oncogene, and/or will inhibit the survival of B-cell lymphoma cells, by inhibiting cell growth induced by mTOR. We conditionally disrupted Raptor using gene targeting strategies in a mouse model of Burkitts Lymphoma whereby the c-Myc oncogene is expressed under control of the immunoglobulin heavy chain enhancer. We found that Raptor deficient mice took significantly longer to develop B-cell tumors when bred to c-Myc transgenic mice; our Kaplan-Meier Survival curve revealed that disruption of Raptor improved survival rates significantly, such that Raptor knockouts rarely showed growth of tumors. To better understand cellular mechanisms by which Raptor may inhibit B-cell lymphoma formation, we utilized flow cytometry to measure cell proliferation and survival of B cell lymphoma cells in the presence or absence of Raptor. Our results revealed a distinct reduction in both cellular proliferation and survival in the absence of Raptor, which suggests Myc driven B-cell proliferation and survival is highly dependent on Raptor and mTOR activity. In the next part of our studies, we will inducibly disrupt Raptor in already established primary Myc-induced B-cell lymphomas to determine whether Raptor and mTOR activity are essential for lymphoma cell survival. Through these results we will be able to determine whether inhibition of Raptor could be utilized as a strategy to inhibit cancer cell proliferation and/or survival.

Mutations in the Presenilin 2 gene, (PSEN2), cause early onset familial Alzheimer’s disease (FAD). PSEN2 encodes the presenilin 2 protein which forms the catalytic subunit of the gamma-secretase complex, responsible for cleaving amyloid precursor protein into fragments. One of the resulting fragments is Aβ_1-42, a major component of brain amyloid plaques and a clinical hallmark of Alzheimer’s disease. Microglia are the resident innate immune cells in the brain clearing the brain of Aß plaque and debris through phagocytosis. Without presenilin 2 protein present, levels of secreted cytokines increase implying a higher immune response, and recent data suggest that hyper-activation of an inflammation state can contribute to development of Alzheimer's disease. We hypothesized that FAD mutations can impair normal presenilin 2 protein function and therefore promote AD through dysregulation of immune function. We have developed a mouse model to study the impact of the most common FAD PSEN2 mutation, the PSEN2N141I, also known as the “Volga German” mutation. Previous experiments in the lab have shown that the absence of wild type PSEN2KO disrupts normal microglial phagocytosis of neuronal debris. My work involves measuring phagocytosis of apoptotic bodies by mouse primary neonatal microglia expressing the FAD PSEN2 mutation to determine if AD associated mutations contribute to AD through altering microglia phagocytosis behavior. Microglia from wild type mice, mice with presenilin 2 knocked out, and mice heterozygous for the human N141I mutation are collected and co-cultured with apoptotic cell bodies. Microglia and apoptotic cell bodies are membrane-labeled and FACS analysis is used to measure percent phagocytosis. My studies will further our understanding of how familial AD mutations alter the normal cellular behavior of brain immune cells and may highlight new areas in pathways appropriate for therapeutic targeting.

Many membranes spontaneously demix into coexisting liquid phases. These include membranes that are as simple as three-component lipid vesicles and as complex as protein-rich vacuole membranes within living yeast cells. The resulting membrane phases are enriched in particular lipids and proteins. Membrane phase separation is one of several mechanisms proposed to explain the observation of heterogenous distributions of lipids and proteins in cell membranes ("rafts"), which are thought to be important in cell signaling, apoptosis, and protein regulation. The overwhelming majority of experiments that probe phase separation in membranes uses only one type of lipid (namely phosphatidylcholine). However, it is known that biological membranes also contain significant fractions of other kinds of lipids (e.g. phosphatidylethanolamine, or PE-lipids). An experimental challenge is that membranes composed of high fractions of PE-lipids are unstable – the lipids assemble in tubes instead of lying in sheets. In my project, I am [1] determining the maximum amount of saturated and unsaturated PE-lipids that can be incorporated into stable membranes, [2] measuring the temperature at which the membranes demix into coexisting phases, and [3] imaging the vesicles by fluorescence microscopy. Results from Aim 1 inform future researchers which lipid ratios they can use to produce stable membranes. Results from Aim 2 provide data against which the scientific community can calibrate molecular dynamics simulations. Results from Aim 3 provide a direct test of a recent prediction that PE-lipids should be anti-registered across fluctuating membranes.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that impairs an individual's social, communicational, and behavioral skills. Males are 4.5 times more likely to be diagnosed with ASD compared to females. One possible reason for the difference in diagnostic rates is that females may be better able to mask their symptoms and therefore go undiagnosed more often than males. If females with autism are better at masking, then it is possible that standard diagnostic tools underestimate their impairment due to the clinician not seeing the full extent of individual's symptoms. The goal of this study is to understand the potential for sex bias in two measures of ASD by examining measurement consistency between parent and clinician report of ASD symptoms. 140 children (70 males, 70 females) aged 8-17 years with a confirmed diagnosis of autism participated in this study. Participants' diagnoses were confirmed via the ADOS-2, a clinician-child interaction measure that scores the child's communication, social skills, and restricted or repetitive behaviors. Parents completed the Social Responsiveness Scale (SRS-2), a survey about their child's social and autism-related impairments. To look at potential bias, we first compared males and females with ASD on both the ADOS and SRS to examine sex differences in autism severity. Second, we examined the relation between parent and clinician report for differences in consistency of ASD symptoms by sex. If females are better at masking their autistic traits, we expect a lower correlation between ADOS and SRS scores in females compared to males, indicating lower parent-clinician agreement. Third, we explore these analyses in children who had clinical diagnoses but who did not meet research criteria for ASD (n = 20). Understanding the relationship between sex and measurement tools should help inform whether traditional diagnostic measures are as effective in diagnosing both males and females.

The midbrain is a critical region of the brainstem that houses the cranial nuclei necessary for coordinating eye movements. It has been observed that neurons in the midbrain express T-box Brain 2 (Eomes/Tbr2), a transcription factor generally associated with development of glutamatergic neurons. Because it has such a diverse role throughout the development of multiple neural structures (i.e. cerebral cortex, olfactory bulb, retina, and cerebellum), Tbr2 is thought to be implicated in the development of certain forms of autism. The purpose of our experiments was thus two-fold: first, we investigated the neurotransmitter phenotype of Tbr2+ neurons in the midbrain. Second, we wanted to identify the function of Tbr2+ neurons in the brain by studying these populations in relation to known, molecular markers of specific, anatomical features. In order to accomplish these aims, we collected tissues at various ages from transgenic mice. We utilized endogenous reporters such as VGlut2-Cre^Ai14 and Tbr2mTnG alongside immunohistochemical staining with the following antibodies: urocortin-1 (UCN-1; which marks the centrally-projecting Edinger-Westphal nucleus, EWcp, in the postnatal brain); cocaine- and amphetamine-regulated transcript (CART, which marks EWcp in the embryonic and postnatal brain); and 2H3 (which marks neurofilaments). We then used a confocal microscope to visualize fluorescence and collect images. We found that Tbr2+ neurons in the midbrain were glutamatergic but not cholinergic. We also found that Tbr2 neurons were not Edinger-Westphal cells, leaving their ultimate anatomical identity unknown. Understanding the identity of Tbr2 cells is a prerequisite for beginning to understand the constellation of features that make up autistic spectral disorder, thereby guiding future clinical and scientific research.

Glioblastoma (GBM WHO grade IV) is the most dangerous brain tumor in adults and comes in two types, primary and secondary. Primary GBMs form de novo and are more common and more aggressive than secondary GBMs, which develop from grade 2 and grade 3 gliomas. Despite primary and secondary GBMs displaying identical tissue, there are significant differences in the genes expressed that affect the GBMs responsiveness to alkylating treatment. Our research focuses on secondary GBMs and why they have an association with higher progression free survival (PFS) than primary GBMs, in hopes to aid the personalization of treatment plans. Our hypothesis that O6-methylguanine-DNA methyltransferase (MGMT) activity is inversely related to PFS. Our method of experimentation includes taking the MGMT content of the extract, which is supernatant liquid that lies above a precipitate of whole tissue lysates. We then use the radioactivity transfer from a DNA substrate containing O6-methylguanine-DNA to protein as a quantitative measure. In secondary GBMs, there are sometimes mutations in the Isocitrate Dehydrogenase 1 (IDH1) gene. These mutations reduce substrate affinity, decreasing the enzymatic activity. Furthermore, the enzyme develops the ability to produce an oncometabolite, D-2-hydroxyglutarate (2-HG). 2-HG synthesis leads to genome wide hypermethylation of CpG islands potentially causing the MGMT gene to be silenced. The MGMT protein repairs DNA and prevents it from breaking. By silencing the MGMT protein activity, DNA gets repaired less and usually results in double strand breaks. The higher PFS associated with secondary GBMs could be affected by the IDH1 mutation, which can reduce MGMT protein activity and lead the tumor DNA to break, making treatment more effective. Establishing this association could lead to personalized treatment because patients with IDH1 mutations respond better to alkylating agents, such as TMZ.

As demand for accurate evaluation of chemicals and their potential to cause effects on humans increases, we have designed in vitro methods to facilitate such assessments. However, models of in vitro cell cultures are limited in their approach typically using a monoculture of limited cell types. Using a 3 dimensional organotypic approach to culture multiple cell types more reflective of a functioning organ can provide a framework for evaluating systematic interactions minimizing the need for in vivo assessment. We utilized a novel organotypic, in vitro model of testicular development that mirrors the development shown by in vivo studies. Our baseline study isolated testes that were harvested from C57BL/6 male mice on post-natal day 9. Testicular co-cultures were maintained for up to 16 days in 24 well plates with data collections occurring at days in vitro (DIV) 3, 7, and 16. Plates were stained with antibodies providing markers that correspond to morphological features of specific cell type markers. Five different primary antibodies were used as markers to visualize the change in cell populations. Markers for Sertoli cells corresponded to Vimentin, Leydig cells with 3b-HSD, Germ cells to DAZL and c-kit, and proliferation with PCNA. For each well, we used an immunofluorescence dual staining technique with Scanning Laser Image Cytometery (iCys) to observe the percentage of cell types and to determine change in phenotypic distribution of the cell population. From our preliminary observations, we were able to observe changes in Sertoli cells, Leydig cells and Germ cells throughout testicular development. Future studies would further develop and confirm the population and morphological changes observed with the testicular cell cultures. With a baseline model created, this in vitro model can be used to test the lethality of chemicals without the need of in vivo alternatives.

Adaptive learning technologies are regarded as an innovative approach to personalize student learning in large college classrooms where interaction with instructors is limited. Such software allows students to learn at their own pace, and to concentrate their efforts on topics which need improvement. However, studies on adaptive learning systems across disciplines have yielded mixed results as to whether they have an impact on improving course mastery. Our research focused on testing how use of adaptive learning reading quizzes in the final quarter of an introductory biology series impacted student exam performance. Students (n=576) enrolled in this course were distributed across 25 lab sections. Students were randomly assigned by lab section to either static or adaptive learning pre-lecture reading quizzes. Both types of quizzes were provided to students through Macmillan’s LaunchPad portal, and points were awarded on a completion/ no completion basis. We used multiple linear regression with model selection to compare student performance between the two treatments, controlling for student characteristics (e.g., GPA, gender). When student performance on in-class reading quizzes and course exams was compared, no statistically significant differences were found between treatments. Similarly, through the administration of three surveys throughout the quarter, we were able to measure students’ attitudes toward different course components. Our findings show that there was no statistically significant difference as a result of adaptive learning reading quizzes on how prepared students felt for class, how much they enjoyed the course, how much they valued reading quizzes, or if they preferred the reading quiz format in this course to those in previous courses. Our findings suggest that given the additional financial cost of adaptive learning technologies to students, instructors should first verify that adaptive learning significantly impacts mastery of course material prior to fully integrating it into their course structure.

New techniques in mapping genome structure have revealed a high level of spatial organization in the nucleus. The 3D location of a gene within this high order structure is thought to impact its transcriptional state. Long-range DNA loops could link distal regulatory elements to their gene targets. To test this hypothesis we plan to develop a programmable system that allows us to loop DNA loci together, bringing them in spatial proximity. We hope to explore whether we can create or disrupt interactions between genes and regulatory elements by engineering new DNA loops. We will use programmable CRISPR-Cas DNA binding domains to target specific sites in the genome. The CRISPR-Cas complex will be tethered to an interaction domain (ID) that can dimerize with another ID at a distant DNA locus. A main challenge faced when building DNA loops is free IDs are likely to outcompete looping interactions between complexes bound to DNA. To favor interactions between complexes bound to DNA, we will use protein switches designed in the Baker lab to sense whether the CRISPR-Cas complex has engaged its DNA target. I am currently testing prototype protein switch designs and measuring their ability to activate when assembled on DNA. Initial results have shown that our switch designs can activate upon DNA binding, but that our system still needs optimization. After our switch design is optimized, we can move on to the full-fledged looping system and begin to assess the regulatory consequences that spatial repositioning has on gene regulation.

Microtubules are critical for cellular and developmental functions, including neurite outgrowth and maintaining stable wiring of the nervous system. Tau, an intrinsically disordered protein, plays important roles in microtubule assembly and stabilization. However, it dissociates from microtubules and forms amyloid fibrils in a set of neurodegenerative diseases termed tauopathies, which includes Alzheimer’s disease, and frontotemporal dementia. Mutations in the sequence of tau cause the disease FTDP-17T (frontotemporal dementia with parkinsonism linked to chromosome 17 and specially characterized by tau pathology), providing evidence that alterations in tau alone can cause tauopathies. Most mutations occur in or near the microtubule-binding domain of tau, which suggests that mutant protein may be deficient in its capacity to stabilize neuronal microtubules, yet efficient at speeding up the process of aggregation. In particular, disease-associated mutations such as ΔK280 and P301L appear to enhance tau’s propensity to aggregate into fibers, and alter its pathological activity. Molecular chaperone proteins are responsible for maintaining protein solubility, promoting proper folding, and preventing atypical aggregation. HspB1, a member of the small heat shock proteins (sHSPs), has been shown to delay wild-type tau fibril formation by weakly interacting with early species in the aggregation process. Hsc70 is a constitutively expressed chaperone that inhibits tau fibrils formation substoichiometrically. Both chaperones are able to recognize aggregation-prone motifs within the microtubule binding repeat region of wild-type tau. Therefore, our interest is to test the affinity and activity of HspB1 and Hsc70 against tau constructs containing the ΔK280 and P301L mutations, to see whether these mutations alter interaction with these chaperones. We will use various biophysical methods, including fluorescence spectroscopy and electron microscopy to analyze these interactions. This will extend our understanding of the characteristics and behaviors of disease-associated mutations of tau and the ways in which chaperones target aggregation-prone tau species.

Synthetic biology allows us to use an organism’s metabolic pathways to produce new, useful metabolites cost-effectively. Growing organisms in a culture allows the production of these metabolites on a commercially-viable scale. However, management of these cultures is time-consuming and labor-intensive, such as HPLC. Our project aims to provide a low-cost, automated system that analyzes the culture in real time at low cost. Our device, a turbidostat bioreactor called the Chromastat, uses images and other optical data to measure the current state of a culture. If these measurements show metabolite production outside specifications, the system modifies the culture’s metabolic expression by adding inducers. To make this system cost effective, we focused on using affordable materials, an open-source controller and analysis system, and visible color outputs instead of the traditional fluorescence. To test our system, we used the violacein metabolic pathway in yeast. By regulating gene expression with inducible promoters, this pathway yields up to four visually distinct pigments. In a production environment, these pigments could provide a proxy measurement to produce useful substances which are costlier to detect. Pigment production is measured by the popular low-cost and open-source Raspberry Pi computer running a program displayed in Java Swing. Optical sensors collect color and opacity information about the culture. Our software, installed on the Raspberry Pi, analyzes the color, and in response, the hardware introduces inducers to stabilize the culture’s production. Data recorded by the Pi over time reveals the relationship between gene expression and metabolite production rates. By combining biological, software, and hardware systems, our unique design can generate previously unavailable visual data in certain biosynthesis processes, such as those involved in antibiotic production or fermentation.

The PTEN (phosphatase and tensin homolog) protein negatively regulates growth-promoting PI3K-Akt signaling in cells. Due to its function as a tumor suppressor, PTEN is often mutated in diverse cancers. Unfortunately, most PTEN variants have not been individually studied, making it difficult to ascertain their functionality within cells. Our lab recently demonstrated that thousands of PTEN missense variants exhibit decreased steady-state abundance when expressed in human cell lines and likely have reduced function. However, the mechanism behind their lower abundance is currently unknown. We hypothesized that many low-abundance PTEN variants are thermodynamically unstable and possess a reduced melting temperature. To test this, we fused EGFP, Enhanced Green Fluorescent Protein, to a panel of PTEN single amino acid variants, and these fusion proteins were expressed within human cell lines. Then, we cultured these cells at their standard growth temperature (37°C) and two lower temperatures (33°C and 30°C). We found that a subset of variants of intermediate abundance at 37°C exhibit WT-like abundance at decreased temperatures, while variants of extremely low-abundance remain unchanged. These results suggest that we can identify missense variants with reduced thermodynamic stability using this method. Next, we will repeat this experiment at high throughput to identify hundreds of temperature-dependent PTEN variants. We will determine biochemical properties shared by partially stable variants and compare them to computational predictors of protein folding. We will also identify variants of intermediate abundance that are not temperature-dependent, which may reveal other mechanisms by which variants lower a protein’s abundance. Our results demonstrate that we can characterize the thermodynamic stability of PTEN variants by measuring their abundances in cells grown at different temperatures. These results might be more physiologically relevant because the variants were studied in a cellular environment. Furthermore, our methods may be applied to other proteins that cannot be studied as purified protein.

Familial caregivers often serve as the primary coordinator of care for children with medical complexity (CMC). However, they currently lack a central tool to organize their child’s health information from multiple fragmented information sources and support their unique information needs. The primary objective of this study was to develop a user-centered, care organization tool by incorporating the information needs and preferences of CMC caregivers. Following a user-centered design methodology, we first conducted interviews with CMC caregivers between 3/16-11/16 to understand their information needs and elicit their preferences for a care organization tool. Next, we conducted design sessions with caregivers between 8/17-12/17, which involved content categorization activities, drawing exercises, and scenario-based prototype testing. Between design sessions, we applied content analysis to interview and design session transcripts, and incorporated findings into the ongoing development of a functional prototype. We conducted interviews with 17 caregivers and design sessions with 6 caregivers. We grouped findings into two categories: design and implementation. An overarching theme of both categories was the need for customizability, as the primary purpose of the tool differed based on the caregiver’s main information needs. In terms of design, caregivers voiced a desire for both mobile and web interfaces, with a customizable home screen, containing templates to track medications, symptoms and major events, and outline communication preferences based on their child’s developmental status. In terms of implementation, caregivers were agreeable to the idea of a cloud-based tool which would provide easy access to emergency information. However, they wanted control over customizable permission settings for others to view and/or edit other content within the tool. We developed a novel, user-centered, cloud-based care organization tool for CMC families by engaging caregivers in the design process. Next steps include examining prototype usability, effectiveness, and integration with provider health records.

Inferior olive (IO) is part of the brain stem that projects their climbing fibers in the cerebellum's Purkinje cells and receives input from the deep cerebellar nuclei DCN to regulate the strength of electrical coupling in the IO. The structure of the IO contains a high density of gap junctions that are responsible for weak spontaneous subthreshold oscillation (STO). The weak electric potential STO can depolarize the neuron and open the N-methyl-D-aspartate receptor (NMDA) channel that is located close to the gap junction by removing its Mg ion. The activation of the NMDA receptor can play a role in strengthening the STO, increasing its synchrony, and inducing a new STO. Increase the synchrony in IO may contribute to the precision learning of the motor skills. Therefore, our study was done on mice in vivo, and extracellular recording technique was used to record from IO before and after the addition of NMDA. It was found that adding NMDA changed the firing of the neuron relative to itself. Second, NMDA decreased the power spectra density's raw power in both groups 2 Hz and 3-6 Hz, however, there was some increasing in the PSD at 5 Hz in the group of neurons that fire with 2 Hz. Third, the coherence value between the neuron and the local field potential (LFP) decreased after the addition of NMDA. There was an exaption in which the coherence value increased at specific frequences. Finally, we found that a neuron showed phase locking its firing activity with the LFP, which was in the negative firing phase. In conclusion, NMDA receptor plays a role in increasing the synchrony of neuronal electrical synapses in IO, involves in the IO rhythmogenesis, and may add temporal precision to coordinate the muscle activity.

Cancer stem cells are thought to play a role in relapses and metastasis in numerous cancers. The inability of traditional cancer therapies, such as chemotherapy, to eradicate these cancer stem cells prompted a search for small molecules that induced apoptosis in cancer stem cells. Drosophila germline stem cells can be used as a model system to emulate cancer stem cells. Upon irradiation, Drosophila germline stem cells are able to survive apoptosis through a molecular signal released by the apoptotic daughter cells via the TIE receptor. We have conducted an in vivo drug screen of 512 compounds in Drosophila melanogaster to find drugs that would disrupt this protective mechanism and induce apoptosis. In particular, Camptothecin, NSC 125197, and NSC 127458 were effective in killing germline stem cells. To test the effect of the small molecules we fed female flies the compound for 3 days, dissected and fixed the ovaries and stained for an apoptosis marker, activated caspase, and the GSC marker, adducin. We quantified the effect of the drug by analyzing the number of germline stem cells and caspase signaling on a confocal microscope. Through this system we aim to find potentially new, and more powerful anti-cancer drugs that can affect cancer stem cells. Future directions include attempting to understand the mechanism of action of these apoptotic compounds in hopes that they might be useful in the fight against cancer stem cells.

The ability to recapitulate the dynamic presentation of signals in a stem cell's microenvironment remains a major hurdle in tissue engineering. By controlling cell growth and differentiation in 4 dimensions (i.e., time and 3D space) through biochemical and biomechanical cues, heterogeneous synthetic tissues could be produced that match the complexity of their native counterparts. By combining strategies in light-programmable hydrogels and recombinant protein engineering, we have demonstrated the ability to control the cellular microenvironment with a UV light mediated reaction that allows a covalent bond to form between a modified protein and the hydrogel. While covalent interactions form strong bonds, they are generally irreversible using mild, cytocompatible chemistries and pose potential temporal limitations. In addition, this technique uses UV light, which may be harmful to cells. Instead, by incorporating LOVTRAP into the photopatterning system, we introduce a greater amount of control by allowing for full reversibility using blue light. In the dark, LOV2 and Zdk1 dimerize to form a protein-protein bond with similar stability to an antibody-antigen interaction. Upon exposure to blue light, the dimer dissociates, allowing for fully reversible control over conjugation. A protein of interest can be tagged with the Zdk1 domain using recombinant protein engineering, while still maintaining its binding affinity for LOV2. LOV2 can then be conjugated throughout a hydrogel to make a light responsive protein binding material. By utilizing a light controlled dimerization event between proteins, the LOVTRAP system is fully reversible using only blue light. Thus far, I have been able to illustrate the photopatterning ability of LOVTRAP and am working towards demonstrating full reversibility. If successful, this technique could be used for tissue engineering applications that require mild treatment conditions and full dynamic control over the presentation of biochemical cues.

East Coast Fever (ECF) is a tick-transmitted disease caused by Theileria parva in cattle that is detrimental to the economic well-being of Eastern and Southern Africa. Current disease control involves pesticides, antibiotics, and a commercial live parasite vaccine, but they are insufficient and thus developing an effective, affordable, and protective vaccine for ECF is a pressing need. Studies by the Nene group at International Livestock Research Institute have demonstrated that the T. parva sporozoite stage-specific surface coat protein p67 provides partial protective immunity to cattle when used as a subunit vaccine. Within the King group at the University of Washington, I generated novel p67C nanoparticle immunogens intended to induce more potent and durable immune responses in immunized cattle. I genetically fused the p67C epitope to a variety of self-assembling protein nanoparticle subunits and screened for stability and expression levels. Based on these data, I selected three best-performing p67C nanoparticles for larger-scale expression and purification: I32-19, I32-28, and I53-50. Each of them, with 60 copies of the p67C, was expressed, purified, and extensively quality-controlled to confirm monodispersity, purity, and low endotoxin levels for immunization studies. Immunogenicity data from the ILRI show that the nanoparticles with p67C induce a similar level of p67C-specific antibodies as a combination of HepB core antigen and mesoporous silica nanoparticles containing more than twice as much p67C antigen, and far higher antibody levels than p67C alone. It is an outstanding vaccine candidate to help those suffering from ECF. These preliminary results will be confirmed by parasite challenge studies in 2018.

The default mode network (DMN) changes with Alzheimer’s disease (AD), mild cognitive impairment (MCI), and normal aging, but these changes are not well characterized. The posterior cingulate cortex (PCC) is a critical hub in the DMN. Using data from the Alzheimer’s Disease Neuroimaging Initiative database, we modeled longitudinal change in PCC connectivity in three subject groups: healthy controls, MCI, and AD, and tested whether connectivity changes were linear or quadratic with time. The HC group had no evidence of Aβ accumulation (N=56, 25 male, 75.7±6.23 years). The MCI group pooled all MCI and significant memory concern subjects with no signs of dementia (N=127, 59 male, 72.6±7.14 years). The AD group fulfilled the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related Disorders Association (ADRDA, now known as the Alzheimer’s Association) criteria for probable AD (N=34, 16 male, 74.2±7.47 years). We processed resting state fMRI images using a workflow that represents best practices for removal of noise, censoring motion outliers. We measured individual connectivity to a 10mm sphere in the PCC (2,-52,28) and transformed the Pearson correlations using a Fisher Z-transformation. We used a software developed in our lab called neuropointillist to fit a mixed effects growth model in R to each voxel, comparing linear and quadratic forms of longitudinal change. Data were cluster corrected for multiple comparisons at p=.05, α=.05. A linear model was superior to a quadratic model for all groups across the brain. All three groups showed decreased connectivity with age of the PCC to the medial frontal wall. However, we found increased connectivity to the dorsal attention network only in the MCI group. We identified distinct regions of the brain that increased and decreased in connectivity to the PCC with age in the three subgroups. These differences likely represent the different pathophysiological processes occurring in normal aging, MCI, and AD.

Auxin is a hormone that is crucial for plant growth and development. Auxin promotes the activity of auxin responsive transcription factors (ARFs) that induce transcription of target genes. Though, we know a lot about auxin response in the model plant Arabidopsis, very little is known about ARF behavior in maize. It has been previously shown that auxin and ARFs mediate many diverse developmental responses in Arabidopsis and maize. We hypothesize this diversity of response may be caused by different ARFs binding to different promoters, allowing different genes to be activated. The goal of the project is to examine the activity of maize ARFs on different promoter structures. We tested if maize ARFs show similar rules of ARF-promoter interactions as Arabidopsis ARFs. By comparing Arabidopsis and maize ARF activity, we are be able to understand how the two species have evolved over time, and if ARF function and auxin response are conserved in the two species. Working on maize is also important because it such an essential food crop for people and animals and can help us resolve issues such as food shortage. To investigate how ARFs regulate the activation of genes, we used a synthetic yeast system to quantify transcriptional activation. We engineered yeast to express both a maize ARF and a synthetic auxin-responsive promoter regulating transcription of a fluorescent reporter. We quantified fluorescence, a readout of ARF activation of the promoter, by flow cytometry. We systematically tested how activation is impacted by the sequence, orientation, and number of cis-elements within a promoter sequence, and how these effects differ among different ARFs.

It is critically important that eukaryotic cells possess the capacity to undergo intracellular change in response to both internal and external cues. Arabidopsis thaliana F-box (FBX) and 14-3-3 proteins are two distinct protein families that regulate intracellular change and have important roles in regulating responses to environmental stress. FBX proteins are variable substrate adaptors within the Ubiquitin 26S Proteasome System (UPS), a system the selects proteins to be degraded by the 26S proteasome using selective ubiquitylation. 14-3-3 proteins recognize and bind unique phosphorylated residues in client proteins to regulate subcellular localization, inhibit protein interactions, and act as a scaffold to facilitate protein interactions. Thus, these two protein families independently have the ability to drastically alter cell physiology. 14-3-3 proteins have been shown to interact with components of the Ubiquitin 26S Proteasome System in only a few instances, including their interaction with the F-box protein F-BOX STRESS INDUCED 1 (FBS1). However, the biological consequences of this FBS1:14-3-3 interaction are completely unknown. FBS1 belongs to a four-member FBX protein family, along with FBS2-4, and we hypothesize that 14-3-3 proteins more broadly interact to at least regulate this family of four proteins. A Yeast 2-Hybrid assay has been employed to test for interactions between FBS1-4 and five of thirteen different 14-3-3 proteins. Full length and truncated constructs are currently being tested to probe the extent of interactions between these two important protein families. Interaction between multiple members of these two important protein families could point to a more broadly used scheme in regulating intracellular events in plant cells.

Posttranslational modification with the small protein ubiquitin (Ub) is an essential process in various regulatory pathways of eukaryotes, such as protein degradation, DNA repair, and the cell cycle. Misregulation of ubiquitylation has been associated with diseases such as cancer and neurodegenerative disorders. Ubiquitylation is accomplished by an enzymatic cascade that involves a family of ~40 ubiquitin conjugating enzymes (E2), which form thioester intermediates with Ub, called E2~Ub conjugates. In most ubiquitylation reactions, it is the E2 that covalently attaches Ub to substrate proteins. While the overwhelming majority of E2s ubiquitylate the ε-amino group of substrates’ lysine residues, important exceptions have recently been discovered. For example, Ube2W has been identified as the only human E2 that exclusively ubiquitylates the α-amino group of substrates’ unstructured N-termini. Knockout of Ube2W in mice leads to severe developmental abnormalities, suggesting its importance for early postnatal survival. Little is known about the molecular mechanism of Ube2W’s unusual specificity and structural insights to the catalytically-relevant species, the Ube2W~Ub conjugate, are lacking. Here, I report the formation and purification of the first stable Ube2W~Ub conjugate mimic. Using a combination of biochemical techniques and nuclear magnetic resonance (NMR), I characterized atomic-level changes within the Ube2W~Ub conjugate during substrate recognition and catalysis. This investigation will expand our mechanistic understanding of E2s and establish insights to a biochemically fascinating and biologically relevant enzyme.

Dementia is a neurological condition that impairs memory, cognitive functions, and the performance of daily activities. It is estimated that 5.5 million Americans suffered from dementia in 2017. Currently, the predominant method used to treat persons with dementia is through pharmacological means. However, through a review of current research we have found evidence to suggest that cognitive behavior therapies (CBT) may be more beneficial to patients, with the potential to reverse some symptoms of dementia. Our study aims to improve CBT through virtual environments. Virtual Reality (VR) is not merely a visual platform but an immersive environment that stimulates the brain in the same way real environments do. Patients with dementia living nursing homes are rarely exposed to different environmental interactions or thought inducing stimuli, resulting in an escalated progression of their condition and a loss of “sense of self.” CBT, specifically reminiscence therapy, has shown promising results in improving cognitive functioning as well as morale. We hypothesize that if we use visual and audio equipment as well as tangible artifacts, we could create an immersive therapy to stimulate cognitive functions. We track certain markers like head and eye movement to track the progression of the disease as well. In collaboration with a UK based game development company we have identified a software with both therapeutic and a diagnostic capabilities. We have made connections with senior centers in the area to identify persons wishing to participate VR therapy sessions. This project will offer understanding of how implementing VR therapy can improve patients’ morale and cognitive capabilities, as well as exploring the use of VR as a diagnostic tool.

Active learning has often been described as the key to increase retention rates among undergraduate STEM majors because students are engaged in meaningful learning. Common activities associated with active learning include completing worksheets in groups, clicker questions, or discussion questions followed by reporting out by random call during class. Random call differs from cold call as the instructor allows discussion time where students can work together to formulate an answer prior to random call. However, previous research has demonstrated that implementing active learning principles may increase student anxiety. Specifically, answering questions in class, either by volunteering or random call, may cause the highest level of anxiety for students. We have conducted a study investigating how student demographics impact the likelihood of students responding to random call. Our study was conducted in an Introductory Biology course with 576 students, in a lecture-hall style classroom. The instructor utilized random call, then recorded which students either gave a response or passed the question when called upon. A binary logistic regression was performed to create a best fit model in which student demographics could predict which students were more likely to answer or pass. Students who are the first in their family to go to college (1st generation students) were 57% less likely to respond to random call questions compared to other students, even when controlling for incoming GPA. This result indicates that students with diverse demographics participate differently in some active learning activities and further investigations are necessary to determine if this has an impact on their learning. To bridge this “responding” gap between 1st generation and Non-1st generation students, instructors should implement additional techniques such as group random call as opposed to individual random call to increase student comfort during random call, thereby increasing the likelihood of students responding regardless of student demographics.

Oil-spills are a widespread hazardous environmental issue, often caused by mistakes when drilling or transporting crude oil. The effects are devastating to the ecosystem. There are very few ways to quickly clean up an oil spill efficiently, at low cost and without additional environmental damage, as often they are cleaned by using expensive chemical dispersants. We conducted research on an environmentally friendly and inexpensive solution to this problem by using pearl oyster mushroom mycelium. The pearl oyster mushroom has a lignin decomposing basidiomycotina that produces a set of extracellular ligninolytic enzymes that have bioremediation properties. Our hypothesis is that, oyster mushrooms can be used to clean oil spills. We cultivated the mushroom mycelium in straw on trays with different amounts of oil on them. We grew this in a greenhouse with controlled temperature and allowed 4-6 weeks for the mycelium to eat the oil. We are currently observing the results. After 6 weeks, we will filter out the remaining hydrocarbons with a solvent and determine if the mycelium reduced the amount of hydrocarbons or consumed them entirely. Our anticipated results are that the mushroom will clean up the oil in six weeks. For future work, we plan on conducting this same experiment but in a salt water environment to explore applications for mycoremediation in a simulated ocean environment.

Probiotics are an area of significant clinical research, as they have been shown to aid digestion, fight infections, and even mitigate irritable bowel syndrome; however, probiotic colonies must grow and thrive in the extreme pH environment present in the human digestive tract. Previous experiments focused on the production of the surface protein Elongation Factor Thermally Unstable (EF-Tu), which attaches to glycoproteins lining the intestines and has been used as a biomarker for probiotic health. The growth of several strains of probiotics (Lactobacillus bulgaricus, Lactobacillus acidophilus, and Bifidobacterium longum) in both lysogeny broth (LB) and De Man, Rogosa, and Sharpe broth (MRS) was evaluated. The fractions of secreted, cytosolic, and membrane proteins were quantified using a Bradford assay. Finally, the relative amount of EF-Tu was determined by tryptic digestion and liquid chromatography-mass spectrometry (LC-MS). LB was found to be a more effective growth medium for the range of bacteria tested, with 24 hours at 37°C the ideal incubation period. To isolate the secreted proteins, the supernatant was drawn off the broth culture after centrifugation. Freeze-thaw lysis was used to extract cytosolic proteins, with three cycles determined as optimal for protein recover. Sodium deoxycholate was used to separate proteins from the membrane. All three protein samples were separated and run through a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the EF-Tu bands (roughly 43 kDa) were excised and sent for analysis by LC-MS. Using the information obtained from this research study, pharmaceutical companies can create more suitably tailored probiotic products, and make them more accessible and understandable to the general population. 

Retinal pigment epithelial cells (RPE) normally reside on the photoreceptor side of the retina; however, they can become dislodged as a result of trauma and surgery. A condition called proliferative vitreoretinopathy (PVR) can occur when these cells undergo an epithelial to mesenchymal transition (EMT) and form contractile scar tissue that results in retinal detachment. PVR is a common complication of retinal detachment surgery occurring in about 10% of cases, and there is currently no effective non-surgical treatment. In-vitro models of PVR have previously been created from surgically excised tissue, but these cultures are difficult to create and maintain. However, one recently isolated primary cell line, fRPE #51, has been noted to spontaneously form contractile bands similar to those found in other PVR models. The goal of this study is to characterize this cell line to determine if it has the potential to serve as a reliable in vitro model of PVR. To fully characterize this fetal RPE cell line, we stained the cells for known markers of PVR. We also completed several assays for proliferation ability of the cells to assess for functional changes consistent with PVR. Finally, in order to characterize changes to the structure of these cells, we assessed the formation of tight junctions by measuring transepithelial resistance (TER) of cells grown on filter membrane inserts and performed transmission electron microscopy to look for other structural changes. Our preliminary data indicate that the candidate cell line fails to establish normal tight junctions. In addition, an initial proliferation assay demonstrates enhanced ability of the candidate cell line to proliferate and migrate in comparison to control lines. Establishing an in vitro model of PVR will allow for screening of potential drugs and other small molecules that may inhibit the formation of PVR.

Oysters support a multi-million-dollar aquaculture industry in Washington State and provide important ecosystem services in estuarine habitats. Sustaining aquaculture and restoring native oysters depends on oyster health. Food availability is a driver of oyster health that interacts with environmental effects of global climate change. These global changes may be altered by the effects of local species interactions like association with eelgrass. Measuring essential fatty acids, which are necessary for survival and physiological processes of oysters, can show the level of food availability to oysters in environments with and without eelgrass. We tested the hypothesis that eelgrass alters oyster health by growing oysters inside and outside of eelgrass at 5 sites in Washington state. We used fatty acid composition of tissue from Pacific and Olympia oysters to look at changes of assimilated food in relation to habitat and environment. We predict that eelgrass will slow water flow, resulting in a decrease in food availability and thus essential fatty acid concentration in oysters grown in eelgrass. Looking at essential fatty acids of oysters from different environments, specifically with or without eelgrass, provides a better understanding of environmental impacts on oyster health and contributes to a collaborative effort in Washington state focused on sustaining the valuable food production and ecosystem services of oysters in our ever-changing climate.

Interactions between HIV-1 and dendritic cells (DCs) have been suggested to play a role in HIV-1 pathogenesis. DCs are antigen presenting cells that take up, process, and present foreign material to T-cells. Previous research suggests that HIV-1 can exploit this process by binding to DCs, allowing HIV-1 virions to relocate to lymph nodes where infection of T-cells can occur. HIV-1 mediates this interaction through binding of the HIV-1 envelope glycoprotein (Env) to DC-SIGN, a glycan binding protein located on the surface of DCs. Env contains a high amount of oligomannose glycans, enabling Env to act as a binding partner for DC-SIGN. Due to high sequence variation, HIV-1 strains exhibit variability in glycan presentation. A gap remains in our knowledge of where DC-SIGN binding occurs and how glycan presentation across various isolates modulates these interactions. In this study, we applied a combination of biophysical and structural approaches to characterize Env:DC-SIGN interactions across multiple HIV-1 strains. Biolayer interferometry experiments measuring binding affinity demonstrate that DC-SIGN binds with strong affinity towards Env from different isolates. This suggests that DC-SIGN is capable of recognizing different oligomannose glycan presentations and may not have a well defined epitope. However, preliminary results using electron microscopy illustrate that Env glycoprotein structure can be disrupted and altered via binding to DC-SIGN. This appears to be an isolate-specific response, as some strains remain unchanged. Using single particle approaches, we identified potential epitopes in heavily glycosylated regions of Env where additional densities are attributed to DC-SIGN. We infer that in addition to binding HIV Env, DC-SIGN may shield key neutralizing epitopes within these glycosylated regions. Our results provide further insight into the interactions that occur between HIV Env and DC-SIGN, and suggest a mechanism where HIV-1 hijacks DC’s normal immune function allowing HIV-1 trafficking while shielding neutralizing epitopes.

The development of neurodegenerative diseases, such as Parkinson’s, Huntington’s, and Alzheimer’s affect millions of people every year. Previous studies have shown that an enzyme found in the endemic Herpes Simplex Virus (HSV-1); UL12.5, degrades the mitochondrial genome, which we hypothesize may predispose patients to neurodegenerative conditions. A majority of the human population is infected with HSV-1 and other herpesviridae, which encode similar enzymes, and their reactivation is commonly seen throughout life. We have found that the degradation of mitochondrial DNA by UL12.5 activity is widely conserved and when UL12.5 is expressed in the model organism, Caenorhabditis elegans (C.elegans), mitochondrial DNA content drops nearly 10-fold. Interestingly, we find that activation of the HSV-1 UL12.5 enzyme alters cellular proteostasis; the cell’s mechanism for maintaining properly folded and functional proteins. This change in proteostasis alters the aggregation of misfolded proteins, especially those involved in the cause of neurodegenerative diseases, and leads to neurological phenotypes like paralysis, which we can quantify in the worm. To study the enzymatic activity of the UL12.5, we used modified worms expressing the protein using an inducible transgene that can be verified by fluorescent microscopy. The effects of the UL12.5 activity is determined by tracking lifespan of worms, performing paralysis assays, and counting protein aggregates with controls that use a catalytically inactive version of UL12.5. We have also found that Emodin, a compound found in traditional Chinese medicine, which has previously been shown to inhibit UL12.5, alters the activity of UL12.5 in a worm model of Huntington’s disease. We hypothesize that activation of this enzyme may cause similar pathologies in the human brain and may be a causative factor in human neurodegenerative disease. Further, inhibitors of UL12.5 like Emodin may prove useful therapeutics for treating neurodegenerative diseases.

There are several approaches to treating Acute Myeloid Leukemia (AML) relapse after an allogeneic stem cell transplant. We were curious about the efficacy of the different types of treatments commonly deployed for patients with post-allograft AML relapse. Eligible patients included those who underwent allogeneic stem cell transplant for a diagnosis of AML and then relapsed between 1/1/2005 and 12/31/2015. We abstracted information about their treatments, GVHD status, immunosuppressive drugs, response (ie relapse or remission) events and vital status from their medical records. Among 120 patients, we found a median number of 2.6 and a mean of 2.9 relapse-directed lines of treatment, while 21% received no treatment. The types of treatment deployed were tightly correlated with the patient’s malignant blast count and the time that had elapsed since first transplant. There were 74 induction chemotherapy attempts, 64 hypomethylating therapy prescriptions, 19 donor lymphocyte infusions (DLI), and 16 second transplants, for example. Ongoing work includes completing data entry on the remaining 186 subjects, and analyzing the relative efficacy of the different treatment options. Importantly, we found that patients whose relapse presented with very low-level disease burden (<5% malignant blasts) still had a dismal prognosis, with only 20% being alive in remission at last follow-up. By contrast, patients relapsing with >5% blasts had an even worse prognosis – only 7% alive in remission at last follow-up. Closer surveillance for post-transplant relapse might catch more patients relapsing at low disease burdens. Initiating treatment when the disease burden is low may be more effective. Our ongoing work will identify the best sequences of treatments (leading to the longest relapse free survival), taking into account the blast burden, time elapsed since transplant, and other predictive factors.
 

Chemically induced dimerization (CID) systems, which are systems that involve the dimerization of two proteins only in the presence of a specific molecule, have shown a great potential in studying and controlling cellular signaling pathways. However, current methods for identifying CID systems for any given target based on small molecule-protein interactions remains a challenge in the area of protein design. Our research team proposes a general approach for identifying new CID systems for which may have clinical and therapeutic applications. To do this, we aim to create nanobody-based CID binders. Using a technique known as “Trinucleotide-Directed Mutagenesis,” our team has been able to create a diverse nanobody library of over 109. Phage display allows us to screen this library for specific nanobodies that can bind with high specificity and affinity to a specific molecule. Currently, we are using cannabidiol (CBD) as a target molecule. After performing several rounds of selection, in order to validate our system’s specificity and selectivity, we use ELISA assay tests as well as Bio-Layer Interferometry to measure the binding affinity and binding rate of our system. In order to ensure the specificity of our CID system we tested it against, tetrahydrocannabinol (THC), a structural homolog. To date, we have successfully found three different nanobodies that can bind with high affinity and specificity to our small molecule of choice. As we move into phase two of our project, we are currently working on identifying the second nanobody in order to form a complete CID system. Designed CID systems will not only provide personalized choices for patients showing different drug responses, but also serve as orthogonal chemical control of different cell subpopulations and cell behaviors to improve therapeutic efficacy and safety.

Studies have shown that both Notch signaling and the transcription factor Sox2 are involved in development of hair cells and support cells in the inner ear, which are essential for hearing and balance. To determine when and where the Notch signaling pathway and Sox2 interact in cells of the cochlea, we observed location of Sox2 and the Notch pathway proteins, Rbpj (transcription factor) and Jagged 1 (ligand of Notch receptors), in mice cochlea at different developmental stages using immunofluorescence labeling. We have observed that Rbpj levels are possibly higher in the floor of the cochlear duct--where the prosensory cells (Jag1+/Sox2+) are--than in the roof of the cochlear duct. This could suggest activation of the Rbpj gene by Sox2 activity or vice versa. To determine whether Sox2 could activate the Rbpj gene or Rbpj could activate the Sox2 gene, we will predict Rbpj- and Sox2-DNA-binding motifs in the open chromatin near Sox2 and Rbpj genes using informatics and our lab’s ATAC-seq data. If Sox2 binding sites are found near the Rbpj gene, then that could suggest activation of Rbpj by Sox2. If Rbpj binding sites are found near the Sox2 gene, then that could suggest activation of Sox2 by Rbpj. These findings will help us understand how Sox2 and the Notch pathway interact to influence development of hair cells and support cells.

Developmental Epileptic Encephalopathy (DEE) is a severe form of epilepsy in children and infants characterized by prominent EEG (electroencephalography) abnormalities disrupting brain function. Identifying the genetic causes of DEE will help researchers develop medical treatments for affected patients. Approximately 5% of DEE cases involve a copy number variation (CNV), where a region of DNA has been duplicated or deleted in a gene, causing the disease. Historically, this type of mutation has been difficult to detect using sequence data. To analyze the large amount of sequence data available in the Mefford Lab, I am creating an algorithm that will analyze smMIP (single molecule molecular inversion probe) data to identify CNVs in patient DNA that are likely causal for DEE. The algorithm is a two-step process. First, z-scores are assigned to the number of smMIP reads at each target site of each patient sample to numerically assess how many standard deviations it is from the mean. Next, regions of contiguous significant z-scores will be extracted as areas of top interest to verify as being a CNV. I will run the completed algorithm with smMIP data from the Mefford Lab's unsolved DEE cases. Since CNVs have historically been difficult to detect, it is unclear what percent of these unsolved cases will contain a CNV. It might be that, due to previous lack of detection, CNVs play a more significant role in DEE than previously thought. The opposite might also be true if a large majority of unresolved cases are due to other types of mutations, e.g. point mutations. Regardless, the algorithm will shed light on new causative CNVs and allow the Mefford Lab to solve more patient cases. Additionally, a successful CNV calling algorithm can be shared with other labs to aid them in their research.

Atherosclerotic vascular disease and its resulting myocardial infarction (MI) is the leading cause of death in subjects with diabetes. Additionally, patients with a history of a MI are more likely to experience a recurrent MI, however the mechanism is not yet understood. Previous work from our laboratory, and others, has demonstrated that diabetes accelerates atherosclerosis partly by affecting monocytes and macrophages, resulting in increased inflammation and macrophage accumulation in the artery wall. Work from others has shown that experimental MI can also accelerate atherosclerosis in non-diabetic mice by increasing the number of blood monocytes, which enter the artery wall to become inflammatory macrophages. Thus, we wanted to ask if diabetes and experimental MI have synergistic effects on macrophage accumulation in the artery wall. Experimental MI, or sham surgeries, were carried out in an atherosclerotic-prone, transgenic mouse model of type 1 diabetes that relies on viral mimicry (Ldlr-/-Gp+) and in non-diabetic controls. At the end of the study, cross sections of the dissected brachiocephalic artery were used to quantify macrophage accumulation by measuring areas of Mac-2 immunoreactivity on sections adjacent to the largest cross-sectional lesion. If macrophage accumulation is synergistically increased with diabetes and experimental MI, attempts will be made to understand the underlying mechanism. Atherosclerotic macrophage accumulation is a dynamic process that involves monocyte recruitment and local proliferation. Macrophage proliferation and monocyte recruitment will be analyzed by staining with proliferation marker Ki67 and estimating the number of previously labeled monocytes that enter the atherosclerotic lesion, respectively. To mechanistically test the role of monocyte recruitment on macrophage accumulation, Mac-2 staining will be quantified in mice (diabetic and non-diabetic with and without MI) deficient in a key adhesion molecule (CD49D) required for monocyte adhesion to activated endothelium. These results should yield important information on potential synergistic effects of diabetes and MI.

Connecting patients to the appropriate health or social service intervention is critical for their long-term health and well being. The 911 system is increasingly being utilized for non-emergency medical situations that originate with non-acute health issues and poor health care access. This diverts resources away from the core mission of fire departments which is to respond to emergencies in a timely manner. This study utilized data from South King County’s 911 dispatch center, Valley Communications, and the American Community Survey to identify socioeconomic factors that may correlate with non-emergent 911 responses. Ultimately, this demographic study identified areas with high primary health needs that are ineffectively addressed and characterized their demographic profiles. Of particular interest are high frequency non-emergent callers that rely on the 911 system for chronic and reoccurring health issues. This information was mapped and related to health care facility locations to determine how health access played a role in this behavior. Researchers and policymakers can expand upon the conclusions of this study to create more targeted, effective, and efficient alternatives to the emergency medical system that can serve regions with the greatest health access and health delivery needs.

 Developmental and epileptic encephalopathies (DEEs) are an early-onset form of epilepsy that result in aggressive and recurring seizures, possibly leading to severe cognitive and developmental impairment. While most genetic variants that cause DEE reside in the coding regions of genes, splice site variants can also be pathogenic. Splice site variants are changes in the DNA close to, or on, the exon-intron boundary, which can cause aberrant splicing, resulting in exon exclusion or intron inclusion within spliced RNA, generating a protein that is non-functional, partially functional, or aberrantly expressed. If aberrant splicing occurs, it could have pathogenic consequences, but predicting which variants near splice sites will have an effect on splicing is difficult. This raises the need to investigate splice-site variants. My work is concentrated on potential splice site variants in three genes associated with DEE: SYNGAP1, SCN1B, and WWOX. SCN1B codes for voltage-gated channels within the brain, SYNGAP1 is associated with postsynaptic signaling, and WWOX acts as a tumor suppressor and is vital in brain development. DEE genes are primarily expressed in the brain, a tissue that is unavailable from most patients, making splicing studies in these genes difficult. I was able to successfully amplify targeted regions of SCN1B, SYNGAP1, and WWOX in RNA isolated from blood and fibroblast, demonstrating the efficacy of using these more easily accessible tissues in my studies. The lab has now collected fibroblast samples from DEE patients with potential splice site variants in WWOX and SCN1B. I will use RNA extracted from these fibroblasts to confirm whether the splice site variants in WWOX and SCN1B cause aberrant splicing, and what the specific consequences of this aberrant splicing is on the protein-coding sequence of these genes. In the future, we also plan on collecting fibroblasts samples with variants from SYNGAP1 as well.

As the average population lifespan increases in many countries, study into age-associated diseases and the basic biology of aging has become even more important. Studying age-associated changes and lifespan-altering genes in the budding yeast has revealed fundamental insights into the aging process. To measure replicative lifespan of budding yeast cells, we image hundreds of isolated yeast cells trapped in a microfluidic device over the aging process. Using fluorescently labeled strains allows the measurement of protein expression and localization during aging.We observe that Msn2, a general stress-response transcription factor, becomes increasingly activated with age in the budding yeast. Paradoxically, knockout of Msn2 and its homolog Msn4 results in increased lifespan, indicating that the Msn2-driven transcriptional stress response is detrimental to longevity. This effect is mediated by the inappropriate upregulation of a cohort of genes associated with the glucose starvation response despite replete glucose conditions. Deletion of these genes—glucokinase (Glk1), phosphoglucomutase (Pgm2), and glycogen synthase (Gsy1)—also results in increased lifespan. These genes are associated with the accumulation of glycogen during glucose starvation, and by staining old cells trapped in our microfluidic device, we find that glycogen content increases with age. We see that overexpression of the glycogen catabolism gene glycogen phosphorylase (Gph1) increases lifespan, indicating that the mechanisms underlying the detrimental effects of the Msn2-driven age-associated transcriptional program may be driven, at least in part, by the build-up of glycogen in aged cells. Accumulated glycogen is seen in the aged cells of a number of evolutionarily distant species including bacteria and human brain tissue and is implicated in multiple human diseases. Thus, our work may elucidate the details of a fundamental frailty of the metabolic network during aging.

Diabetes is increasingly prevalent in the world, and there are currently over 29 million people in the United States with diabetes. Type 1 diabetic patients depend on insulin injections to maintain their blood insulin within a certain level so that their cells are getting enough sugar for energy production. Type 2 diabetic patients mostly rely on diet treatments and combinations of insulin and other drug treatments to avoid hyperglycemia. Moreover, type 2 diabetes often leads to different complications such as cardiovascular disease, blindness, and kidney failure. The goal of our study is to investigate insulin effects on the activation of macrophages, which are immune cells with important roles in inflammation. Since a pro-inflammatory state of macrophages can enhance insulin resistance and lead to cardiovascular diseases, it is crucial to understand if insulin changes the macrophage activation states that can cause off target effects leading to other medical complications. The experiments for this project are performed using RAW 264.7 cells, an immortal cell line derived from mouse macrophages. A dose experiment of insulin stimulation was performed to test if phosphorylated STAT1 (pro-inflammatory or M1) and phosphorylated STAT6 (anti-inflammatory or M2) were detected via western blot. Additional experiments determined the intensity of the macrophage response by measuring downstream signaling proteins (PI3K or IRS2) via immunoprecipitation and western blot. Also, RNA extraction and qPCR were performed to see if the gene transcription of macrophage polarization is activated by the above transcription factors. For example,  TNFα and IFNγ were measured for M1 polarization and IL-4 and IL-10 were measured for M2 polarization. Finally, NF-κB, another transcription factor was measured to determine if phosphorylation occurred, which should lead to M1 gene transcription. Together, these experiments revealed whether insulin affects key pro-inflammatory and anti-inflammatory signaling pathways in macrophages.

In recent years, researchers have dedicated much effort to overcoming the ~250nm spatial resolution limit of light in order to reveal biological details that have been obscured by diffraction. A new form of super-resolution microscopy called expansion microscopy (ExM) has enabled ~65nm image resolution or better by physically expanding fixed specimens in a swellable hydrogel polymer. While ExM allows researchers to achieve high resolution with standard microscopes, the technique requires the use of water-immersion lenses, rather than higher-resolution oil-immersion objective lenses, when imaging more ~5 µm deep due to spherical aberrations caused by refractive index mismatch with oil lenses and water-based hydrogels (n=1.5 vs n=1.3). Oil-immersion lenses also collect more signal than water-immersion lenses, enabling higher signal to noise with oil-immersion lenses under the same excitation conditions. I have developed an index-matching technique that significantly reduces spherical aberrations when imaging with oil lenses and expanded specimens, facilitating improved resolution (~1.12x lateral improvement and 1.5x axial improvement) for depths of up to 25 μm. Expanded specimens are index-matched by simply incubating the specimen in a high-index iohexol solution which equilibrates with the specimen in approximately 18 hours. A modest ~17.5% shrinkage of the hydrogel is produced as a result in the current implementation, which I can in principle compensate for by tuning the hydrogel recipe to achieve a slightly higher expansion factor. I found that the iohexol solution on its own accelerated the rate of fluorophore bleaching and led to some fluorophore quenching. However, the addition of the triplet-state quencher n-propyl-gallate effectively mitigated this chemical bleaching. Ultimately, this iohexol-facilitated index-matching procedure not only combines the utility of ExM and the high collection efficiency of oil immersion objectives but allows researchers to use common chemicals and readily available instruments to obtain images that reveal a previously inaccessible wealth of information.

Previous research on JNK-mediated stress signals demonstrated that stem cells in the posterior midgut of Drosophila Melanogaster only undergo compensatory proliferation or apoptosis. However, our group discovered that the stem cells can also undergo the process of basal extrusion and dissemination, resulting in the cells being eliminated from the tissue into the hemocoel, the blood containing intertissue body cavity. The JNK signal promotes the cells to exit the epithelium of the gut, move through the muscle layer, and be released to the hemocoel, which resembles the process of metastasis in human cancer. In order to understand the mechanism of this extrusion process, we carried out an RNA Interference (RNAi) screen and sought to find genes that are necessary for the stem cell extrusion in the JNK activator, HepCA, expressed flies. We used the ESG-GAL4, UAS-GFP, TUB-GAL80TS(EGT) genetic system to study the knockdown effect from the RNAi of each gene. We selected 215 lines of kinases and phosphatases of flies to test if the knocked-down gene results in suppression of cell elimination by the JNK stress signal. After 4 days of inducement, which is the sufficient time for the JNK signal can promote complete extrusion of the intestinal stem cells, the intestines were dissected, fixed, mounted, and examined with a fluorescent microscope for any presence of stem cells. Through this screen, we could find 33 lines that had strong suppression of cell elimination, 39 lines that had a moderate effect. Our results suggest that the knocked down genes with strong suppression of cell elimination are involved in the mechanism of basal cell extrusion, and future research dictates an investigation into the molecular function of each of these genes.

Prior research shows adolescents’ negative pain memories predict greater subsequent postsurgical pain. However, understanding of the content of memories and whether memories change over time is limited. We applied emotional stress theory, which proposes that negatively perceived events are recalled with more central (relevant) than peripheral (irrelevant) details, to examine how post-surgical pain memories change in adolescents and their parents. We hypothesized that 1) central memories would contain more negative emotion than peripheral memories, 2) ratio of peripheral to central memories would increase over time as memories become less negative, and 3) parent and adolescent memories would be associated at each time-point. Nineteen adolescents (12-18 years;63% female) who underwent major surgery and their parents were asked individually at 2-months and 12-months after surgery to recall “everything you remember” about being in the hospital and the initial weeks at home following surgery. Linguistic Inquiry and Word Count software was used to identify negative emotion words and N-Vivo software was used to code central and peripheral memories. Central memories included specific (“going through the doorway was…sheer pain”) and general (“kinda got better”) memories of post-surgical recovery. Peripheral memories ranged from inside (“clowns…visited me”) to outside details (“dad split his finger open”). As expected, central memories contained more negative emotion words than peripheral memories (p’s<.05). Use of negative words and ratio of peripheral to central memories were stable over time for parents and adolescents. In regression analyses, controlling for adolescent age, parent peripheral to central memory ratio was significantly associated with the adolescent peripheral to central memory ratio at 12-months (β=.57;p=.02), but not 2-months (β=.04;p=.88) after surgery. Findings suggest that memories are stable after two months. Associations within parent-child dyads were stronger at later follow-up. Future studies should assess earlier memories to increase knowledge of temporal association between memories and subsequent pain experiences.

Brain tumors are the most common form of pediatric cancers after leukemia. Deregulation of the family of MYC (avian myelocytomatosis viral oncogene homolog) transcription factors in some forms of pediatric brain cancer correlates with very poor prognosis. It is believed that overexpression of MYC may play a role in sustaining aggressive brain tumor cell populations in diffuse intrinsic pontine glioma (DIPG), medulloblastoma, and atypical teratoid/rhabdoid (AT/RT) pediatric brain tumor types. A targeted therapy capable of inhibiting MYC transcription factors may be a promising option for children with MYC-upregulated brain tumors. We addressed the role of MYC genes in the tumorigenesis of several patient-derived pediatric cell lines with upregulation of MYC transcription factors. Via a lentiviral delivery agent, we attempted to knock down MYC transcription factions through the expression of doxycycline induced omomyc—a 90 amino acid, MYC-derived, polypeptide that interferes with normal MYC activity by outcompeting for the enhancer binding sites that MYC normally binds to. We found that MYC inhibition, using omomyc, halted the growth of tumor cells in over half of the patient-derived primary cell cultures with MYC overexpression. 6 out of 11 lines showed significant inhibition of tumorigenesis through the induction of omomyc. This suggests that the mechanism of action of omomyc, as a MYC inhibitor, may serve as a model for drug development against MYC for pediatric brain cancer.

Rett Syndrome (RTT) is a neurodevelopmental disorder that is first recognized in infancy and almost exclusively affects females. The syndrome is characterized by normal development in the first 6 to 18 months of life, followed by a regression of developmental milestones, particularly the loss of mobility skills and purposeful use of the hands. Additional features of RTT include severe disturbances in cardiac and respiratory functions. These are characterized by prolonged QT intervals as well as increased respiratory rate and occurrence of abnormal respiratory events such as breath holds. RTT results from mutations in the gene encoding the protein MECP2, which helps to regulate gene activity. The location and type of the MECP2 gene mutation influence the course and severity of the syndrome. Previous animal studies and clinical case reports have suggested that ketamine administered at a low dosage can reduce deficits in brain activity and improve neurological function in RTT. In this pilot study, we sought to determine the efficacy of ketamine in reducing the respiratory and cardiac phenotype observed in RTT. Electrocardiography (ECG) and respiration patterns were recorded before and after treatments of four RTT patients with low doses of ketamine. Examination of these recordings showed that ketamine decreases heart rate variability, respiratory rate, and the number of abnormal respiratory events within the 20 hours post drug administration. These results suggest that low-dose ketamine treatment may potentially serve as an effective future treatment for the cardiac and respiratory symptoms of RTT.

Familial Platelet Disorder with Predisposition for Acute Myeloid Leukemia (FPD/AML) is an autosomal dominant human disorder caused by germline, heterozygous mutations in RUNX1. RUNX1 is a master regulator of hematopoiesis and has specific roles in megakaryocyte maturation and the production of platelets. Monoalellic mutations in RUNX1 result in haploinsufficiency of RUNX1 protein, leading to thrombocytopenia and impaired platelet function before leukemic transformation later in life. RUNX1 is controlled through direct positive auto-activation and its half-life is tightly regulated through ubiquitin-mediated proteasomal degradation. Tightly regulated auto-regulatory circuits are known to have capacity as centers for epigenetic cellular memory. Cellular memory effect offers the opportunity to transiently manipulate steady state product levels and observe maintenance that continues the newly attained levels.The goal of this work is to determine if the positive feedback loop controlling RUNX1 expression has capacity for epigenetic cellular memory that is responsive to transient RUNX1 overexpression. We hypothesize that increasing the presence of endogenous RUNX1 in RUNX1-deficient cells will correct megakaryocyte maturation and platelet formation. To initially determine if an increase in the half-life of RUNX1 will correct megakaryopoiesis, a series of ubiquitylnation and proteasome inhibitors were administered to HEK 293t cells for 6hr, 12hr, and 24hr periods to determine their effect of RUNX1 production and stability, showing increases in RUNX1 expression at RNA and protein levels. iPSC's derived from FPD/AML patients will be ultimately differentiated and analyzed via flow cytometry to observe the inhibitors' effects on platelet production. Anticipated results from this work hope to confirm a dose-dependant response in the epigenetic programming of RUNX1 that could illuminate novel therapies for individuals with FPD/AML.

Aging is a phenomenon that brings about the onset of many diseases and increases mortality. Understanding aging mechanisms can help us increase longevity and delay the onset of multiple chronic diseases. It has been previously observed that dietary restriction is a method that increases lifespan and delays the loss of function caused by age-associated pathologies. We used the roundworm, Caenhorbabditis elegans, to dissect the mechanisms of dietary restriction-induced longevity. Specifically, we focused on the phenomenon of adult reproductive diapause (ARD). ARD is induced when pre-reproductive juvenile larvae are subjected to starvation. Upon reintroduction to food after prolonged starvation, ARD worms undergo a morphological rejuvenation and resume a normal lifespan. Mechanisms that control post-diapause recovery are still quite unknown. We have found that the TGF-β/BMP signaling pathway is required for post ARD rejuvenation. The TGF-beta signaling pathway is a critical factor for growth processes including cell growth, differentiation, embryonic development, and much more. We aimed to characterize the role of this signaling pathway in ARD maintenance and post-ARD recovery. We hypothesized that TGF-β/BMP mutants accumulate excessive amounts of cellular damage during ARD and are unable to recover from ARD. To address this hypothesis, we analyzed markers of cellular aging among diapaused animals using fluorescent microscopy, in both wild type and TGF-β/BMP mutants. Using transgenic fluorescent reporter strains we examined pH, nucleolar morphology, mitochondria, and cytoskeleton and nucleoskeleton integrity for aging-associated phenotypes. These insights can help us understand the role of the conserved TGF-β/BMP pathway in dietary restriction induced longevity.

Aging is a major risk factor for the most common causes of death in the developed world which include cancer, heart disease, and neurological disorders. Because of this, therapies that target the hallmarks of aging are a promising area of study. The mechanistic target of rapamycin (mTOR) kinase is a major regulator of growth, aging, and survival in cells. Inhibition of the mTOR signaling pathway is associated with increased longevity in organisms including yeast, nematode worms, fruit flies, and mice. Inhibition of mTOR can be achieved through pharmacologic intervention, which is a promising strategy to extend healthy lifespan. To better understand how mTOR inhibitor response varies between genetically-diverse populations, we utilized a set of 90 wild and domesticated yeast strains. We tested a set of mTOR inhibitory compounds on these strains to identify strains with increased sensitivity or resistance to mTOR inhibition. We have identified genetic variants that drive drug sensitivity or resistance in these strains. We then engineered mutations into laboratory strains to confirm that these mutations recapitulate the phenotype of interest. By understanding drug response across genetically-diverse populations, we will create the framework for a precision medicine approach to developing healthspan-enhancing compounds that can be translated to humans.

Understanding how extracellular environments facilitate the differentiation of stem cells holds much promise for the field of regenerative medicine. Non-muscle myosin II (NMII) plays an important role in cell morphology and also generates forces that alfter biochemical signalling. It has been shown that the activity of NMII controls mechanoreceptors and integrins facilitating stem cell fate. More specifically, mechanotransduction of transcriptional coactivators YAP/TAZ facilitate differentiation of mesenchymal stem cells (MSCs). YAP and TAZ are responsible for upregulating genes associated with stem cell differentiation. Under external stresses mesenchymal stem cells differentiate into osteocytes whereas softer extracellular conditions favor the differentiation into adipocytes through mechanotransduction of YAP and TAZ.To further explore the role of physical forces determining the fate of stem cells, we used a computationally designed self-assembling homo-polymer called 2E01, generated by the Institute for Protein Design (IPD). Mechanical stress and the physical activity of NMII can be mimicked by controlled expression of the length of the E01 protein fiber. We introduced the 2E01 gene under a doxycycline-inducible promoter into the AAVS1 "safe harbor" locus of the induced pluripotent stem cells (iPSC). Expression of the E01 fiber in iPSCs lead to changes in colony morphology and subsequent differentiation of those cells, suggesting that changing the morphology of cells can change their fate. I want to see if I can use the 2E01 fiber as a tool to accelerate differentiation from IPSCs to neuronal cells and cardiomyocytes. In addition, we expressed 2E01 in mesenchymal stem cells, a cell type currently used in lab, and observed the activity of YAP and TAZ as an indicator of differentiation. Utilization of the 2E01 fiber could be benefical to the field of regenerative medicine as a directed agent of physical manipulation towards desired stem cell fate.

Bisphenol A (BPA) is a lipophilic toxin and an endocrine disruptor that has been shown to interrupt normal hormonal functions in invertebrates. BPA can bioaccumulate in filter feeders like marine mussels, which are both economically and environmentally important species. Previous studies have shown that mussels are sentinel organisms because they can detect toxin contamination in their aquatic environment by producing fewer byssal threads. Byssal threads are necessary for their survival in tidal zones as well as in suspension-culture mussel farming. It was hypothesized that when exposed to high concentrations of BPA, the mussel Mytilus trossulus would produce fewer byssal threads and have higher concentrations of BPA in their tissues. Using a single-subject design, 32 mussels were placed in seawater microcosms for 24 hours and exposed to different BPA concentrations (control and 1-9 ppm BPA). Their byssal threads were counted afterwards. High performance liquid chromatography-photodiode array (HPLC-PDA) analysis was used to determine the BPA concentrations in both the mussel tissue samples and the microcosm seawater. Mussels exposed to high concentrations of BPA produced significantly fewer byssal threads and had higher concentrations of BPA in their tissues compared to the controls. BPA’s lipophilicity allows it to bind to food particulates and mussel gills, resulting in the compound to bioaccumulate in the organism. Without byssal threads, mussels exposed to BPA would experience high mortality in tidal zones as they are more easily dislodged by waves and predators. There would also be massive economic losses in the shellfish aquaculture industry that use suspension-culture farming.

Primary sclerosing cholangitis (PSC) is a rare liver disease associated with inflammatory bowel disease (IBD) that causes strictures of the biliary tree and could result in the need for liver transplantation. One disease mechanism is upregulation of the mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) protein and subsequent increased lymphocyte migration to the biliary epithelium. The purpose of this research project is to compare the levels of enzymes (especially alkaline phosphatase, ALP) that represent surrogate markers of liver inflammation in PSC patients before and after infusion of vedolizumab (VDZ), a drug to treat IBD that interferes with this lymphocyte migration. We conducted a retrospective observational study to collect information on PSC patients from their electronic medical records and enter it into Excel. Information collected before and after VDZ infusion included: demographics, medical and social history, laboratory blood tests, information from first clinical encounters, and histology and radiology reports (e.g. ERCP, CT, MRI, endoscopies). Subsequently, we wrote a script in R to import the dataset, visualize the enzyme levels before and after VDZ infusion, and perform statistical inference using a matched-pairs t-test to determine if there was a significant difference in mean ALP levels in PSC patients before and after VDZ infusion. We obtained the following results (n = 13, α = 0.05): mean ALP difference = 10.67 U/L, t = 0.23, df = 12, p = 0.82, 95% CI = (-88.41, 109.76). Since our p-value was greater than 0.05 (and our confidence interval contained the value 0), we failed to reject our null hypothesis and could not conclude that there was a significant difference in mean ALP levels before and after VDZ infusion. Thus, more information is needed to determine if VDZ may have a beneficial effect on liver function.

Rhabdomyosarcoma (RMS) is an aggressive malignant pediatric cancer characterized by pathological skeletal muscle development. Due to a small accumulation of genetic mutations, RMS tumor progression is believed to be driven by epigenetic regulators, which modify chromatin structures to control gene expression. Through gene expression and knockout experiments, our lab identified components of the Switch/Sucrose non-fermentable (SWI/SNF) complex as being potentially involved in RMS growth. SWI/SNF is a family of chromatin remodeling complexes that may function in histone binding and chromatin organization to regulate gene expression. To identify any possible interactions and understand the role SWI/SNF genes in RMS, we developed a large-scale multiplex CRISPR/Cas9 genetic screening system that targets 50 epigenetic regulators, including all the SWI/SNF components. These genes are targeted individually and in every possible two-gene combination. CRISPR, a gene editing technology which uses guide RNA (gRNA) sequences to direct double-stranded cuts in DNA, allows us to knock out genes in cells to gain insight into their function. I have made the CRISPR targeting DNA constructs for the SWI/SNF genes to be included in the library of epigenetic regulators for the genetic screen. We are currently introducing the CRISPR library into human RMS cells to assess the effects of all possible combinations of single-gene and dual gene-knockouts on tumor cell growth. Using this large-scale multiplex genetic interaction screen, we hope to identify genes and gene combinations that are essential to the growth of RMS cells. If two genes have cooperative or redundant function in promoting RMS tumor growth, a dual-gene knockout is expected to result in a more significant reduction in cell growth compared to targeted disruption of either gene alone. The study will provide valuable insight into genetic interactions among key epigenetic regulators in RMS and potentially identify novel therapeutic targets for the treatment of RMS.

Zika is a virus in the Flaviviridae family, along with other established viruses such as Dengue virus and West Nile virus. These viruses have been shown to infect humans, often with dire consequences. Zika, specifically, has been linked to fetal microcephaly as a consequence of infection during pregnancy. While the mechanism Flaviviruses employ upon infecting cells has been researched, many details of this process remain unknown, especially for Zika. Flaviviruses cause endoplasmic reticulum stress in cells, which in turn elicits the cellular unfolded protein response. This is a common cellular response to Flavivirus infection and provided the basis for the experiments conducted in this research. One of the components of the unfolded protein response is the activation of IRE1, which is an enzyme in the membrane of the endoplasmic reticulum that is activated by endoplasmic reticulum stress. Activated IRE1 functions as a nuclease, which is responsible for processing the mRNA encoding a transcription factor called XBP1. IRE1 elicits a cascade which results in splicing of the mRNA encoding this transcription factor and leads to the expression of a variety of downstream gene products. The mRNA of these genes can be measured through laboratory techniques such as quantitative PCR. Our research goal was to identify whether Zika virus activates this cascade, and whether its activation aids in viral replication. Our experiments have shown that spliced XBP1 mRNA is present in increased levels in Zika virus infected cells. Our experiments have also detected the induction of XBP1 target genes, suggesting that IRE1 activation is present in cells infected with Zika virus. Additionally, we have found decreased levels of Zika virus RNA in cells which are genetic knockouts for the enzymatic activity of IRE1. These data suggest that IRE1 is a potential drug target for suppressing viral replication.

In humans, CYP2J2 is involved in both drug metabolism and converting arachidonic acid to epoxyeicosatrienoic acids (EETs), which then act as signaling molecules. EETs are involved in angiogenesis, vasodilation, opening of ion channels, and general protection against cardiotoxicity. Currently, the Totah lab is testing the hypothesis that CYP2J2 has a critical role in the heart as one of the main contributors to EETs production. This research project seeks to determine whether CYP2J2 regulation is similar among different tissues, specifically between the heart and the liver, under oxidative stress conditions. A critical function in the heart would mean that under times of stress, CYP2J2 expression will increase. In contrast, CYP2J2 expression in the liver is expected to decrease similar to other CYP proteins under oxidative stress. To test this, reagents that increase reactive oxygen species (ROS) levels have been incubated with hepatic cells (HepG2) and the levels of CYP2J2 mRNA were measured and compared to untreated control cells. In the heart, CYP2J2 has a more important function in creating protective EETs and the benefits from increasing production of EETs have been shown in our lab to protect against the harmful effects of ROS. A few of the conditions being tested are: varying concentrations of hydrogen peroxide, cobalt chloride, and hypoxic conditions. MTT, a tetrazolium based dye, assays were used to quantify cell viability and rt-PCR was used to determine gene upregulation or downregulation of CYP2J2. Afterwards, the results were compared to similar treatments and conditions in cardiomyocytes. If transcription in HepG2 cells is downregulated but increased in cardiomyocytes, it supports the hypothesis that CYP2J2 has a critical role in the heart compared to the liver, specifically under times of ROS stress.

Eukaryotic ribosome biogenesis is an essential and energetically demanding cellular process that must be tightly regulated. In S. cerevisiae, this regulation is partially achieved via ubiquitination of RPA190, the largest subunit of RNA polymerase I, and further mediated by the deubiquitinating enzyme (DUB) Ubp10. Human USP36, the ortholog of Ubp10, rescues RPA190 stability in ubp10 delta yeast cells, indicating DUB activity may be conserved across eukaryotes. As such, there is considerable interest in deciphering the mechanism by which RPA190 and human RPA194 are ubiquitinated. Previous work identified three lysines (K405, K408, and K410) as possible ubiquitination sites. However, whether one or a combination of these lysines are ubiquitinated has yet to be determined. We created single lysine to arginine mutant RPA190 plasmids for each site and transformed them into rpa190 delta cells. These constructs may be used to help determine which lysine(s) are targeted by Ubp10 and whether those site(s) are functionally analogous in RPA194. This will yield a more complete understanding of the importance of DUB activity in the context of eukaryotic ribosome biogenesis.

Viruses are nonliving entities and therefore lack their own metabolism, but they do have the ability to alter the host cell's metabolic pathways for their own gain. Kaposi's Sarcoma-associated herpesvirus (KSHV), a human herpesvirus, is the etiological agent of Kaposi's Sarcoma (KS), the most common tumor in AIDS patients worldwide. In KS tumors and cultured endothelial cells, KSHV is predominantly in the latent state, with only a small portion of viral genes and a microRNA cluster expressed, and no virion assembly. Our lab has shown that KSHV dramatically activates many host cell metabolic pathways during latent infection, including glycolysis. A global metabolomics screen suggested that glycolytic intermediates might be shuttled through the pentose phosphate pathway, (PPP) during KSHV infection. Our hypothesis is that the PPP is important for viral perseverance and the survival of human endothelial cells latently infected with KSHV. Infected endothelial cells were treated with the glucose-6-phosphate dehydrogenase (G6PD) inhibitor 6-aminonictotinamide (6-AN) at different concentrations and the amount of cell death was quantified. The inhibition of the PPP by 6-AN significantly increases cell death in KSHV infected TIME cells, but not in matched uninfected cells. KSHV microRNAs are expressed during latent infection and have been shown to be sufficient to induce glycolysis. Interestingly, a KSHV mutant, ∆miR, which lacks the microRNA cluster is less sensitive to inhibition of the PPP. This suggests that the microRNA cluster may be responsible for the reliance of KSHV infected endothelial cells on the PPP. The data suggest that inhibitors of the PPP provide novel therapeutic agents for KS tumors.

Palatal shelf elevation is an essential morphogenetic process during closure of the secondary palate. It has been proposed that palatal shelf elevation results from an intrinsic elevating force while also relying on extrinsic factors associated with development of other orofacial structures, such as the lower jaw and tongue. While in vitro palate culture is commonly utilized to study palate shelf elevation, it requires removal of the tongue or mandible prior to culture, which prevents any determination of the role of extrinsic factors in regulating palatal shelf elevation. Therefore, the aim of our project was to improve upon palate culture methods to better mimic in vivo conditions of palate development for studying the mechanism of palatal shelf elevation. We modified culture conditions to maintain the integrity of the oral cavity by leaving the tongue and mandible in place and cultured these mouse palate explants in suspension in dynamic conditions. Using this technique, the palatal shelves successfully elevated after 24-48 hour culture and displayed morphology like that of in vivo elevation, suggesting that our method improves upon previous culture techniques for investigation of the roles of both intrinsic and extrinsic factors during secondary palate development.

Conventional farming techniques involve large quantities of chemical fertilizers, which often leach into bodies of water causing eutrophication. The influx of excess nutrients from fertilizer results in rapid increase of aquatic algal populations followed by dissolved oxygen depletion. This process creates regions of low oxygen that negatively impact the water quality of many major lakes and coastal regions. To address this problem, the sensitivity of plant growth-promoting bacteria (PGPB) to excess fertilizer was investigated. We hypothesize that the addition of high amounts of fertilizer will result in smaller, less productive plants and diminished rhizosphere colonization of the PGPB, Bacillus subtilis. A modified Kirby-Bauer disk diffusion test was performed as an initial assessment of the effect of varied fertilizer concentrations on B. subtilis. Roots of romaine lettuce seedlings were then inoculated with B. subtilis and grown in soil treated with the same range of fertilizer concentrations. The effects of each treatment on plant growth were determined using total leaf area, quantification of the rhizosphere colonization by B. subtilis, and carbon assimilation measured with a LI-6800 Portable Photosynthesis System. These results are an important step towards establishing guidelines for appropriate application of agricultural fertilizer in order to mitigate the frequency and severity of eutrophication events in aquatic systems.

In the US more than 100 million people are living with diabetes or pre-diabetes. The economic burden caused by these conditions, including medical costs, is approximately $322 billion annually as of 2013. Conventionally, transgenic Escherichia coli has been the primary source of commercial insulin production, a process that requires extensive purification to ensure shelf stability and complete removal of contaminants. This study seeks to establish an alternative mode of insulin production using polyethelyne glycol (PEG) to transform the oyster mushroom, Pleurotus ostreatus, with the human insulin gene. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins and fully sequenced genome. P. ostreatus was transformed using PEG with a plasmid containing the human insulin gene and a carboxin resistance gene. Transformed cells were selected using carboxin, extracted, and regenerated on plates composed of yeast extract, malt extract, and glucose (YMG). Integration of the human insulin gene in to the mushroom genome was confirmed through PCR analysis of the transformants. Successful PEG transformation of P.ostreatus offers a new avenue for insulin production, potentially diversifying the market and treatment options for diabetics.

Leigh syndrome is a severe pediatric mitochondrial disease for which no effective treatments currently exist. One of the defining features of Leigh syndrome is the appearance of progressive necrotic lesions in the brain, specifically in the brainstem, cerebellum, and olfactory bulb. In humans, the disease is caused by mutations in Ndufs4, a subunit of the mitochondrial electron transport chain complex I. However, lesion development is not directly linked to impaired mitochondrial function. At the Seattle Children’s Research Institute, I am assisting as an undergraduate researcher in a project aimed at uncovering the pathogenesis of these brain lesions. Homozygous loss of Ndufs4 in mice closely resembles the human disease, making mice a valuable model for study. For my project, I have been longitudinally tracking circulating metabolites in knockout and control mice, measuring bulk tissue metabolite levels via biochemical assays and examining brain slices using immunological and histological staining techniques to analyze glycogen (stored glucose) and lipid localization in lesion areas. Using these techniques, we have identified a developmental shift in circulating metabolites at the age where Leigh syndrome mice first develop disease (around post-natal day 35). Starting at this age, glycogen and lipid droplets accumulate at lesion sites, co-localizing in regions where loss of neuronal marker expression is evident. Because astrocytes (neuron supporting cells,) play a large role in neuron metabolism and survival, we also utilized fluorescent markers to assess the localization of accumulated astrocytes at areas of high lipid concentration. Using 3-D microscopy, we found that the smaller accumulated lipid droplets co-localize with astrocyte processes. Our findings support a role for altered circulating and tissue level metabolism in the pathogenesis of Leigh syndrome. This study is not only helpful in understanding the pathogenesis of Leigh syndrome, but also in linked metabolic and neurodegenerative diseases, leading us closer to effective treatments.

Diagnostic tests in mental health can often lead to inconclusive diagnoses, or diagnoses that take months to come to. The focus of this study is to identify neuropsychiatric conditions earlier and more accurately using voice analysis. Our study tested the ability of a speech analysis software to determine whether a patient suffers from a given mental illness, since mental illness can affect speech patterns. For example, one standard tested against in depression is a slower speaking rate. This study examined the relationship between voice as an indicator of those illnesses by analyzing short, random speech samples from patients with a diagnosed psychological or neurological condition. Our large team of interns was divided into several partnerships that focused on collecting samples of one specific disease. These samples were then analyzed for speech markers related to that condition by initially extracting audio, linguistic, and prosodic features and then using various statistical tools, mainly in python, to build predictive models using these features. These samples, along with controls from unafflicted patients, were then cross-referenced against an objective scale rating of the given condition, such as the PHQ-9 questionnaire for depression. The degree of correlation revealed by this analysis was used to determine if specific voice features were predictive of illness. We hope to make this software accessible to individual practitioners in a mobile application, with the potential to allow an individual practitioner to identify mental and neurological conditions much sooner, more accurately, and less invasively, allowing for earlier and more effective treatment.

Joubert syndrome (JS) is a rare recessive genetic disorder characterized by hindbrain malformations appearing as the “molar tooth sign” on axial brain imaging. Causal genetic variants for JS need to fulfill three criteria: They need to be 1) rare, 2) deleterious, and 3) present in both alleles in the patient. Patients in our cohort are sequenced for rare predicted-deleterious variants (RDVs) to identify variants that satisfy the first two criteria. To determine whether the segregation pattern of the variants satisfies the third criterion, we sequence both parents, each of whom should carry only one of the variants. My research uses Next-Generation Sequencing to determine the segregation pattern for families in whom RDVs have already been identified. Specifically, I performed targeted-sequencing of putative causal variants in the parents using molecular inversion probes, and then I used the sequencing data to determine the segregation pattern. In most families, one RDV will be present in each parent, indicating that they affect both alleles in the child, because these variants are robust candidates that have passed the filters for being rare and deleterious. If both RDVs are present in one parent, then they have to be on the same allele and cannot be the cause of JS in the family. In these families, we will have to consider other variants or perform additional sequencing to identify the causal variants. Ultimately, the goal of my research is to elucidate the genetic basis of JS to improve prognostic and risk recurrence information, provide diagnostic, carrier and prenatal testing, and delineate the biological mechanisms of JS that will be the targets of future precision therapies.

Human genetic variation is ubiquitous and its impacts are often not fully understood. The purpose of this study is to examine and catalog genetic variation in the specific context of the Fanconi Anemia disease family. Fanconi Anemia (FA) results from mutations in any of 22 FANC (Fanconi) genes. FA disease-associated pathogenic variants lead, in almost all instances, to loss of Fanconi protein expression and/or function: this is consistent with the recessive Mendelian inheritance of FA as a clinical disease phenotype. Recent work has identified aldehyde damage as an important driver of FA pathway function, and an important contributor to FA disease pathogenesis. Aldehydes are ubiquitous, potentially toxic molecules that are generated as part of normal cellular metabolism. Aldehyde damage is limited by the actions of aldehyde (ALDH) and alcohol (ADH) dehydrogenases, two gene families encoded by 26 different genes. To systematically characterize base pair-level pathogenic and human population-level genetic variation in the FANC, ADH and ALDH gene families, we built new databases of human genetic variation for clinically-ascertained FANC gene pathogenic variants (n = 1,015 variants), and for population genetic variants in the 22 FANC genes (n = 13,306 variants) and 26 ADH/ALDH genes (n = 8,388 variants). Population genetic variation was sourced from over 60,000 individuals included in the Exome Sequencing (ESP), the 1000 Genomes (1KGP) and the Exome Aggregation Consortium (ExAC) projects data. In order to interrogate potential pathogenicity we used CADD (Combined Annotation-Dependent Depletion) scoring, which provides a robust assessment of the predicted deleteriousness of base substitution and short indel variants by objectively integrating many diverse annotations into a single measure (C score) for each variant. This project tested the implementation of CADD scoring as a model and identified new potential pathogenic candidates in the ADH/ALDH gene families for future experimental confirmation.

Ca²⁺, or Calcium ions, are very important signaling molecules that are essential to normal eyesight. A light-sensitive layer in the eyes, called the retina, contain photoreceptors which require proper concentrations of Ca²⁺ for correct function. Mitochondria in the retina may help store Ca²⁺. To transport Ca²⁺, the mitochondria rely on Mitochondrial Calcium Uniporter proteins (MCU) and Mitochondrial Calcium Uptake 1 regulators (Micu1). This study hypothesizes that the Micu1 to MCU ratio influences the levels of Ca²⁺ in the photoreceptors and that an abnormal Micu1 to MCU ratio may compromise photoreceptor health. To analyze the Micu1 to MCU ratio, quantitative polymerase chain reactions (qPCR) are conducted. Retina cells are compared to other cell types, such as brain and heart cells, to determine the ratio of Micu1 to MCU relative to other tissue types. qPCR is also used to determine the Micu1 to MCU ratio in cells designed to express Micu1 or MCU abnormally. Current results suggest that the retina and brain express higher amounts of Micu1 than the heart, but the retina expresses the least MCU. It is hypothesized that most of the MCU channels in the retina are regulated by Micu1. On the other hand, heart tissue has more MCU and less Micu1, suggesting that the MCU channels are not regulated to the same extent. This makes sense because the retina and brain both contain large amounts of neurons, while the heart largely contains muscle. Therefore, different cell types with different functions express unique Micu1 to MCU ratios. Cells which are designed to express Micu1 or MCU abnormally have different ratios that may be insightful in addressing visual disorders. Further work may include manipulating the Micu1 to MCU ratio and observing the consequent effects on the photoreceptors.

Antibiotic resistance is a growing threat across the planet. This resistance, coupled with a dearth of new antibiotics, makes development of new antibiotics of critical importance. Antibiotics derive from microbial pathways that synthesize complex natural products, and engineering these pathways to produce unique products is an exciting prospect. We will use such an engineering strategy to produce variants of the antibiotic Microcin J25 (MccJ25) in E. coli. MccJ25 belongs to a unique set of compounds known as lasso peptides. It is produced by three biosynthetic enzymes that modify the precursor peptide, McjA, and then export the mature lasso. MccJ25 functions by blocking the nucleoside triphosphate (NTP) uptake channel of RNA polymerase, interfering with transcription. Moreover, a single amino acid change in RpoC, one component of RNA polymerase, confers resistance to MccJ25. Extensive studies on the MccJ25 biosynthetic pathway suggests that the biosynthetic enzymes are tolerant of mutations in McjA. We plan to identify variants of MccJ25 that overcome resistance due to rpoC mutation. We will construct a library of protein variants of McjA and transform this library into a strain of E. coli carrying the rpoC T931I mutation. After induction of the MccJ25 biosynthesis pathway, cells with biologically active variants of MccJ25 should drop out of the population. By deep sequencing the population before and after selection, we can infer which variants successfully inhibited growth. This strategy of altering a precursor peptide to create novel antibiotic structures may provide a framework for future antibiotic engineering efforts.

The microbial community residing in the gastrointestinal tract, called the gut microbiota, plays an important role in host energy homeostasis. Since energy homeostasis is essential for survival, it is plausible that neural circuits involved in maintaining host energy homeostasis would evolve to shape the composition of the gut microbiota in alignment with the energy needs of the host. In humans and mice, neurons in the arcuate nucleus of the hypothalamus that express agouti-related protein (AgRP neurons) play an important role in stimulating food intake and maintaining energy balance. AgRP neurons are maximally active during starvation conditions; thus, we are investigating whether they help conserve energy under these conditions by altering the composition of the gut microbiota to a state with a greater capacity for energy harvest. To test this hypothesis, we expressed an excitatory designer receptor, the hM3Dq receptor, in the AgRP neurons of mice and then analyzed the bacterial composition and energy harvest capacity of the gut microbiota of these mice following pharmacological activation of the hM3Dq receptor relative to unstimulated control mice. We assessed the bacterial composition of the gut microbiota using 16S rRNA amplification, sequencing, and characterization, and we assessed the energy harvest capacity of the gut microbiota by determining the energy content of the mouse fecal matter using bomb calorimetry. Our results show how AgRP neuronal activity influences the composition and energy harvest capacity of the gut microbiota. These results reveal that the central nervous system can shape the gut microbiota to promote energy balance. This finding contributes to a better understanding of why and how gut microbiota changes occur, which may be relevant to understanding metabolic diseases such as obesity.

The budding yeast is a popular model organism for aging research. Amino acids are a fundamental building block of biology with crucial roles in metabolism and intracellular signaling, but effects of amino acid levels and ratios in the growth media on yeast aging has never before been tested. We measured the effects of supplementation of different amino acids on yeast replicative lifespan by imaging hundreds of trapped mother cells in a microfluidic device during the course of replicative aging. We find that increased absolute levels of threonine can increase yeast lifespan through reduction of toxic biosynthetic intermediates. Conversely higher relative levels of cysteine depress lifespan. These effects are regulated by the status of the vacuole, which becomes less acidic with age. Thus, we find that longevity promoting optimal amino acid composition can optimized in an age-specific manner.

Gene amplifications are an often-overlooked source of genetic variation frequently associated with genetic disorders, including cancer and developmental delays. However, the mechanism by which they arise is still unclear. There are currently two competing models for how gene amplifications arise. The first model requires a double-stranded break in DNA that results in chromosome recombination. In the second model, an error in DNA replication results in the formation of an extra-chromosomal DNA intermediate that can reinsert into the genome. To distinguish between these two models, I am using CRISPR/Cas9 to induce double stranded breaks in a strain of yeast with two fragments of the URA3 gene on separate chromosomes. I then select for strains with restored URA3 function, indicating that the two fragments have recombined to form the full URA3 gene. If the genome rearrangements are occurring through the double-stranded break mechanism, inducing these breaks should increase the frequency of recombination events that result in functional URA3 genes. However, the preliminary results suggest that these events are occurring through the replication-error mechanism rather than through double-stranded breaks. Clarifying the mechanism of gene amplifications is a significant step toward understanding the how these genetic disorders arise and suggests further research in preventing disease-causing gene amplifications.