While many brain malformations at birth are caused by de novo variants-- genetic mutations that arise early during fetal development-- in some cases deleterious mutations are inherited from a parent unaffected by the mutation. If the parent harbors the mutation in a small amount of cells, it is possible for them to lack a disease phenotype despite being able to pass on the mutant gene to their offspring. This genetic variability in somatic cells is called mosaicism. Mosaicism is challenging to detect with traditional methods of sequencing, as it is difficult to determine if a small read number of mutant DNA is an error in sequencing or if it is a biologically real low-level sequence variation. In order to confidently identify low-frequency mosaic mutations, we utilize deep-coverage next-generation sequencing methods with very low thresholds for mutant DNA detection. Digital-droplet PCR, or ddPCR, is a promising new sequencing technique ideal for detecting low-level mosaicism. As this is a recently developed technology, we first optimized the protocol to determine the level of attainable coverage in mosaic samples and compare this with other high-depth methods. When this was successful, we tested samples from families of children affected by serious brain malformations. We selected families with healthy parents and multiple affected children, as this is a strong indication that one of the parents carries a mosaic mutation. We also tested dysplastic brain samples from affected children who underwent epilepsy surgery to quantify the level of mutant cells. Overall, we were able to optimize ddPCR to detect a mutant allele frequency of 0.2%, which is substantially better than other high-depth methods. This work provides a promising new avenue into the detection of low-frequency mosaicism, which can be used to present clinically-relevant information for the diagnosis and genetic consultation of children affected by various genetic conditions.

Juvenile Idiopathic Arthritis (JIA) is the most common rheumatologic disorder found in children, affecting millions worldwide. It has no known cause, prevention, or cure, and can lead to debilitating destruction of the joints. In adult Rheumatoid Arthritis (RA), previous research suggests a connection between RA and oral bacteria that cause inflammatory diseases. Specifically, Porphyromonas gingivalis, found in patients with periodontitis, expresses an enzyme that modifies the amino acid arginine to citrulline, which the immune system recognizes as foreign, reacting to produce anti--citrullinated protein antibodies (ACPAs), which attack otherwise healthy proteins in the body. Therefore, we hypothesize that in JIA, the unique bacterial colonies of the mouth induce a chronic ACPA production that leads to arthritis. We use enzyme-linked immunosorbent assays to assay patient plasma for ACPAs with 6 peptides. We have tested plasma from 79 patients with JIA, 56 children recruited from dental clinic, and 29 healthy children for antibodies to citrullinated filaggrin, a known autoantigen in RA. Compared to healthy controls, JIA patients had a significant increase in anti-citrullinated filaggrin antibodies (relative levels 0.88 vs. 0.45, p=0.001 by t-test). Anti-filaggrin antibodies were elevated in patients with oligoarticular JIA, (mean 0.62, p=0.0012) and extended-oligoarticular JIA, (mean 0.88, p=0.007), but not polyarticular JIA (mean 0.49, p=0.47). In addition, there is a higher prevalence of bleeding on probing (BOP, an indicator of gingival inflammation) in JIA, with an average BOP score of more than two times that of children from dental clinic (p=0.008). Our next step is to see if there are any specific microbes associated with higher BOP scores and ACPA levels. If we find that oral pathogens are associated with inflammatory disease, prevention can be designed to eliminate plaque-associated pathogens through various dental hygiene practices.

Acute myeloid leukemia (AML) affects people of all ages and, despite aggressive treatment, more than a third of patients relapse, creating the necessity for novel therapies. Immunotherapies had successes in treating other cancers, but have not yet been fully developed for AML. Dendritic cells (DC) are potent antigen-presenting cells that can stimulate cytotoxic T cells and can be derived from leukemic blast cells (AML-DC). AML-DC have the potential to prime and expand leukemia-specific cytotoxic T cells for use in immunotherapies. DC can be generated in the laboratory using a standard protocol in which CD14+ monocytes are exposed to inflammatory cytokines (CD14-DC). However, most AML blast cells are CD14- and an alternative selection process must be used. CD33 is a myeloid marker present on monocytes and on AML. To develop an appropriate control for AML-DC, we hypothesize that CD33 selected cells from normal donors can be used to generate DC (CD33-DC) with similar properties to those from CD14+ monocytes. CD14-DC and CD33-DC from normal donor peripheral blood mononuclear cells showed similar expression of the DC maturation markers CD80 and CD86 (97% vs. 95% and 99% vs. 90% respectively for CD14-DC vs. CD-33) but only 63% of CD33-DC expressed CD83 vs. 87% of CD14-DC, which could indicate that CD33-DC are less capable of stimulating T cells. To compare functionality of CD33-DC, we used CD33-DC and CD14-DC to present known antigens to CD8+ T cells and evaluated expansion of antigen-specific T cells by the DC. Additionally, we compared cytokine production by CD33-DC and CD14-DC. Determining if CD33 selection is capable of producing DC in normal donors gives us the necessary controls to use for comparison to AML-DC. We can use endogenously expressed antigen from AML-DC to find leukemia-specific T cells to be developed for T cell immunotherapies to cure AML.

Chronic kidney disease currently has no cure but continues to plague our population, demanding new effective strategies to treat kidney disease through the study of biochemical mechanisms of fibrosis. Kidney fibrosis begins with the loss of function of parenchymal cells that leads to excessive scarring in the kidney due to excessive extracellular matrix formation. Several studies in both human and experimental mice models have shown that increased oxidative stress plays a critical role in renal fibrosis due to the generation of reactive oxygen species. However, the mechanisms by which oxidative stress promotes kidney injury or fibrosis remains unclear. PRX2 is an antioxidant enzyme that normally protects the cell against oxidative stress by the reduction of hydrogen peroxide and oxidized proteins. Normally, an increase in oxidative stress has shown to increase an inflammatory response, potentially leading to fibrosis. Unfortunately, the exact mechanisms by which this occurs is unclear. In contrast to its role as an antioxidant, recent studies suggest that PRX2 may be an important mediator of the proinflammatory response as a direct result of oxidant stress. Based on these studies and our preliminary data, the Okamura Lab currently hypothesizes that the loss of PRX2 leads to a significant decrease in renal fibrosis by attenuating the inflammatory response. Our preliminary data suggests that PRX2 is expressed in tubular epithelial cells in normal kidneys and expression decreases with progressive injury by unilateral ureteral obstruction (UUO). Despite the decrease in PRX2 during UUO, PRX2-deficient mice had a significant reduction in fibrosis with a significant decrease in the proinflammatory cytokine TNF-alpha. Methods to further study the mechanism of PRX2 include western blotting, immunostaining, and quantitative PCR. As this study continues, the information on PRX2 will be a valuable asset to developing antioxidant treatment therapies to find a cure for chronic kidney disease.

Youth with T1D, although encouraged to exercise as part of their treatment plan, are not implementing recommendations to adjust insulin around exercise. Little is known as to whether insulin delivery method impacts these behaviors. Our aim was to compare behaviors surrounding exercise between youth on insulin pump therapy and those on insulin injections, specifically focusing on evening behaviors after exercise. We recruited 50 youth aged 10-18 years with T1D who completed the Type 1 Diabetes Report of Exercise Practices Survey (T1D-REPS), a questionnaire designed to assess specific practices around insulin management, nutrient intake, and glucose monitoring. 30 were on insulin pump therapy and 20 on injection therapy. We measured frequency of glucose checks and hemoglobin A1C (HbA1c). HbA1c measures average blood glucose level over the preceding 3 months. Higher HbA1c indicates worse blood glucose levels. Pump users were more likely to be male (73%), had lower HbA1c (8.5% ±1.3 v. 9.8% ±2.7, p<.05), and reported more glucose checks per day (5.1±3.0 v. 3.2±1.9, p<.05). Pump users were less likely to check their blood glucose prior to exercise (37% of pump users reported often or always checking v. 60% of non-pump users; p=.07). Pump users were also less likely to adjust insulin for bedtime snacks following exercise (8% of pump users v. 31% of non-pump users, p<.05).  Overall, pump users reported less glucose monitoring before exercise and were less likely to implement interventions to prevent overnight hypoglycemia, though they displayed more frequent monitoring around exercise. Understanding populations at risk for glycemic excursions around exercise will allow for the development of intervention strategies to improve adherence to these recommendations.

Synaptic plasticity influences many functions in the body, some of which include the mechanisms of learning and memory. However, isolating these cells for further study is inefficient as grinding up a portion of the brain yields a mixture that includes synaptosomes, neuronal and glial contaminants, and free myelin as well as mitochondria. One way to sort synapses from the mixture is to use FACS (fluorescence-activated cell sorting). However, this method is limited as it is very time consuming. Instead, our lab is developing methods to sort specific synapses based on immunology cell-sorting techniques that can isolate activity-dependent synapse species from the assortment of synapse types in the brain. We have created TAG proteins that targets glutamate synapses, therefore potentially enabling us to utilize the more efficient MACS (Magnetic Cell Sorting) method that is proven to isolate cell types at higher rate. My project is to develop a method to ensure that the TAG RNA is translated only in specific neurons. I utilized Cre-lox recombination methods so that the TAG RNA is translated only when a CRE protein removes the lox-stop-lox gene. To do so, I used a plasmid with an AS-LSL-TAG insert previously made by the lab. I then used HEK 293 cells, each with a different condition to evaluate how well the CRE protein will be able to remove the LSL portion of the tag construct: one with untransfected cells (control), another with the CRE-RFP plasmids only, a third with only AS-LSL-TAG plasmids, and the last with both CRE-RFP and AS-LSL-TAG plasmids. If I achieve HEK 293 cells with the AS-LSL-TAG and the CRE-RFP that express the both the green fluorescence from the AS-LSL-TAG and the red fluorescence from CRE-RFP, I will then insert the LSL TAG construct into neurons and analyze the results by western blots and microscopy.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation in either of the genes PKD1 or PKD2. This disease has been the focus of ongoing research due to its high prevalence and lack of curative treatments. How does ADPKD develop? To understand cyst formation in ADPKD, we are utilizing the murine model PKD1 p.R3277C (RC). Human dominant expression of the APKD phenotype is not similarly found in mice; null mutations of PKD1 or PKD2 are embryonic lethal, and the cystic phenotype of mice is observed only in conditional deletion mutants. However, the RC murine model recapitulates the late onset cystic growth of human ADPKD. The morphology of the pre-cystic kidney was characterized by formalin-fixed paraffin embedded (FFPE) sectioning in combination with H & E staining performed on RC homozygous mutant and control kidneys at post-natal time points of p20 (days), p40, p60, and p80. In the future, RNA from RC mutant and wild-type kidneys at p20 will be collected and sequenced. RNA sequencing will be utilized to determine pre-cystic expression changes between affected and unaffected phenotypes. Although less advanced, cyst formation has been shown to occur as early as p20. Because of this, the earlier time point of p10 was also assessed. By investigating these time-points we aim to identify the earliest differences that we hypothesize are more likely to be causal for cyst formation, versus changes that are a secondary consequence of PKD pathology.

Muscular dystrophy affects millions of people across the globe; the disease leads to the progressive degeneration of skeletal and cardiac muscle tissues. The goal of this project is to identify a pharmacological treatment for muscular dystrophy in zebrafish. The disorder results from a mutation in the DMD gene, which encodes the instructions for producing the dystrophin protein. Dystrophin both protects and strengthens muscle fibers. Because of their genetic similarity to humans and the relative ease of introducing genetic mutations, we used zebrafish as a model. The lab utilizes a method that renders the dystrophin-encoding gene non-functional, eliminating the dystrophin protein in zebrafish larvae and producing a DMD zebrafish mutant strain. Without the protective effects of dystrophin, these zebrafish develop a severe form of muscular dystrophy. Previous results indicated that Trichostatin A (TSA), a selective inhibitor of some histone deactylase enzymes, is an effective treatment for salvaging deteriorating muscle fibers in mutant larvae. TSA disrupts the removal of acetyl groups and thus alters gene expression. We hypothesized that we could use the mutant DMD zebrafish to identify additional new pharmacological interventions. Upon testing new drugs, we looked for fish exhibiting less muscle deterioration than mutant DMD zebrafish with no drug treatment. After zebrafish larvae from fish carrying the DMD mutation were treated with novel drugs or control media, we performed genotyping on the larvae to identify the DMD homozygous mutants. Once animals were genotyped, we scored the animals for muscle defects using microscopy. We predicted that effective drug treatments for zebrafish DMD would show improved muscle in the larvae when compared to the untreated control DMD larvae. This research may provide a foundation for drug therapy and treatment possibilities in humans with muscular dystrophy. 

Mast cells are bone marrow-derived, granule-containing immune cells that are widely distributed in connective and mucosal tissues. Although mast cells are known to store and release histamine and other inflammatory agents in IgE-mediated allergic reactions, the specifics of their extracellular signaling and regulatory mechanisms are not fully understood. Therefore, this study aimed to further characterize the mast cell surface proteome to better understand the signals that promote mast cell activation. Murine bone marrow-derived mast cells were cultured and their membrane proteins were purified using hydrazide chemistry to isolate galactose and sialic-acid moiety-containing glycopeptides; these peptides are involved in post-translational modifications and anchorage of proteins to cell membranes. Post-cell lysis and purification, both liquid chromatography and mass spectrometry were used to classify and quantify detectable proteins. Use of online databases identified characteristics of these proteins, such as the presence of transmembrane domains, glycosylphosphatidylinositol (GPI) linkage, or target sites. As the cell surface proteome of bone marrow-derived macrophages is well characterized, they were subjected to the same treatments as mast cells, serving as a control. By comparing the mast cell and macrophage cell surface proteomes, we distinguished which proteins are mast cell-specific. Results show the presence of 69 transmembrane proteins including proteins known to be expressed by mast cells such as receptors for stem cell factor (c-kit) and interleukin-33 (ST2). Additionally, we detected and confirmed the expression of a potential cell surface histidine transporter, solute carrier family 15A4 (SLC15A4), with quantitative polymerase chain reaction (qPCR). The identification of mast cell-specific proteins can help us further understand their effects on mast cell activation and subsequent degranulation.

Studies using primary airway epithelial cells from asthmatic and healthy donors have reported differences in gene expression of a variety of genes between cells from asthmatic and healthy donors. However, few studies have investigated the stability of gene expression by primary cells with increasing cell passage number, or how viral infection might affect the stability of gene expression with increasing cell passage number. This research investigated: 1) whether expression by primary bronchial epithelial cells (BEC) of genes IFIH1, which codes for a viral sensor protein with an important role in the innate immune response, and TGFβ2, a pro-remodeling cytokine, from asthmatic and healthy children, is stable with increasing cell passage number, and 2) if BEC infection with respiratory syncytial virus (RSV) alters the stability of IFIH1 and TGFβ2 expression with increasing cell passage number. To answer these questions, we differentiated passage 1, 2, 3, 4, and 5 BECs from asthmatic and healthy children at an air-liquid interface for 3 weeks. RNA was then harvested from BECs and RT-PCR was performed for TGFβ2 and IFIH1. To assess the expression stability across increasing cell passage number for each gene, we compared expression at passages 2-5 to expression at passage 1. Preliminary data collected shows that expression of TGFβ2 and IFIH1 from asthmatic and healthy children was stable with no significant differences between passages 1, 2 and 3. However, expression at cell passages 4 and 5 was significantly greater and more variable than by passage 1 BECs. We anticipate gene expression to be even more variable for RSV-infected passage 4 and 5 BECs. These observations illustrate the importance of using BECs from passage ≤ 3 when comparing gene expression between asthmatic and healthy primary BECs, and of characterizing the expression pattern across increasing cell passage number for each gene studied.

Congenital Heart Disease (CHD) is the most common inborn defect in babies and is responsible for 1/3 of all major congenital anomalies. With its continuous prevalence in the human population, the genetic causes remain poorly understood. Previous findings have shown that Pbx transcription factors control gene expression during development. Studies in zebrafish shows that pbx4 has significant requirements for heart muscle differentiation. Our hypothesis states that mutations in human Pbx genes can lead to heart defects. The purpose of this study is to acquire further insight about the role of Pbx genes in the context of CHD and analyze cardiac dysmorphic patterns in pbx gene mutants. By using the genomic editing system CRISPR/Cas9 (Clustered regulatory interspaced short palindromic repeats), we have been able to make exact genetic changes in Pbx genes in zebrafish (Danio rerio) animal models. In particular, we have engineered a Pbx gene mutation found in humans with heart defects, known as Pbx4 SNP (single nucleotide polymorphism). For our experiments, samples of zebrafish DNA are obtained and genotyped with various assays for mutations. We use microscopy and transgenic markers to analyze heart defects in mutant embryos. Pbx4 mutant zebrafish display distinct phenotypessuch as precardial edema and bulged ventricles, but it is not clear at what stages these morphogenesis defects occur. To further investigate the dysmorphic phenotypes, our next step is to conduct cardiomyocyte counting analysis in Pbx4 SNP and Pbx2/Pbx4 double mutant fish and gather data at 24 and 48 hours of development. This experiment will assist in better understanding the heart defects previously seen in the single Pbx4 mutant samples. Understanding the mechanisms and discovering the genes of CHD, as well as cardiac morphogenesis patterns, will contribute to finding therapies and improving genetic screenings for patients with lifelong heart defects.

Investigation of the phenotypic features of a putatively unaffected individual, in a family diagnosed with a multiple congenital contracture disorder called Distal Arthrogryposis Type 2B (DA2B), identified joint hypermobility, a phenotypic trait not commonly associated with DA2B. Review of the family history revealed joint hypermobility in multiple other family members extending back four generations. Analysis of exome data from the family, identified two candidate variants, one in exon 1 of the gene SRL and another in exon 8 of NPR3, which may explain the joint hypermobility observed in this family. Recently, we used Sanger sequencing to genotype five family members with hypermobility and four unaffected family members for the two candidate variants to see if either variant tracks with the hypermobility phenotype. Briefly, we amplified exons via polymerase chain reaction and verified product sizes using gel electrophoresis.  We then sequenced the amplicons using di-deoxy chain termination sequencing and generated electropherograms using an automated DNA sequencer. Using the computer program CodonCode, we aligned and examined the sequences. Analysis of the data is underway. Through pinpointing mutations that cause hypermobility, characterization of Distal Arthrogryposis syndromes will become more accurate which may lead to better patient outcomes for individuals with additional features like severe hypermobility.

Pierre Robin Sequence (PRS) is a human congenital disorder manifesting as a combination of several orofacial anomalies that include a small lower jaw (micrognathia), posterior placement of the tongue (glossoptosis), and high incidence of cleft palate. The resulting airway obstruction in affected infants and young children can be life-threatening. While lower jaw growth is thought to permit tongue descent to allow palatal shelf elevation and fusion, it remains unclear how tongue malposition and cleft palate results from lower jaw malformation in PRS. The aim of this study is to analyze the physical interactions between the tongue and other orofacial structures, such as the palatal shelves and lower jaw, to determine if dysregulation of these interactions plays a role in the pathogenesis of PRS. We used a Wnt1-Cre driver, a method to specifically knockout genes in the craniofacial mesenchyme, to disrupt hyaluronic acid synthase 2 (Has2), an enzyme crucial to the extracellular matrix and overall craniofacial development. This allowed us to produce conditional knockout (CKO) mouse models that developed the entire spectrum of PRS phenotypes including micrognathia, cleft palate, and a high-arched tongue interfering with the nasopharynx. Combining histology with 3D imaging techniques, we analyzed morphological changes of the palatal shelves, tongue and lower jaw in Wnt1-Cre, Has2 CKO mice. Our results indicated that reduced lower jaw growth affects tongue growth and its interaction with the nasomaxillary complex, while reduced palatal shelf growth affects tongue shape changes. This suggests the abnormal shape and positioning of the tongue in PRS may result from abnormal orofacial tissue interactions due to defects in the lower jaw and palatal shelves. The implications of this research will allow us to better understand the regulatory mechanisms of orofacial development and the pathogenesis of PRS in humans, which is essential for diagnosis and treatment of this disorder.

Systemic Lupus Erythematosus (SLE) is one of the complicated chronic autoimmune diseases that can involve many different organs including liver, kidney and skin. There is evidence that proliferation and apoptosis (cell death) of autoreactive T lymphocytes are defective in SLE, sustaining the inflammatory response. One specific protein that may contribute to T cell dysregulation is CD30. The main goal of my project is to study the expression of CD30 on T cells from SLE patients, specifically Lupus Nephritis (LN) patients. CD30 is expressed on activated memory T cells and it regulates communication between T and B cells along with initiation of apoptosis. Previous studies suggest that there is elevated soluble CD30 in LN patients. There is an FDA approved drug that causes apoptosis by targeting CD30 expressing cells, which is used to treat lymphoma patients. The drug Brentuximab Vedotin could potentially replace chemotherapy for those who cannot tolerate its toxicity and used as a primary treatment. Results of this study provide principle data to support the approach of therapeutically targeting chronic T cell activation in SLE via CD30. I hypothesize that the LN patients with more severe disease will have higher numbers of CD30 expressing cells depending on their diagnosed classes. Patient samples are stained and evaluated by immunofluorescence. The expression of CD30 was correlated to disease status at the time biopsies or urine samples collection. We expect that the higher classes such as III and IV are more likely to have higher number of CD30 positive cells compared to classes I and II as well as non LN patients. Therefore, I compared the frequency of CD30 expressing cells in SLE patients to those without SLE and those with different classes of LN. This research has great implications for SLE patients and could replace previously expensive and highly toxic treatments.

Infantile Spasms (IS), classified as an epileptic encephalopathy (EE), is a severe epileptic syndrome that mostly occurs in newborn babies. Because these seizures are hard to treat, IS can lead to other forms of epilepsy and is often associated with intellectual disability. Many IS cases have an underlying genetic cause – specifically, mutations acquired during DNA replication that disrupt key genetic pathways of synaptic function and brain development. However, many IS cases remain without a genetic diagnoses due to the genetic heterogeneity of the disease. Because IS shares many phenotypic traits with other EEs, my research uses a candidate gene approach to identify genetic variants in the coding regions of 51 known EE genes in a cohort of 105 patients with IS of unknown cause. Sequencing these candidate genes from a population of patients with IS will determine if mutations in these EE genes cause IS. I used next-generation DNA sequencing to screen for variants in 51 known EE genes. I filtered the data to identify 1) variants that result in an alternative amino acid sequence, which may lead to the disrupted function of a protein, and 2) variants that are unreported in a healthy population – identifying 16 variants. IS is most commonly the result of de novo mutations, where the patient has a pathogenic genetic mutation that was not inherited from either healthy parent. I performed segregation testing to determine the inheritance of the 16 identified variants. Of the 105 patients with IS, I identified 4 de novo, pathogenic mutations in 3 genes and am currently waiting on parents to determine the mode of inheritance of 4 other variants. My lab has solved 10% of the IS cases in total. These new genetic discoveries will contribute to a better understanding of IS and lead to more accurate diagnoses.

Iron sufficiency is essential for optimal brain growth and development, particularly among preterm infants. Current guidelines for iron supplementation of these infants (2-4 mg/kg/day) may be inadequate to maintain iron sufficiency. Our objective was to evaluate zinc protoporphyrin-to-heme (ZnPP/H) ratio and ferritin as measures of iron status in neonates. Iron sufficiency over time was also assessed. All UWMC NICU inpatients who received paired ferritin and ZnPP/H measurements between October 2014 and May 2016 were evaluated retrospectively. Concordance of iron measures were evaluated, as were effects of sepsis, transfusion and iron supplementation over time. There were in total of 601 paired samples from 228 patients and the paired values were assessed during culture-positive sepsis, defined as positive blood culture within 2 days before or 1 day after ZnPP/H or ferritin measurement. Mean ZnPP/H in infants with and without sepsis were not significantly different whereas ferritin values were significantly higher during sepsis. Ferritin values were also more affected by red blood cell transfusion given within seven days prior to ferritin and ZnPP/H measurements. A trend toward worsened iron sufficiency in infants born to diabetic mothers was noted. Small for gestational age infants also showed a trend towards poorer iron status. The mean iron supplementation at 30, 60 and 90 days was 5.4 mg/kg/day, 6.9 mg/kg/day and 7.4 mg/kg/day. Despite this, ferritin values decreased with advancing postnatal age, with 66% of ferritin values <76 ng/dL. In conclusion, ferritin is more significantly affected by sepsis and transfusion, and therefore ZnPP/H may be a more reliable marker of iron status during proinflammatory states. Infants showed worsening iron sufficiency over time despite supplementation above AAP guidelines suggesting a potential need for more aggressive iron supplementation.

Joubert syndrome (JS), a rare recessive genetic disorder, is characterized by abnormalities in the cerebellum and brainstem. Notably, JS is a ciliopathy because the known causative genes have products that localize to the primary cilium, a vital organelle acting as the nexus between many signalling pathways. Understanding more about JS will provide insights into this emergent group of disorders revolving around the cilium. Causal mutations for JS need to fulfill three criteria: They need to be rare, deleterious, and present in both alleles (copies of a gene) in the patient. Patients in our cohort are sequenced for rare 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 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 am using molecular inversion probes (MIPs) to target-sequence putative causal mutations in the parents and then using the sequencing data to determine the segregation pattern of these variants. If the MIPs data show RDVs for a causative gene are on the same allele, then the variants cannot be the cause of JS in the family and we will have to consider other variants or perform additional sequencing to identify the causal mutations. The data will likely show that the mutations are in both alleles because the mutations targeted with MIPs are mutations that have passed the filters for being rare and deleterious and are robust candidates for being causal. Ultimately, the goal of my research is to elucidate the genetic basis of JS in hopes of improving risk recurrence analysis, prenatal diagnosis, and help discover biological mechanisms of JS.

Alcohol consumption among college students has been well documented to increase during school gamedays. While studies have focused on the effects of peer and event specific influences on college drinking, there remains a gap in understanding between behavior and social media content such as Instagram. Instagram’s unique lifestyle and filter driven platform have facilitated the glamorization of alcohol displays. The purpose of this study is to assess whether the use of Instagram and viewing school gameday content amplify the receptivity of alcohol behavior. Undergraduate students ages 18-25 were recruited from a Pacific Northwest University using a convenience sample. The survey was structured using a Likert-scale questioning pattern to assess attitude towards alcohol, and four distinct alcohol scenarios will be shown to participants. Responses were used to assess whether the variations in alcohol attitudes changed while school sports affiliation was present in some scenarios. Further questionnaire regarding past experiences of viewing alcohol displays and Instagram use were also included to support findings. This survey will then aim to capture whether undergraduates favor alcohol depictions on Instagram with greater probability when the school team is made visible. Findings could be used to spread social media messages in proximity with sporting events to prevent problematic alcohol behavior. Future studies could incorporate a multi-university study focusing on school sports polls to determine whether school rankings correlate with alcohol consumption.

Alcohol is used among 45% of college students, and this behavior is associated with up to 1800 deaths per year. Social Media has been shown to influence student's alcohol use. Little is known about the role of alcohol-related Facebook events in promoting safe or problematic alcohol use. The purpose of this study was to investigate the presence of safety messages in alcohol-related Facebook events, as well as students' perceptions of these posts. This mixed methods study involved content analysis and qualitative interviews. Researchers will use the Facebook Discover feature, which enables the user to search for events meeting given criteria, to identify events in the Seattle, Washington area categorized under "Nightlife". The search was be conducted at 4 time points 1-2 days apart, each weel, and the top 4 events will be included, yielding a total of 100 events for analysis. Researchers evaluated events for the following variables: alcohol-related public event, number of people attending, interested, and invited, and inclusion of alcohol safety related messaging. Then researchers identified ten 18-22 year old college students to participate in qualitative interviews by approaching them at popular campus locations. Interviewees viewed a slideshow of common themes present in alcohol-related Facebook events content analysis findings and answered questions about their attitudes about attending such events. These interviews were recorded, transcribed, and undergo thematic qualitative analysis. The results will quantify the proportion of Facebook events with alcohol-related content on Facebook events available under a specified public search. The results from this study can be used to consider ways that safety messaging can be improved on alcohol related Facebook events.

Empathy is the capacity to understand or feel what another person is experiencing from within the other person’s frame of reference. Empathy has been shown to be steadily declining in college students. Use of various types of media has been shown to increase empathy if the media is prosocial, relating to a positive behavior, while violent or antisocial media is shown to decrease empathy. College students are highly connected through social media. There has been little research on the impact of mobile app usage on empathy. The purpose of this study is to compare smartphone app usage to the Interpersonal Reactivity index, a measure of empathy. A sample of approximately 30 college students with iPhones from two universities were recruited through class Facebook pages and email listservs. The study was conducted through an online survey that uses an established measure (Interpersonal Reactivity Index) to determine perspective taking, empathetic concern, fantasy, and personal distress of each participant. Additionally, participants were asked to submit a screenshot of their weekly app usage that shows time spent on apps. A codebook was used to identify these apps as prosocial or antisocial. Analysis included comparing scores from the Interpersonal Reactivity Index to the amount of time spent on apps according to the screenshot. From these sets of data it will become more clear what kind of smartphone app usage is prosocial, what kind is antisocial, and the effects of app usage on perspective taking, fantasy, empathetic concern, and personal distress amongst college students.

Adolescents without college attending parents and from minority communities have lower post-secondary enrollment rates. Non-profit college preparedness organizations targeting these adolescents can provide support to help increase these enrollment rates. An important aspect of reaching adolescents is outreach through social media, given its prevalence among this population. However, pilot evaluations show infrequent social media use by college preparedness organizations, and it remains unclear which online outreach approaches yield the highest user engagement. The purpose of this study is to understand approaches used by college preparedness organizations in social media outreach, and which factors are associated with user engagement. In this content analysis study, seven college preparedness organizations listed as partner organizations on the Seattle government website were randomly selected. A codebook was developed to evaluate outreach approaches using components derived from the Facebook Influence and non-profit organization models, which will use variables for how organizations communicate information, build communities, create immersion, and facilitate action in their social media posts. The codebook was used to evaluate each organization’s posts from their Facebook and Twitter profiles for a period of a year. Likes, Shares, and Retweet totals were also collected for each post. Associations between social media outreach approaches and user engagement were examined. Organizations may use more community and immersive orientated posts to generate more likes and followers. The results of this study could potentially be used to develop strategies that will allow other college preparedness organizations or organizations in other outreach based fields to increase their user engagement.

Research has shown there are potential benefits to social media use in reaching underserved populations such as runaway homeless youth. Social media use in professional social service practice has often been seen as a taboo subject, and anecdotal reports suggest it is something to refrain from because of the potential for ethical dilemmas. However, studies have shown that many social work students feel the National Association of Social Workers Code of Ethics is vague in addressing correct professional social media use. The purpose of this study is to gain practitioner perspectives on professional social media use. The study recruited 20-30 practitioners working in youth-serving social service agencies who regularly come into contact with clients, using snowball sampling as the primary source of recruitment. A 10-20 minute phone or in-person interview was conducted with each participant gauging perspectives on social media use, agency policies on social media, whether ethical codes of the profession adequately address social media use, and how a practitioner would address an online ethical dilemma. The data included qualitative and quantitative components and was analyzed for differences of opinion regarding education level, years in the field, and familiarity with social media. The researchers anticipate that a majority of the participants will report inadaquate training on social media use and that a majority of participants will have encountered clients on social media networking sites before. Furthermore, the researchers anticipate a majority of participants will feel the ethical codes of the profession do not adequately address social media use. This study could inform further social work research into the efficacy of professional social media use as a benefit to the helping relationship.

A growing number of parents are opting out of vaccinations for their children, which presents a prominent public health risk. Studies have shown that parents are now more likely to look to Web 2.0 communities, such as blogs, than rely on medical expertise for health advice, especially on vaccination. However, there is scant literature outlining how parents contribute to online discussion of vaccination. The purpose of this study was to explore parent involvement in online vaccine communication, by analyzing content in a setting that is informal, unmediated, easily accessible, and intended to express vaccination opinions to the non-descript, lay, parent population. Content analysis was conducted to evaluate a sample of online comments on popular, parenting blog posts on vaccination. Investigators entered the search terms “top parenting blog vaccination posts” into the Google search engine, and included posts that met specified inclusion criteria, such as parent-focused and parent-written. This sample was analyzed using an adapted codebook, vetted through inter-rater reliability testing. The codebook included variables for participant discussion role, examples include providing information or asking questions; stance: pro, con, or neutral; and argument rhetoric, including emotionalization or fact-based. The comment dates were also mapped over a timeline of relevant, vaccine-related events (i.e. Measles outbreak at Disneyland). Investigator analysis compared types of rhetoric and content by stance of the commenter. Statistical and qualitative analysis of this data was performed to reveal trends towards better understanding of anti-vaccination sentiments, and inform efforts to improve vaccination coverage. A divergence is anticipated in the rhetoric and discussion contribution between the two sides of the conversation. Preliminary results favor a tendency of pro-vaccination comments to rely on scientific evidence, and anti-vaccination comments to rely of emotionalization.

On Twitter, there are over 52,000 tweets containing the word “antibiotic”, many of which portray inaccurate information. Young adults are social media’s most avid users and may view such content. It remains unclear if and how college students decipher information in inaccurate tweets. The purpose of this study is to investigate how youth perceive and assess the accuracy of Twitter posts about antibiotics. College students were recruited from the Seattle area through snowball sampling and participated in focus groups. Four focus groups were conducted with a range of 3-5 students in each group. Researchers developed a mock Twitter feed composed of real tweets discussing antibiotics. The tweets chosen conveyed accurate information (e.g., “Antibiotics don’t help viral infections…”), made inaccurate claims (e.g., “Doctor gave me some antibiotics for my flu…but a least I’ll recover”), or embodied disconnect from health assertions (e.g., “Finished antibiotic Tue...”). The facilitator read a vignette about being a college student, sick during finals weeks, who turned to Twitter, and searched “antibiotics”. Students were asked to imagine that they are this person when answering questions throughout the focus group. The mock Twitter feed was presented. Students discussed initial perceptions of the tweets, followed by being asked to group the tweets in any way they saw fit. Then students were asked to categorize the tweets again, but this time into accurate or inaccurate claims about antibiotics. After, a discussion was held about the categorizing processes. The focus groups were recorded, transcribed, and analyzed for reoccurring themes throughout the discussions and categorizing process. This study helps inform health professionals of a need to improve education about antibiotics particularly around evaluating the accuracy of social media content.

Joubert syndrome (JS) is an autosomal recessive neurodevelopmental disorder, characterized by the "molar tooth sign" on axial brain MRI. JS is caused by mutations in 35 known genes, all of which encode proteins that localize to the primary cilium, a microtubule-based organelle that projects from the surface of most cells. Our current understanding of the underlying molecular mechanism causing JS involves lack of ciliary localization of ARL13B and INPP5E, two JS-associated proteins. ARL13B is required for the ciliary localization of INPP5E. Normal INPP5E localization maintains a phosphatidylinositol subtype distribution in the ciliary membrane. My research investigates ciliary phenotypes in cell lines harboring causal mutations in C2CD3, which encodes a protein that localizes to the primary cilium’s basal body, using existing images of patient cell lines. Loss of C2CD3 function in mouse models has presented lower ciliation rates and shorter cilia lengths. Previous research in our lab on two other JS-associated basal body proteins, OFD1 and KIAA0586, identified normal ciliary ARL13B-INPP5E levels. To determine ARL13B and INPP5E localization, cells from two control and two different C2CD3 patient cell lines were grown and serum-starved, inducing ciliation. Cells were stained with antibodies to acetylated tubulin (marking cilia), and INPP5E or ARL13B (marking protein of interest). Using Fiji, an image analysis software, I “painted” each cilium by outlining the signal in the acetylated tubulin reference channel to define a mask. Using that mask, I quantified ciliary ARL13B and INPP5E. I measured ciliation rates using the number of masks divided by the number of nuclei, and cilium lengths using the “skeleton length” function. Based on data from mouse models, we predict that C2CD3 patient lines will have fewer and shorter cilia, but normal ARL13B-INPP5E localization. These expected data would exclude the hypothesis that INPP5E mislocalization is an obligate part of the biological mechanism underlying JS.

Joubert syndrome (JS) is a genetic neurodevelopmental disorder with medical features including a distinctive hindbrain malformation and developmental delay. All 35 JS-associated genes to date encode gene products localizing to the primary cilium but the mechanism behind JS is still unknown. The primary cilium serves as a cellular antenna for signaling, and when dysfunctional, can lead to the above features and more. One JS-associated gene, INPP5E, is an enzyme that controls phosphatidylinositol (PI) subtype distribution in the ciliary membrane. We hypothesize that abnormal distribution of PI subtypes PI(4,5)P₂ and PI(4)P in primary cilia is central to the JS mechanism, and therefore should be present across genetic causes. To test this hypothesis, I used fluorescently-tagged PI biosensors, domains from naturally occurring proteins that bind to different PI subtypes, and a domain from serotonin receptor (5-HT₆) that marks cilia. The PI biosensors were already tagged with GFP (green fluorescent protein) so I replaced GFP for RFP (red fluorescent protein) on the PI(4,5)P₂ biosensor, and inserted BFP (blue fluorescent protein) on the 5-HT₆ domain, allowing me to distinguish all three molecules in the same primary cilium. To do this, I removed the GFP sequence from our PI(4,5)P₂ plasmid using restriction enzymes that cut DNA then inserted RFP (amplified from a template plasmid carrying RFP) using DNA ligase. I confirmed the RFP insert by restriction enzyme digests specific for RFP and by direct DNA sequencing. PI(4,5)P₂-RFP plasmids were transformed into cells with PI(4)P-GFP and our BFP ciliary marker, then live-imaged. These tools will make it possible to determine if abnormal PI subtype distribution in primary cilia is a unifying cellular defect in JS patient cell lines. If so, we can then explore the effects of altered PI distribution and determine whether modulating PI distribution has the potential for mitigating the effects of Joubert syndrome.

Targeted therapies designed to inhibit hyperactive oncogenic signaling have demonstrated some encouraging clinical responses. However, many of these responses are not durable as tumors may develop new mutations to “work around” pathway inhibition leading to drug resistance. Technologies that enable more efficient discovery of “suites” of drugs that inhibit multiple pathway nodes are needed. The SABRE (Splice Acceptor Brilliant Reporter) platform was developed to provide an unbiased method to screen large compound libraries and dramatically improve the speed and efficiency for which novel targeted therapeutics can be identified. SABRE is built on the premise that oncogenic changes in gene transcription can be harnessed as powerful reporters of pathway activation status. SABRE utilizes gene trap technology coupled with a drug selection process to isolate cells that generate a robust “off to on” luciferase signal in response to pathway inhibition. Via massively parallel comparative analysis, multiple traps let nature provide the best reporter for further analysis and drug discovery. We employed the SABRE technology to identify insertion sites specific to the MAPK (mitogen-activated protein kinase) pathway, an oncogenic pathway that is implicated in a plethora of cancers. Experiments were performed using the human melanoma cell line A375, containing BRAFV600E mutation. SABRE lentiviral transduced A375 cells were treated with trametinib, an inhibitor of MAPK pathway, and clones were isolated that emitted a positive luciferase signal upon drug treatment. To determine if these reporters were MAPK pathway specific, the platform was used to screen a 6000+ compound library. Results from the screen found that 70% (28/40) of the top drug hits were known to directly modulate the MAPK pathway revealing the power of the SABRE gene trap technology to generate a panel of reporters specific to an unlimited number of cancer signaling pathways. SABRE holds the promise for discovering untapped drug therapies for cancer.

Fundamental understanding of how effector T-cells are transformed into quiescent memory T-cells is vital for developing immunotherapeutic treatments for autoimmune diseases and cancer, and for the development of vaccines against diseases such as AIDS, tuberculosis and malaria. Previous research on conversion of memory precursor effector cells (MPECs) into memory T cells has found evidence of Treg cell involvement in the process. Treg cells are regulatory helper T cells which selectively inhibit effector T cells to prevent and suppress overly strong immune responses and autoimmune responses. Research has also shown that MPECs produce their own IL-2, a cytokine involved in immune tolerance and activation. Based on these data, we hypothesize that direct interaction between MPECs and Treg cells, possibly via MPEC-produced IL-2, is required for conversion of MPECs into memory T-cells. We also addressed colocalization of Treg cells with short-lived effector cells (SLECs). SLECs are IL-2 non-producing effector cell counterparts of MPECs and do not form memory. Potential MPEC or SLEC interaction with Treg cells was visualized through immunofluorescence microscopy of the secondary lymphoid organs (spleen and lymph nodes) of experimental mice injected with lymphocytic choriomeningitis virus (LCMV). To bypass the low precursor physiological frequencies of MPECs we employed the P14 LCMV GP33-specific TCR transgenic mouse system, which is a well-regarded model system for studying memory differentiation. To test the role of IL-2 in cell-cell interactions, we used IL-2 agonist and antagonist antibodies. A detailed understanding of how Treg cells are involved in the process of MPEC to memory T cell conversion is key to developing novel immunotherapeutic treatments.

Nearly 94% of adolescents report daily online smartphone use; thus, understanding specifics regarding time spent on smartphone apps is important. Self-reported time spent on apps has been shown to be unreliable. New methodology is needed to better understand app use among highly connected adolescents. The purpose of this study was to test a novel methodology to investigate smartphone application use among older adolescents. A sample of freshman at a large university was recruited through freshman interest groups and university forums. For 9 sequential weeks participants submitted weekly screenshots of their mobile application use showing their most-used apps including social media and messaging tracked by battery usage. Incentives were given at both week 5 and week 9. Analysis included paired t-tests. We evaluated a total of 134 screenshots (82.7% response rate) submitted by 18 participants who were age = 18 (SD=1.1), 72% female. The average number of apps listed on battery usage was 16.1 (SD=1.6). Average battery use of social media apps was 16% (SD = 6.2) and average battery use of messaging apps was 11.8% (SD =6.8). Among social media, Snapchat and Facebook were among the most popular apps used with all participants having both/one consistently in their top 5 battery usage for at least 8 weeks. A total of 9 participants had “home/lock screen” among their top 5 battery usage for 3 or more weeks. These 9 participants had an average of 30.2% of battery usage for their most used app on weeks when home/lock screen was among top 5 apps, compared to 18.2% on weeks without home/lock screen in their top 5 apps (p=0.01). Phone tracking and screenshot data collection offers a novel and feasible methodology to understanding app use among highly connected populations. Additionally, frequent home/lock screen may be an indicator of compulsive phone checking.

Social media plays a significant role in the lives of college students today. Most young adults (82%) use Facebook while 59% use Instagram. Research has begun to examine the effects that social media usage has on mental health issues, but conclusions have been mixed. Anxiety is the most common mental health issue among college students, with 17% of undergraduates having been diagnosed with an anxiety disorder in the past year. Few studies have focused on social media’s effect on anxiety in young adults. The purpose of this study was to produce a stakeholder-driven conceptual model of the relationship between social media and anxiety. This qualitative focus group study examined whether college students perceive a relationship between social media and anxiety and what factors play into the relationship. College students ages 18-22 were recruited through student organizations and class settings. Participants were asked questions about their perceptions of social media and anxiety and to construct a consensus-based conceptual model. During the conceptual modelling activity, participants arranged notecards representing factors derived from the literature, including social comparison and approval, self-esteem, stress, and idealized selves on social media. Participants also had the opportunity to write in additional factors on blank notecards. Then, participants collaborated with the other focus group members to construct a collective model on a whiteboard. Analysis included comparing models that the participants created and synthesizing them into one final model. Additionally, the themes and key points of the focus group discussions were analyzed using qualitative methods. The results of this study will inform future research by creating an innovative conceptual model of the relationship between social media and anxiety. Future studies will be able to form targeted research questions based on the model or more closely examine contributing factors and outcomes.