Parent-teacher relationships are important in supporting young children’s social and emotional development. Especially in preschool, strong parent-teacher relationships can support a preschooler’s development across home and school. Despite the importance of parent-teacher relationships, parents from racial and ethnic minority backgrounds report having lower-quality relationships with their child’s preschool teacher. In this qualitative study, we sought to evaluate the voices of parents from racial and ethnic minority backgrounds, to understand barriers to and strategies for creating strong parent-teacher relationships. As part of a community-based partnership, we partnered with local preschools that serve a majority of underrepresented students. I assisted in conducting interviews with nine parents, in which three identified as Asian, one identified as Black/African American, three identified as white, and two identified as more than one race. During the interviews we asked parents questions about barriers at the individual, center, and systematic level that stand in the way of establishing close relationships with their child’s teacher, as well as potential solutions. My team is currently in the process of coding and analyzing all transcripts to explore barriers and solutions in more detail. Preliminary results reveal that parents brought up several barriers including time, limited face to face interaction, lack of communication, and feeling unwelcome in their child’s school. These barriers were described as impediments to the parent’s ability to form relationships with their child’s preschool teachers. We are currently analyzing and working with our community partners to identify solutions to improving parent-teacher relationships. The findings of this study are important in understanding how to support parents from racial and ethnic minority backgrounds in forming and maintaining strong relationships with their child’s preschool teacher.

Building strong relationships between parents and teachers is critical to supporting young children in developing key social, emotional, and pre-academic skills. Especially in preschool, parent-teacher relationships can support a young child’s development across home and school. Communication is an important aspect of successful parent-teacher relationships; however parents and teachers face interpersonal, intrapersonal, and structural barriers to communicating with one another. This study aims to elevate the voices of racial and ethnic minoritized parents of a preschooler and preschool teachers to understand barriers to communicating and strategies to overcome these barriers. Using a qualitative approach, we conducted semi-structured interviews with 9 parents of a preschool child and 7 preschool teachers at two local early childhood centers. Using a codebook, I am currently analyzing all interviews to answer the following research questions: 1) What type of communication do parents and teachers want? 2) What barriers do parents and teachers face when communicating? and 3) What are some strategies for improving communication between parents and teachers? Preliminary results indicate that both parents and teachers desire open, honest communication. Parents expressed wanting daily communication related to how their child was doing in school. Teachers expressed a desire for parents to understand more about their kids and to be able to speak to parents when they have a concern about their child’s behavior. Despite a desire for communication, both parents and teachers describe feeling unsatisfied by their current level of communication, citing how COVID-19 has limited their ability to communicate. Some strategies discussed included increasing face-to-face contact, having more events at school, and creative ways for daily communication (e.g., interactive platform, daily notes, etc.). Through listening to parents and preschool teachers about their current experiences, we hope to identify ways to improve communication between parents and teachers, ultimately improving young children’s outcomes.

During neonatal transport, specialized pediatric transport teams closely monitor the status of critically ill newborns. Hyperspectral imaging, a method of manipulating light, can be used to measure the vital signs along with video of the patient’s physical appearance for remote monitoring. Appropriate light intensity is critical for clear visibility of the newborn and hyperspectral imaging accuracy, but this must be balanced with safety for sensitive eyes. In June 2022, an experiment using light bars and a photometer was conducted at Seattle Children’s Hospital using a newborn manikin in a transport incubator to measure the amount of light needed to view the manikin and the potential light exposure to the eyes of the newborn. Eleven images depicting the visibility of the patient model in the incubator were taken in controlled amounts of light. Upon my work analyzing the experimental results, preliminary light testing in the range of light intensity (0.5 - 1400 Lux) showed that the amount of light that reached the patient’s eyes was significantly lower than the maximum intensity of the light source and did not increase linearly with the increasing light intensity. I found that visibility of the patient relied on the light level, increasing as the light measured increased. My research of optimizing light levels for visibility and safety will inform approaches to remote patient monitoring during neonatal transports. Next steps include: to determine minimum acceptable lighting conditions for patient visibility, to establish minimum lighting conditions for hyperspectral imaging, to compare the spectral properties of measured light with existing data from ophthalmology literature on the safety of the light for newborn eyes to regulate the safest amount of light required for visibility.

The Developmental Origins of Health and Disease (DOHaD) hypothesis evaluates how the prenatal environment affects health after birth. The placenta is a multi-faceted organ that sustains life during human development and is key to evaluating the DOHaD hypothesis. Glial Cells Missing Transcription Factor 1 (GCM1) is a transcription factor that plays a critical role in placental development. Our goal is to understand the downstream effects of GCM1 on various genes necessary for placental development by evaluating gene expression after GCM1 knockdown. The BeWo choriocarcinoma cell line is a model of placental syncytiotrophoblasts cells which undergo a cell fusion process called syncytialization in the placenta to form multinucleated cells that help exchange nutrients. Previously, we knocked down GCM1 in full-term primary placental cells that spontaneously syncytialize and assessed gene expression using RNA sequencing. We identified 10 differentially expressed genes. Based on those findings, we hypothesized that GCM1 plays a greater role during early pregnancy leading us to repeat the GCM1 knockdown in BeWo cells. BeWo cells were treated with 20µM, 50µM and 100µM forskolin (FSK) for 48hr to induce syncytialization which was confirmed via qPCR of syncytialization markers GCM1 and Syncytin-2 and through fluorescence microscopy. GCM1 expression increased 3.15, 1.3, and 1.2 fold respectively after treatment with 20µM, 50µM, and 100µM FSK, whereas Syncytin-2 increased 78.1 fold after 50µM FSK treatment. We then performed an siRNA knockdown of GCM1 in unsyncytialized BeWo cells with two concentrations of siRNA (25nM and 50nM) for 24hrs and observed a 70% and 80% reduction in GCM1 expression, respectively. Next steps include optimizing the siRNA procedure for syncytialized BeWo cells and comparing these results to our previously conducted experiment. Overall, this will improve understanding of how GCM1 coordinates gene expression in the placenta during pregnancy.

Human brain organoids (HBOs) are multicellular, three-dimensional tissue structures, created from human donor stem cells. Journalists and scientists commonly refer to HBOs as “mini brains” when communicating to lay audiences. Given the brain’s central role in our sense of personal identity, it is important to be thoughtful about the terminology used to describe this research. In this project, I am seeking to understand how terminology impacts lay perceptions of organoid research. We interviewed 59 participants who donated biospecimens for HBO research using the term “brain organoid,” to query their perspectives on HBO research. They were then asked about the alternative term “mini brains.” I qualitatively coded interview transcripts for key themes. Analysis revealed the majority of participants preferred the term “brain organoid.” Perceived benefits of “brain organoid” included sounding more scientific, accurate, and respectful; drawbacks included overcomplication and necessity of explanation. Perceived benefits of “mini brain” included sounding more relatable and matching mental imagery; drawbacks included inaccuracy, over-exaggeration, and feeling misled. Of those who indicated a preference for “mini brain,” most had misconceptions about the level of sophistication of organoids. “Mini brain” was more commonly associated with fears about cloning or misuse. These results suggest the terminology used to describe a novel neurotechnology like HBOs could shape public perception of research. Although “mini brains” may seem like the more accessible term, we believe that properly explaining “brain organoid” promotes scientific literacy and accessibility to neuroscience for the general public. Use of accurate terminology conveys respect for lay audiences and discourages misinformation which could raise undue ethical concerns. Future research will include analyzing data from a survey where participants are randomized to “mini brain” or “brain organoid” versions to assess a large population for differences in responses based on terminology.

Joubert syndrome (JS) is a neurodevelopmental condition diagnosed by the appearance of the “molar tooth sign” on axial brain magnetic imaging (MRI). Patients display hypotonia, abnormal eye movements, and ataxia. Substantial progress has been made on identifying the genetic causes of JS, which typically displays recessive inheritance. Nonetheless, the cause cannot be identified in ~25% of our cohort of JS-affected families. The contribution of variants that impact RNA splicing remains unknown. Our goal is to evaluate the role of noncanonical splice variants in the pathogenesis of JS. Canonical splice variants impact RNA splicing by disrupting the splice site directly, whereas noncanonical splice variants may affect it through alternative mechanisms, which need to be validated by RNA analysis. We previously identified genetic causes in 520 of 679 families with JS. To identify additional causes, we used SpliceAI (SpliceAI score >0.5) to identify candidate variants that impact splicing. We extracted RNA from patient cell lines and converted it into complementary DNA (cDNA). Then we used polymerase chain reaction (PCR) to amplify the affected exons with two sets of primers flanking the relevant splice junction. We evaluated PCR product size and sequence using gel electrophoresis and Sanger sequencing. We found 74 families with ≥1 canonical splice variant. An additional 34 families have ≥1 candidate noncanonical splice variant. We confirmed the pathogenicity of two of the candidate noncanonical splice variants by demonstrating an abnormal splicing event in AHI1 and MKS1 in two patient samples. By extrapolation from our data in JS, noncanonical splice variants may contribute as much as 10% to the genetic causes of recessive conditions. A precise genetic diagnosis informs prognosis, avoids unnecessary work-up, guides monitoring for associated complications, and opens the door to gene-specific treatments.

Chronic non-bacterial osteomyelitis (CNO) is an autoinflammatory disease that predominantly affects children. Osteoclasts are cells that carry out normal bone degradation, and their hyperactivity contributes to pathogenesis of CNO. We do not yet understand how the environment and precursor cells contribute to hyperactivity. We are examining the effects of serum on osteoclastogenesis, the differentiation of osteoclast precursors, from peripheral blood mononuclear cells (PBMCs). Serum from patients with active CNO, inactive CNO, juvenile idiopathic arthritis (JIA), and healthy control participants was collected and used at different concentrations (0.1%, 1%, 10%) to determine their respective effects on osteoclastogenesis. With IRB approval, we sorted PBMCs from ZenBio for monocytes and plated them with media, receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). Cells were then incubated and media exchanged twice. Tartrate-resistant alkaline phosphatase (TRAP) was used to stain osteoclasts and DAPI was used to stain nuclei. All TRAP positive osteoclasts with three or more nuclei were counted using ImageJ. We then compared counts across serum types and concentrations to identify trends in osteoclastogenesis. Over the course of this project, we encountered issues with the TRAP stain quality and imaging. In response, we modified the procedure at various steps to normalize staining quality across all plates to facilitate the quantification of osteoclast formation. Our results thus far suggest there is a trend of increase in osteoclasts as serum concentration increases with the exception of inactive CNO serum. Given that this study is ongoing and methods are frequently improved, we expect to gain a more comprehensive view of the trends as we progress. The goal of this study is to gain a better understanding of the pathogenesis of CNO, and its results have the potential to guide treatment of CNO and future research.

Currently, nearly 10% of Americans have Type 2 Diabetes (T2D) and a further 34% have prediabetes - placing it among the most common chronic diseases in the United States. The capacity of the brain to elicit sustained remission of diabetic hyperglycemia in rodent models of T2D following intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) is now well established. Previously, we reported that prolonged (>24 hr) hypothalamic signaling by extracellular signal-related kinases 1 and 2, members of the mitogen-activated protein kinase (MAPK) family, is required for the sustained antidiabetic action of FGF1. Recent studies have now revealed that in addition to FGF1, FGF17 also stimulates the MAPK/ERK signaling pathway, which promotes oligodendrocyte proliferation and overall neurocircuit remodeling. Based on this observation, we hypothesize that FGF17 would have potent antidiabetic actions by activating hypothalamic MAPK/ERK signaling and remodeling hypothalamic neurocircuits. To test this hypothesis, cohorts of adult male C57BL/6J wild-type mice recieved a single icv injection of either FGF17 or saline vehicle. Analysis revealed that FGF17 induced sustained hypothalamic MAPK/ERK signaling, though the magnitude of this induction was less than that obtained with FGF1. We then injected FGF17 or a saline vehicle icv in a cohort of adult, male, diabetic ob/ob mice (diabetic and obese due to mutation in the leptin gene) and took daily measurements of blood glucose, food intake, and body weight. We found that FGF17 induced a transient reduction in food intake and body weight, but was able to normalize diabetic hyperglycemia for 30 days. This data shows that FGF17 is able to induce sustained activation of hypothalamic MAPK/ERK signaling and has antidiabetic actions. This is significant because sustained remission of hyperglycemia can reduce the risk of developing associated chronic diseases such as cardiovascular disease, diabetic nephropathy, and diabetic retinopathy.v Currently, I am performing immunohistochemical studies to identify the specific hypothalamic neurocircuits that FGF17 acts upon to produce these effects.

 Cystic fibrosis (CF) is a progressive, life-threatening disease, that results from the formation of thick mucus that builds up in the lungs, digestive tract, and other parts of the body. It leads to severe respiratory and digestive problems as well as other complications including opportunistic infections and diabetes. CF is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Recently, the initiation of highly effective CFTR modulators including Trikafta (a combination of the medications elexacaftor, tezacaftor, and ivacaftor) has significantly improved the quality of life and life expectancy of patients with CF. However, recent clinical studies have shown that CF patients taking Trikafta have an increased risk of developing obesity and diabetes, though the underlying mechanisms remain unknown. In addition to being expressed in peripheral tissues, the Cftr gene is also expressed in the brain in the arcuate nucleus (ARC), a key brain area involved in metabolic regulation. To begin testing the hypothesis that Trikafta predisposes to metabolic syndrome by altering the activity of signaling of neurocircuits that regulate metabolism in the ARC, I investigated the ability of Trikafta to activate neurons in the ARC of mice (based on histochemical detection of c-Fos, a marker of neuronal activation). Following a single intracerebroventricular injection of Trikafra, compared to vehicle-treated mice, I found that mice treated with Trikafta had significantly increased activation of neurons in the ARC. I am now conducting studies to identify the phenotype of the neurons in the ARC that are activated by Trikafta and predict that successful completion of these studies will advance our understanding of the pathogenesis of obesity and metabolic impairment induced by Trikafta and inform the development of strategies that can avert these deleterious side effects.

This project examines developmental atypical patterns of visual attention in infants in relation to Autism Spectrum Disorder (ASD). Research in this area could help identify additional, specific risk groups or factors that could facilitate focused research that translates to real-world applications. Specifically, this project examines how cognitive development relates to visual attention to faces versus activities at 12 and 24 months of age among different birth weight groups. Developmental scores will be evaluated through data collected using: the Mullen Scales of Early Learning (Mullen), a developmental test measuring cognitive and motor development, and the Vineland Adaptive Behavior Scales (Vineland), a caregiver-interview measuring child adaptive skills. Visual attention will be quantified using eye tracking data which measured proportions of looking towards faces versus activities in social scenes. Participants in the lab were split into two groups, low birth weight and regular birth weight, and were seen by researchers at both 12 months and 24 months. Mullen, Vineland, and eye tracking tests were conducted at both timepoints. Science shows that as infants grow, they focus less on faces and more on the activities they are doing. I anticipate similar effects in eye tracking, with increasing age associated with a higher mean difference in preference for activities versus faces. Uniquely, I hypothesize that the strength of the relationship between looking at activities and developmental skills will be greater at 24 months than it will be at 12 months, and the opposite will be true for looking at faces. We will test our hypotheses on a linear regression model that predicts developmental skills from factorial effects of time point, birth weight, and region of eye tracking preference. This project hopes to seek to understand the interaction between birth weight, age, and attentiveness to faces versus activities as they relate to developmental skills.

Hypoxic-ischemic encephalopathy (HIE), a brain injury that occurs when infants do not get enough blood flow or oxygen to the brain, is a leading cause of neonatal mortality and morbidity worldwide. Therapeutic hypothermia (TH) is the current standard of care for newborns with HIE, but TH is only available in high-resource settings and only provides partial neuroprotection. Thus, the search for additional neuroprotective treatments is critical. The ferret provides an excellent model for investigating novel treatments for HIE as, unlike rodents, it has a gyrified brain and gray-to-white matter ratio that is like humans. Previous research has shown that the ferret brain is resilient to brain injury, requiring additional hypoxia periods and increased pro-inflammatory stimuli to create the same injury as in rats. However, no previous studies have evaluated why the ferret brain is so resilient. This study will use live rat and ferret organotypic brain slices to investigate this resiliency. Whole hemisphere brain slices from postnatal day (P)10-12 rats and P21-23 ferret, equivalent to term gestation in humans, will be collected. The slices will be randomized to oxygen-glucose deprivation (OGD) injury, to mimic HIE, or control groups. OGD slices will be in 0% oxygen for 2 hours, resulting in partial, but not total, cell death. Subsequent analyses will assess transcriptomics using NanoString nCounter technology, which provides a neuropathology panel of 770 genes, as well as cell-specific regional death in brain regions affected by HIE: hippocampus, cortex, corpus callosum, subcortical white matter, basal ganglia, and thalamus. Preliminary results show that genes such as UCHL1 and TLR4, both associated with injury, are upregulated in ferret OGD slices, but rat data are currently incomplete. Identifying the pathways associated with the resiliency of the ferret brain to injury at the transcriptome level could inform future therapies to treat infants at risk for HIE.

Birth asphyxia is the inability of a newborn to begin and maintain breathing. Twenty-three percent of neonatal deaths globally are caused by birth asphyxia [1]. Birth asphyxia results in a neurological injury called hypoxic ischemic encephalopathy (HIE). Rapid HIE screening within six hours after birth is crucial to identify neonates at risk. Unfortunately, the diagnostic equipment is impractical for low resource settings because it is costly ($20/test and $5,000 for equipment) and requires technical staff, that are in short supply, to operate. We hypothesize that a cost-effective device can be developed for HIE analysis. pHast Cam quickly screens for birth asphyxia and HIE in infants via a paper-based blood pH sensor. The device combines an inexpensive pH sensitive dye, a smartphone camera, and a fixture that controls the imaging environment to quickly identify acidosis that results from HIE. A low-cost paper-based strip is made with a water-soluble resin doped with a pH-sensitive dye, bromothymol blue (BTB), and a membrane to filter out red blood cells. The fixture removes lighting variation. The smartphone camera records the pH indicator image, and an algorithm captures, reduces noise, and accesses color change. pHast Cam incorporates four features: 1) accurate assessment of acidity within 0.05 pH units, 2) require only a few microliters of blood, 3) use electrical hardware and software only from the smartphone, and 4) affordability. At this stage, we have achieved a regressive linear model that predicts buffered solution acidity. In the future, we will transition from measuring buffered solutions to blood-plasma. Ultimately, we expect pHast Cam to screen for HIE by quantifying plasma pH in neonates so that timely therapeutic interventions and plans to address long-term complications may occur. [1] Diaz-Rosello JGP, Niermeyer S, et al. WHO Basic guidelines on new born resuscitation. 2012.

 Hemophilia A is an inherited bleeding disorder caused by mutations in the human coagulation Factor VIII (FVIII) gene resulting in mild to severe loss of clotting ability when bleeding. While protein replacement therapy has greatly decreased the mortality and morbidity of HA, 30% of patients develop FVIII neutralizing antibodies (inhibitors) that bind to FVIII protein in the blood and initiate clearing of FVIII from circulation leaving patients vulnerable to life-threatening uncontrolled bleeding events. N-linked glycosylation is a post-translational modification known to modulate the functional and antigenic properties of proteins. Deletion of glycans at the N2118 site of FVIII has been shown to significantly reduce the immunogenicity due to the elimination of a potent glycopeptide epitope that can interact with and activate T-cells, however, more investigation is needed to characterize the humoral immune response in detail. Here we describe efforts to optimize a method for the production and isolation of cell-derived mannosylated peptides for use in ongoing humoral immune response characterization experiments. We chose to use cell-derived peptides rather than synthetic peptides so the glycoforms will closely resemble the plasma-derived human FVIII and cell-produced recombinant FVIII proteins used clinically. SDS-PAGE and LC-MS analysis confirmed our peptides contain the desired mannosylated glycoform and preliminary results demonstrate that the peptides were able to generate a robust immune response in HA mice as intended. Going forward, these peptides will be used to generate an immune response in mice so we can characterize the inhibitor binding affinity to our peptides, assess the specificity of splenic CD4+ T cell receptors and determine the T helper cell differentiation status via cytokine assays. The findings of these experiments will hopefully lead to the development of a more immune-tolerant FVIII protein product for protein replacement and gene therapy treatments for HA.

Postpartum depression (PPD), with prevalence rates in East Africa ranging from 17% to 24%, is associated with adverse health outcomes among offspring of affected mothers including emotional and developmental delays and poor growth. The 2022 World Health Organization (WHO) maternal and newborn care recommendations call for routine PPD screening using a validated screening tool. In Rwanda, a low-income country severely impacted by the 1994 genocide, routine PPD screening has not been implemented. This study was conducted to describe the prevalence of PPD among new mothers and determine sociodemographic characteristics and health factors associated with PPD and recent suicidal ideation. Postpartum mothers delivering a live birth at the Kabutare District Hospital (KDH) in Huye, Rwanda between August and September 2022 were recruited for this study. This study was a cross-sectional survey administered via face-to-face interviews conducted in Kinyarwanda. Following written consent, the mothers responded to sociodemographic, and maternal/ infant health questions, and completed the Edinburgh Postnatal Depression Scale (EPDS). Postpartum depression was defined as an EPDS score of > 10. Data collection was approved by the KDH Ethics Committee. Our study population consisted of 66 Kinyarwanda-speaking mothers. Over half (52%) had PPD, and 26% had suicidal thoughts in the past 7 days. Many reported a history of depression (39%), PPD (18%), or anxiety (29%). Mothers with a history of depression, anxiety, or PPD were more likely to have PPD and recent suicidal ideation. There was a greater prevalence of PPD among mothers reporting pregnancy-related complications or a history of mental illness compared to their counterparts (70% vs. 44%, p < 0.05; 67% vs. 36%, p < 0.05). Mothers at particularly high risk for PPD are those with pregnancy-related complications and a history of mental illness. These findings demonstrate a need for routine PPD screening among new mothers, as recommended by WHO.

Inherited retinal diseases (IRDs) are a diverse family of disorders which cause vision loss and retinal degeneration. With only 1-2% of the genome being protein-encoding, genetic variation within the expansive noncoding genome is critical to the development of disease phenotypes in the retina. Macular Telangiectasia Type II (MacTel) is an IRD resulting in disruption of central vision and greatly impacting vision-related quality of life. MacTel has an estimated prevalence of 1 in 1000 individuals, affecting approximately two million people globally. Though MacTel etiology largely remains unknown, accumulation of improperly degraded lipids within the retina is a leading hypothesis in its pathogenesis. Additionally, genome-wide association studies have implicated numerous loci in the development of MacTel, including the novel gene locus ceramide synthase 4 (CERS4). As CERS4 plays a critical role in the synthesis of lipid precursors and is highly expressed in the retina, it stands as a promising candidate for influencing MacTel development. We hypothesize that cis-regulatory element (CRE) mutations are central to the genetic frameworks underlying MacTel. We aim to characterize the sufficiency of putative enhancer regions to drive gene expression. We have identified potential CERS4 enhancer regions through a machine learning approach using adult human retina ATAC sequencing datasets. Sufficiency of candidate enhancer regions will be evaluated by insertion to a barcoded reporter library and electroporation into mouse retinas. Following proof of sufficiency, we will perform saturation mutagenesis on identified enhancers to investigate the impact of all possible single nucleotide variants (SNVs) within these regions. The results of our investigation will aid in identifying SNVs of interest within the CERS4 locus, potentially implicating specific mutations towards the development of MacTel. Greater understanding of CRE mutations will improve early clinical diagnosis and inform future therapies for patients with MacTel.

HIV infection impairs B cell function, in turn, altering immunoglobulin production and function. Immunoglobulins (Igs) exist as isotypes including IgA, IgG, and IgM. Within the IgA and IgG isotypes there are subclasses IgA1-2 and IgG1-4 respectively; all with distinct functions. Previous studies have shown that HIV infection influences Ig isotype and subclass concentrations in serum, but few have explored their concentrations in the breast milk of mothers living with HIV (MLHIV). Widespread use of antiretroviral treatment during pregnancy has led to an increase in the incidence of HIV-exposed and uninfected infants (iHEU). iHEUs have heightened immune activation and inflammation and display high infectious morbidity compared to compared to HIV-unexposed infants. It is plausible that immune factors transferred in breast milk contribute to altered immunity in iHEU. Therefore, knowing whether HIV infection impacts total immunoglobulin concentrations or inflammatory biomarkers in breast milk is important. I used enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of immunoglobulin isotypes and their subclasses and Luminex to profile cytokines and chemokines in breast milk 4 weeks after delivery. Assays were performed according to manufacturers’ instructions and sample values were extrapolated from a standard curve. I compared these factors between MLHIV and uninfected mothers using Mann-Whitney U test. MLHIV had significantly higher mean concentrations of total IgG1 (36.9 ug/mL versus 26.6 ug/mL, p=0.018) and IgG3 (2.9 ug/mL versus 1.3 ug/mL, p=0.0013). There was no difference in concentrations of IgA and IgM between the groups. Furthermore, MCP-1, MIP-1-β, and SDF-F-α were the most abundant chemokines in breast milk in both groups. However, we found no significant difference in concentrations of cytokines and chemokines in breast milk of MLHIV versus controls. Overall, we observed increased breast milk concentrations of IgG1 and IgG3 in MLHIV. Future work will explore implications of these IgG subclasses on iHEU immunity.

Leishmaniasis, caused by various species of Leishmania, is a global public health burden, with the World Health Organization reporting over a million cases in the last 5 years, leading to over 20,000 deaths annually. Leishmania and closely related organisms have the unique ability to glycosylate thymidine (Ts) residues to form a base termed Base J. Previous work by the lab has shown that de novo insertion of J is initiated by a protein called JBP2 that is targeted to specific regions of the genome by J2-TDP, a Tudor domain protein. Tudor domains bind to methylated arginine or lysine residues that are most commonly found in histones. To determine if targeting JBP2 to a genome location was sufficient to initiate formation of J, we wanted to target JBP2 to a region of the genome that does not normally contain J. To achieve this, we took advantage of the ability of the tetracycline repressor (TetR) to bind to the operator (TetO) by fusing JBP2 and J2TDP separately to TetR and expressing the proteins in Leishmania harboring a TetO cassette in a locus that does not contain J. I constructed a cassette with the necessary components which contain in order- a drug selectable marker, TetO sites, and a GFP, as a reporter gene to monitor any possible effects J has on the cells when inserted. Using CRISPR/Cas9, I inserted this construct into Leishmania that expressed the TetR fusion proteins. I then grew these strains in the presence or absence of Tet and then determined if J was present at this location. The results of these findings will give us a deeper understanding of the molecular mechanism(s) responsible for this specificity as well as this will offer potential opportunities for development of novel therapeutic agents against Leishmania.

Adoptive T cell therapy presents a promising possibility of a safe and effective therapeutic for a wide range of cancers through utilizing CD8+ T cell-mediated killing of targets expressing tumor-associated antigens (TAA). However, developing these therapies require knowledge of a T cell receptor’s (TCR) antigenic specificity. My project aims to find the antigen specificity of CD8+ T cells from a patient with an exceptional response to hematopoietic cell transplantation (HCT). We hypothesize that leukemia-specific T cells may have helped prevent early relapse and can be identified in samples from this patient. Bone marrow T cells from the patient post-HCT were previously identified and their TCRs were sequenced. By using genetic engineering to knock out (KO) endogenous TCRs on a healthy donor’s T cells and then transducing the patient’s TCRs into the T cell via lentivirus, TCR-transduced healthy donor T cells can be prepared for functional testing. These TCR KO, TCR transduced lines are expected to provide more consistent responses in functional assays due to the presence of only one TCR on the cell surface. . The Long Killing Assay, a flow cytometry-based cytotoxicity assay requiring the targets to be in co-culture with T cells long enough to ensure T cell-mediated killing, will be utilized for functional testing. This will determine whether target cells presenting a variety of antigens are killed by the TCR-expressing T cells. After the assay, the target cells will be further studied and deconvoluted to determine the resulting TCR antigen specificity. This project will be important in refining our lab’s method of determining antigen specificity of reconstructed TCRs and potentially provide insight on the antigens responsible for antileukemic immune responses. 

Macular telangiectasia type II is a late-onset retinal degeneration disease which causes loss of central vision and a disruption in cell class proportions in the retina. Genome-wide association studies have identified a point mutation in the 5q14.3 enhancer as associated with MacTel. This enhancer has been shown to regulate the activity of microRNA 9-2. To elucidate the function of this particular enhancer on retinal health and cell class composition, both enhancer knockout and miR9-2 mice models were generated. In adult enhancer knockout mice, there were no significant changes in cell class composition compared with wild-type mice. In the miR9-2 knockout mouse model, it was found that at the 5 week time point, there was a significant increase in müller glial cells. Müller glial cell loss has been observed in Mactel patients, and these cells have been shown to play a crucial role in maintaining proper vascular networks. Future experiments to determine the effects of enhancer and miR9-2 loss on vasculature in the retina would help further identify the role of the 5q14.3 enhancer and its targets on retinal health.

The placenta is a crucial fetal organ providing oxygen and nutrients to the developing infant. Researchers typically use placental cell models to study the placenta, which are derived from immortalized cells. The use of in-vitro placental cell models is important because human samples are difficult to obtain, and placental biology is highly species-specific. Despite this, our understanding of the characteristics of these cell lines and how they compare to placental tissue samples is limited. This project aims to determine which placental cell model most directly reflects the gene expression of the human placenta. RNA sequencing data from the placental cell models HTR-8/SVneo, JEG-3, BeWo as well as data from primary trophoblast cells was obtained using the Gene Expression Omnibus (GEO) database or through lab-generated datasets. Data for each cell line was combined into a single dataset of shared genes (n=6835) and individual datasets of genes unique to each cell model. For genes that were unique to each cell model, I performed KEGG pathway analysis and characterized placenta specific genes using the Human Protein Atlas (HPA). BeWo cells expressed the highest number of unique genes (n=355) and shared the highest number of genes with the primary trophoblast cells (n= 1,167). Pathway analysis showed that genes unique to primary trophoblast cells (n=2661) were overrepresented in 24 pathways, unique BeWo genes were overrepresented in 15 pathways, while unique HTR-8/SVneo genes (n=355) were overrepresented for a single pathway- the neuroactive ligand-receptor interaction. Placenta specific genes were expressed within the uniquely expressed genes for JEG-3 (n=1), BeWo (n=1), and primary trophoblast cells (n=17), but not HTR-8/SVneo. Ultimately, the results from this project will provide a tool to evaluate differences in placental cell models and aid the placental biology research community in understanding which cell line is most representative of human placental tissue samples.

The period around birth is when neonates are at the highest risk of neurological injury or death. A common neonatal neurological injury is hypoxic-ischemic encephalopathy (HIE), which occurs after the brain does not receive enough oxygen or blood flow. There is a large disparity in the severity and long-term neurodevelopmental outcomes of HIE between high-income countries (HICs) and low-and-middle income countries (LMICs). In HICs, HIE occurs in 1-4 neonates per 1,000 births. In LMICs, the instance of HIE is at least 2-3 times higher. Furthermore, cases of HIE seen in LMICs suggest a different type of injury - a more prolonged intermittent injury resulting in white matter injury - compared to HIE in high-income countries that is more acute and affects the deep grey matter. Therapeutic hypothermia (TH) has been the standard of care for HIE in HICs; however, TH is not an effective treatment for HIE in LMICs. Thus, the creation of alternative and accessible therapies for HIE in LMICs is crucial. This study will seek to model HIE as seen in LMICs through an in vitro ferret model that may be used to pilot therapies before applying them to in vivo models. Organotypic brain slices from postnatal day (P) 21 ferrets, equivalent to a term neonate, will be cultured and randomized to receive increasing intervals of oxygen glucose deprivation (OGD), with and without serum deprivation. Serum deprivation is defined as culturing in 2.5% serum as opposed to the standard 5% to mimic certain aspects of malnutrition that may be more common in LMICs. Cell death and white matter injury will be assessed 24 hours after OGD. We hypothesize that slices with more rounds of intermittent OGD and serum deprivation will display relatively more cell death and white matter injury, thus serving as a model of HIE in LMICs.

The mammalian target of Rapamycin (mTOR) signaling cascade plays an important role in a variety of cellular processes, such as autophagy, cell proliferation, and protein synthesis. Previous depictions of signaling through the mTOR pathway have suggested linear signal transduction; however, this does not accurately represent the network of interactions between proteins in complexes of this pathway nor their dynamics in response to stimuli. To better characterize mTOR protein interaction network (PIN) dynamics, SEPS lab has developed a panel of antibodies targeting key proteins in mTOR signaling for use in quantitative multiplex co-immunoprecipitation (QMI), a method of detecting changes in protein interactions using flow cytometry. Following QMI of mTOR signaling proteins in serum-starved and serum-refed mouse 3T3 fibroblasts, I validated changes in select interactions from this pool separately via co-immunoprecipitation and western blot analysis. The lab then applied inhibitors of mTOR pathway constituents, including PI3K, AKT, MEK, ERK, and mTOR, to define modules of interactions that comprise the PIN and observe changes in these interactions with stimulation after application of each inhibitor, which I again validated via co-immunoprecipitation and western blot analysis. Finally, to validate antibody specificity in human cells, I prepared human embryonic kidney 293 (HEK293) cells for short interfering RNA (siRNA) transfection and knockdown of mTOR pathway proteins targeted by antibodies from the initial panel. Assuming these HEK293 cells lack any additional proteins with high affinity for these antibodies, I expect flow cytometry data to reflect specificity seen in the 3T3 fibroblasts. Conducting this validation is critical for ensuring the reliability of the PIN changes observed in QMI analysis. These experiments allow us to evaluate coordinated interactions between mTOR pathway proteins and their dynamics during signaling events, which is highly useful in developing treatment strategies for mTOR pathway-associated disorders.