ItemStimulation of NOD2 in Acute Myeloid Leukemia activates Natural Killer Cells and improves survival(The Ohio State University, 2023-05) Murugesan, Rakesh; Tridandapani, SusheelaAcute Myeloid Leukemia (AML) is a hematological malignancy of the myeloid lineage of immune cells in the blood and bone marrow. It is characterized by low five-year survival rates of 27.4%, and high incidence of relapse, indicating a need for novel therapeutic strategies. A signature of AML is the evasion of immune surveillance by Natural Killer (NK) cells, which are immune cells possessing anti-leukemic capabilities. NK cell activating receptors are downregulated in AML-NK cells, and these cells exhibit defective cytotoxic ability. Recently, our lab has found that treatment with MTP-PE (a synthetic ligand for NOD2 used for the treatment of osteosarcoma) along with IFNγ provided a survival benefit in a murine model of AML. Data from this study show that blood-derived NK cells isolated from mice treated with MTP-PE and IFNγ show greater levels of maturation, as indicated by markers CD27 and CD11b. Additionally, we saw increased expression of activation/degranulation markers, CD69 and CD107a, on healthy donor NK cells in in vitro cocultures with AML patient samples. This study is focused on elucidating the mechanism by which NK cell activation occurs when AML is treated with MTP-PE and IFNγ. Preliminary data suggests that ligand-receptor cell-to-cell contact between AML cells and NK cells is necessary to elicit the observed NK cell activation. In this study, we show that MTP/IFNγ treated AML cells strongly upregulate IL-15, an important cytokine involved in NK cell expansion and activation, as well as ligands of the receptor NKG2D. In AML patient samples, we have found the co-treatment to upregulate CD69 and CD107a on suppressed patient NK cells, both in peripheral blood and apheresis. We also observed expansion of NK cells in these patient samples. Further study is necessary to evaluate and understand the potential of MTP-PE and IFNγ to activate suppressed NK cells in Acute Myeloid Leukemia and improve anti-leukemic immunosurveillance. ItemInhibition of Ras-like Protooncogenes A and B, RALA & RALB, Sensitize Soft Tissue Sarcomas to Radiation Therapy(The Ohio State University, 2023-05) Chakravarthy, Prathik; Sizemore, StevenTreatment options for patients with soft tissue sarcomas (STS) have changed very little over the past 50 years. While radiation treatment (RT) improves outcome for patients with many types of cancer, STS are notoriously RT resistant. Understanding the mechanism(s) of this resistance and how to overcome it will improve outcomes for STS patients. Recent approaches to understand radiation resistance in cancers have placed an emphasis on uncovering key genetic alterations that are helping to confer this radiation resistance. RALA and RALB are small GTPases that are differentially expressed in a variety of cancer and have been shown to play a role in conferring radiation resistance. In STS the RALs are also altered in expression making them promising targets to study the issue of radiation resistance in STS. The role of RALA and RALB in STS radiation resistance were investigated through in vitro assays studying cellular response to radiation with variable expression of RALs. Radiation responses analysis included cell survival and sustained DNA damage. RALA and RALB expression was experimentally decreased in STS cell lines to study the roles of the RALs during RT. Colony forming assays were performed to understand how the survival and growth of STS cells were altered with and without the expression of RALA and/or RALB. Additionally, DNA damage, one of the major responses to RT, was analyzed post-radiation in STS cells in which RAL expression was once again experimentally decreased. Assays involved immunofluorescence methods measuring the presence of yH2AX, a biomarker representative of DNA damage. Knockout or depletion of the RAL small GTPases inhibited the radiation resistance of STS cancer cells. Clonogenics assays showed that in vitro when the RALs were depleted there was a decrease in the surviving fraction of STS cells after radiation, particularly when RALB was depleted. This showed that increase in RAL could be responsible for radiation resistance in STS and inhibiting RAL expression could re-sensitize cells to RT. DNA damage post-radiation showed similar results with RAL depletion resulting in increased sustained DNA damage after radiation exposure. This once again shows that when RAL expression was experimentally decreased there was a more effective radiation response as the cells sustained a greater amount of DNA damage. Together these data show the significant role that RALs play in conferring radiation resistance to STS and perhaps a possibility in inhibiting RAL expression to sensitize STS cells to RT. Further investigation will be necessary to understand the mechanisms by which RALA and RALB are conferring this resistance for the eventual use of RALs as targets for more effective RT. ItemCell-type specific transcriptomic signatures of neocortical circuit organization and their relevance to autism(The Ohio State University, 2023-05) Moussa, Anthony; Wester, JasonA prevailing challenge in neuroscience is understanding how diverse neuronal cell types select their synaptic partners to form circuits. In the neocortex, major classes of excitatory projection neurons and inhibitory interneurons are conserved across functionally distinct regions. There is evidence these classes form canonical circuit motifs that depend primarily on their identity; however, regional cues likely also influence their choice of synaptic partners. We mined the Allen Institute's single-cell RNA-sequencing database of mouse cortical neurons to study the expression of genes necessary for synaptic connectivity and physiology in two regions: the anterior lateral motor cortex (ALM) and the primary visual cortex (VISp). We used the Allen's metadata to parse cells by clusters representing major excitatory and inhibitory classes that are common to both ALM and VISp. We then performed two types of pairwise differential gene expression analysis: (1) between different neuronal classes within the same brain region (ALM or VISp), and (2) between the same neuronal class in ALM and VISp. We filtered our results for differentially expressed genes related to circuit connectivity and developed a novel bioinformatic approach to determine the sets uniquely enriched in each neuronal class in ALM, VISp, or both. This analysis provides an organized set of genes that may regulate synaptic connectivity and physiology in a cell-type-specific manner. Furthermore, it identifies candidate mechanisms for circuit organization that are conserved across functionally distinct cortical regions or that are region dependent. Finally, we used the SFARI Human Gene Module to identify genes from this analysis that are related to risk for autism spectrum disorder (ASD). Our analysis provides clear molecular targets for future studies to understand neocortical circuit organization and abnormalities that underlie autistic phenotypes. ItemInhibition of BET proteins by novel inhibitor, PLX51107, regulates Fc-gamma receptor function and reduces inflammation in rheumatoid arthritis(The Ohio State University, 2021-05) Shankar, Divya; Tridandapani, SusheelaMembers of the Bromodomain Extra-Terminal Domain (BET) family — BRD2, BRD3, BRD4, and BRDT — are key regulators of transcription. BET inhibition is associated with reduced immune responses, making the BET family a potential therapeutic target for autoimmune diseases, including rheumatoid arthritis (RA). Autoimmune disease pathogenesis is dependent on the overactivation of immune responses. This includes the heightened production of inflammatory cytokines, like TNFα, and secretion of autoantibodies, such as isotypes of rheumatoid factor and anticitrullinated protein antibody (ACPA), which target healthy tissues. Fcγ receptors (FcγR) expressed on the surface of myeloid cells bind Immunoglobulin G (IgG) immune complexes; FcγRIa, FcγRIIa, FcγRIIIa are activating FcγR, whereas FcγRIIb is an inhibitory FcγR. Recognition of autoantibodies by FcγRs induces an inflammatory phenotype that results in tissue damage and further escalation of the inflammatory response. Here we examine a novel BET inhibitor, PLX51107, and its effect on regulating FcγR expression and function in RA. PLX51107 substantially upregulated FcγRIa and downregulated FcγRIIa, FcγRIIb, FcγRIIIa, and FcϵR1-γ (γ-chain) — FcγRIa and FcγRIIIa's associated signaling protein — mRNA and protein levels in healthy donor and RA monocytes. Consistent with this, PLX51107 treatment decreased phagocytic and rosetting indexes, FcγR-mediated NF-κB, AKT, and MAPK signaling, and TNFα production, indicating the attenuation of the immune response. Our findings suggest that BET inhibition may have clinical relevance in the treatment of RA. Further studies are critical to explore how BET inhibition mechanistically regulates FcγRs to alleviate their mediated inflammatory responses, and to determine if BET inhibition, via PLX51107, is therapeutic in a murine model of RA. ItemAn Evaluation of Porosity and Bone Mineralization and Relationships with Fracture Risk in Human Tibiae(The Ohio State University, 2022-05) Goldsmith, Carla; Harden, AngelaBone quality assessment tools in clinical settings are used to evaluate fracture risk in living patients. Additionally, ex-vivo analyses of individual bones have assessed bone quality through examination of porosity and mineralization.1–3 However, a comprehensive understanding of relationships between demographics (e.g., age, sex, body size) and variables associated with bone quality is lacking. Understanding the factors contributing to bone quality will improve diagnosis of osteoporosis, a disease characterized by bone loss, and identify high fracture risk populations (e.g., older individuals, females). The objective of this study was to investigate the relationships between porosity and mineralization in human tibiae with sex, age, body size, and bone mineral density (BMD) and assess differences in bone quality assessment via methodology (clinical vs histological vs compositional). Sections from human tibiae were collected at the 66% site (n = 14) and the 15% site (n = 16) for porosity and mineralization analyses, respectively. Areal (aBMD) and volumetric (vBMD) BMD, collected via dual x-ray absorptiometry and quantitative computed tomography scans, values were compared to porosity, mineralization, and individual-level variables (e.g., sex, age, body size) to determine if these variables had relationships with these clinical bone quality assessment tools. Overall, few relationships were observed within this study; however, males (M=1.11 g/cm2) exhibited greater aBMD than females (M=0.93 g/cm2), mineralization had a negative correlation with age, and height and weight had positive correlations with vBMD. Future research should investigate porosity and mineralization utilizing a larger sample size, wider age range, and more varied body sizes per sex to better evaluate bone quality. Overall, a variety of bone- and individual-level variables should be used to make accurate assessments of bone quality to inform individuals' fracture risk. ItemOptimization of Novel Lipid Nanoparticle Formulations for mRNA Delivery(The Ohio State University, 2022-05) Bohmer, Margaret; Dong, YizhoumRNA research has heightened in prevalence due to the emergence of mRNA-based vaccines for the SARS-CoV-2 virus, but there is still a need for carriers to deliver future mRNA-based vaccines and therapies. The aim of this study was to develop and optimize formulations for novel lipid nanoparticles (LNP) to deliver mRNA in different cell lines. One novel LNP, CW51, was selected for formulation optimization via an orthogonal array. These optimized formulations were able to transfect JAWS II dendritic and C2C12 skeletal muscle cells with luciferase mRNA. These findings indicate that the lead LNP warrants further investigation into its potential in mRNA-carrying nanoparticles. ItemAssessing the Impact of Adding Scenario-based Augmented Reality Training to Traditional Textbook Training on Accurately Identifying Airway Obstruction Symptoms(The Ohio State University, 2020-05) Mansour, Lauren; Patterson, EmilyRecent developments in the portability, flexibility, and affordability of augmented reality (AR) technology allow it to address some limitations of current medical training. This pilot study aims to determine whether AR technology can enhance the detection and identification of subtle cues used to make accurate medical diagnoses. We randomly divided five participants into a control group, who received diagnostic training by reading a standard textbook, and an experimental group, who received diagnostic training by a combination of reading a standard textbook and AR-based training. Participants then encountered a tension pneumothorax scenario on the AR patient and were asked to list the symptoms they observed before making a final diagnosis. We recorded both the total amount of time participants required to make a final diagnosis as well as the number of correctly identified symptoms, and the results of the control and experimental groups were compared with a t-test. We found no significant difference between groups in the time needed to make a diagnosis. However, the AR-trained participants correctly identified a significantly greater number of symptoms (average: 12 symptoms) compared to the textbook-trained participants (average: 5 symptoms). These results indicate the potential of AR-based training to enhance detection and identification of cues in medical diagnostics. ItemComparative analysis of Salmonella enterica serovar Typhi isolates from acute and chronic infections.(The Ohio State University, 2018-05) Eichar, Bradley; Gunn, JohnSalmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever- a human-specific disease that results in approximately 200,000 deaths each year. With appropriate treatment, most patients recover from the acute stage of the disease. However, 3-5% of S. Typhi infections lead to a chronic infection and asymptomatic colonization in the host gallbladder – allowing carriers to unknowingly infect others despite no outward signs of illness. While it is known that S. Typhi mediates carriage through the formation of biofilms on the surface of cholesterol gallstones in the gallbladder, molecular mechanisms for chronic S. Typhi carriage are not well understood. This project compared genetic, molecular, and functional characteristics of 22 different S. Typhi isolates from confirmed acute and chronic infections, hypothesizing that the components necessary to mediate chronic carriage in the gallbladder may vary and be unique from acute clinical isolates. Biofilms of the isolates were examined utilizing a crystal violet quantification assay and confocal microscopy. The presence and relative abundance of Vi-antigen and lipopolysaccharide (LPS) were confirmed using dot blot assays with specific anti-Vi-antigen and anti-LPS antibodies and subsequent densitometric analysis. Additionally, immunofluorescent microscopy was used to evaluate Vi-antigen and LPS expression. The presence of cellulose was detected quantitatively via a microtiter-based calcofluor binding assay. Finally, the genome-sequence as well as pulsed-field gel electrophoresis (PFGE) patterns of all strains were determined. PFGE and the sequencing data informed phylogenetic relatedness determinations of the strains. Biofilm and extracellular matrix (cellulose, Vi-antigen, and LPS) expression studies revealed unexpected high variability between the S. Typhi strains. Preliminary analyses do not support the uniqueness of the chronic versus acute isolates. Additional analysis will provide a better understanding of how salmonellae enter and persist in the chronic carrier state, which may lead to targeted drug therapies to alleviate the carrier state. ItemKicking in infants with perinatal stroke: can we predict ability and disability?(The Ohio State University, 2016-08) Durbak, Emily; Tanner, Kelly; Heathcock, JillStroke in very young infants is the most common cause of cerebral palsy, a chronic disorder of motor function impairments with lifetime physical, social, and emotional consequences. While stroke is typically diagnosed within the first week of life, cerebral palsy is diagnosed much later, at 18-24 months of age. Spontaneous kicking is one of the few early movements that has been shown to be indicative of motor ability in young infants with motor disorders. This is a longitudinal trial of children 2.5-7.5 months describing spontaneous kick quantity and quality in infants with and without stroke and those who go on to have CP. Infants were tested in 2 conditions: with and without toys. N=29 infants completed this study (n=11 with perinatal stroke (PS); n=18 typically developing (TD)). Medical chart review at 3 years of age determined cerebral palsy status of infants with stroke. Infants were videotaped kicking freely for eight, thirty-second trials, six with toys, two without. Videos were then analyzed by two trained blinded raters with overall agreement of 0.96. Infants with cerebral palsy showed a statistical interaction with infants without cerebral palsy for total kicks (p=0.019) and two of three kick subtypes (p=0.041; p=0.029). The no toy condition yielded more kicks than the toy condition for total kicks (p=0.025) and two of three kick subtypes (p=0.050; p=0.065). Our findings support the possibility that spontaneous kicking may be used to identify infants with motor deficits at much younger ages than is currently possible. The use of toys in lower extremity analyses is cautioned. ItemElucidating the Role of UbE2K in the Post-Translational Regulation of the Tumor Suppressor PTEN(The Ohio State University, 2016-05) Lordo, Matthew; Majumder, Sarmila; Leone, GustavoThe tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN) is one of the most frequently altered genes in human cancer. Germline mutations in PTEN result in autosomal dominant hamartomatous syndromes, including Cowden Syndrome, a disease that carries an increased risk for breast, thyroid, and endometrial cancer. Although genetic and epigenetic alterations account for PTEN loss in many cancers, in breast cancer there is a very low incidence of mutation in PTEN. Loss of PTEN has been shown to significantly increase the rate of tumor progression in multiple mouse models of breast cancer, including ErbB2 induced mammary tumorigenesis, and plays an important role in both the epithelial and stromal compartments. These data also correlate with human disease progression, as our studies demonstrate that patients with low PTEN protein levels have worse disease outcome compared to patients with high levels of PTEN protein (unpublished data). However, PTEN mRNA levels in this patient cohort are not predictive of disease outcome. Our observation that 30-40% of the breast cancer patients have a reduction in PTEN protein levels without significant alteration of its expression suggests that post-translational events plays a key role in regulating PTEN protein level in breast cancer. Thus, a global shRNA screen to identify putative regulators of PTEN protein was undertaken in the laboratory. A subset of the identified PTEN degradome are components of the ubiquitin-proteasome pathway. Treatment of cells with the proteasome inhibitor MG132 results in an increase in total PTEN protein, supporting our observation. We chose to study one of these genes, ubiquitin conjugating enzyme E2K (UbE2K), to elucidate its mechanism of action in regulating PTEN protein levels. ItemExploring the Role of an Adipocyte-Specific Knockout of MHCII on Inflammatory-Driven Complications of Obesity in Female Pro-atherogenic Mice(The Ohio State University, 2021-05) Middendorf, Dana; Hsueh, WillaThe adipocyte, in addition to functioning as an energy storage cell, is an important immune cell that links the innate and adaptive immune system. In obese humans, our laboratory has previously demonstrated that the major histocompatibility complex II (MHCII) pathway is one of the most differentially expressed between lean and obese adipocytes. Further work in high fat diet fed mouse models demonstrates that increased adipocyte MHCII expression resulted in pro-inflammatory T cell differentiation leading to insulin resistance and decreased glucose tolerance. In contrast, loss of adipocyte MHCII expression in aMHCII knockout male mice resulted in less inflammatory adipose tissue characterized by increased adipose tissue immunosuppressive regulatory T cells (Tregs) and prevented the development of insulin resistance and glucose intolerance induced by Western high fat diet (WD). Moreover, these mice had less atherosclerosis and less liver steatosis and inflammation, common complications of obesity. We, thus, aimed to understand the impact of the loss of aMHCII expression in female mice on these and other inflammatory complications. We crossed aMHCII KO mice with proatherogenic low density lipoprotein receptor knockout (LDLR-/-) mice, aged the female animals for one year to accelerate atherosclerosis and hepatic injury, and fed them WD for twelve weeks (n=12 control, n=12 aMHCII KO). Glucose (GTT) and insulin (ITT) tolerance tests, body fat composition and fasting blood were collected prior to WD start and at study end. At the time of sacrifice, visceral adipose tissue was collected for T cell flow analysis and gene expression, liver was collected for liver fat analysis and histology, and aortas were collected for atherosclerotic lesion area measurement. Results showed that the female aMHCII KO mice experienced a significantly lower percent weight gain over 12 weeks than wild-type (control) littermate control mice, despite demonstrating no significant difference in overall percent body fat. The knockout mice additionally demonstrated improved glucose tolerance after 12 weeks of WD, compared to the control mice (AUC control = 23090.0 +/- 520.9 vs. KO= 20442.3 +/- 657.9, P=0.0045), although no difference was observed in insulin sensitivity. No difference was found in circulating plasma triglyceride or cholesterol levels; however, liver fat accumulation was significantly decreased in KO mice (control= 17.14 +/- 0.93 %fat vs. KO= 13.89 +/- 1.04 %fat, P=0.039). Finally, no difference was found in atherosclerosis % lesion area of the aorta. These results support that the adipocyte knockout of MHCII leads to improvement in the metabolic and hepatic phenotype in female mice after 12 weeks on WD. However, as the females demonstrated some variation from the effects seen in male mice, further investigation into the cause of these differences is warranted. ItemCharacteristics of Ohio women that did not receive reproductive and sexual healthcare(The Ohio State University, 2021-05) Foster, Abigail; Norris Turner, AbigailObjective: The American College of Obstetricians and Gynecologists (ACOG) recommends that a person with female reproductive anatomy receive their first Obstetrics and Gynecology (OBGYN) visit at ages 13-17, and continue to have annual women's health visits throughout their lifetime.1 Though this recommendation is well known, whether there is equitable access for all adult Ohioans with female reproductive anatomy is unknown. Previous research has determined that barriers to accessing reproductive healthcare in the United States are disproportionately experienced by marginalized individuals2 and it is critical to determine if this relationship holds true in Ohio, a state with vastly different community spaces from urban, to suburban neighborhoods and rural farming communities. Through this analysis, I analyze the characteristics of Ohio women who experienced reduced accessibility of reproductive and sexual healthcare. Methods: The Ohio Survey of Women is a population-representative survey that captures data about reproductive health and demographics, including age, race and sexuality, among women in Ohio. This survey was conducted by NORC in 2018 through 2019 and sampled among women of reproductive age living in Ohio (18-44 years). Using unadjusted logistic regression, I identified characteristics of Ohio women that are associated with not receiving an annual women's health visit in the past year. Results: A total of 2,613 participants completed the survey. After removing respondents who were missing data on the primary variables of interest, I was left with an analytic sample of 2,434. Race (Black OR= 1.18, CI= 0.75, 1.86; Hispanic OR= 0.91, CI= 0.49, 1,66; Multiracial/other OR=1.44, CI= 0.95, 2.16), living in rural Appalachia (OR=1.05, CI= 0.82, 1.32), and being currently pregnant (not pregnant OR= 1.52, CI= 0.77, 3.03) were not significantly correlated with not obtaining an annual women's visit. Individuals that identified as a sexual minority (OR=1.52, CI= 1.11, 2.08), were young (aged 18-24 OR=2.35, 95% CI= 1.86, 3.27), lacked health insurance at some point over the last year (OR= 4.51, CI= 3.10, 6.56), were not employed (OR=1.50, CI= 1.16, 1.94) and had low socioeconomic status (less than 75,000 U.S. dollars a year, some college or less OR=2.26, CI=1.72, 2.99; income more than 75,000 U.S. dollars, some college or less OR=1.86, CI= 1.28, 2.70; income less than $75,000, bachelor's degree or higher OR=1.43, CI=1.03, 1.99) had significantly higher odds of not obtaining an annual women's health visit in the last year. Conclusion: It is critical to identify which Ohio women are most likely to miss their annual women's health visit. When advocating for a system of nondiscriminatory healthcare access, we must identify the groups experiencing the most barriers to access in order to create interventions that better serve these groups. ItemVAPING WITH NICOTINE REDUCES LEFT VENTRICULAR CARDIAC FUNCTION IN ADOLESCENT MALE MICE(The Ohio State University, 2021-05) Neczypor, Evan; Wold, LorenElectronic cigarettes (ECs) are the most commonly used tobacco product by teens and young adults. The aim of this study was to determine the potential effects of EC exposure on cardiac health in adolescent mice. Four-week-old FVB mice were exposed to EC vapor with 20.2 mg/mL of nicotine or vehicle alone for either 3 weeks or 3 months. Mice were exposed to HEPA- filtered air as a control (FA). Body weight was significantly reduced in mice exposed to EC vapor with nicotine versus mice exposed to the EC vehicle or to the FA control. Echocardiography revealed that male, adolescent mice displayed reductions in fractional shortening and impaired diastolic function after exposure to EC with nicotine, but not in response to vehicle alone. RT- qPCR demonstrated that EC exposure with nicotine caused an increase in left-ventricular COL1A1, and COL3A1 expression after 3 weeks of exposure, but this was reversed after 3 months of exposure. Trichrome staining showed increased vascular fibrosis following exposure to the EC vehicle for 3 months. Male mice exposed to EC vapor with nicotine had elevated serum concentrations of 3 inflammatory cytokines and 2 growth factors. While serum nicotine concentrations in male and female mice exposed to EC with nicotine were similar, concentrations of multiple metabolites were significantly lower in females. Further, the 3- hydroxycotinine/cotinine metabolite ratio, a measure of enzymatic nicotine metabolism (CYP2A5 activity), was significantly higher in females vs males. Further RT-qPCR targeting liver Cyp2A5 also showed increased expression in females vs males, suggesting a more rapid rate of enzymatic nicotine metabolism in females. These results heighten the concern for the dangers of EC use, specifically in youth, and call for further work detailing the mechanistic contributions to the observed reduction in cardiac function. The novel results of this study demonstrate that EC vapor exposure with nicotine can reduce cardiac function in developing male mice. ItemEffects of Sdhd-H50R Missense Mutation on Thyroid Tumorigenesis(The Ohio State University, 2019-05) Chakravarthy, Karthik; Kirschner, LawrenceThyroid cancer is among the most common cancers and is the most frequent type of endocrine malignancy. Increased risk of thyroid cancer has been associated with Cowden syndrome (CS), an inherited endocrine tumor disorder. CS has been associated with germline mutations in PTEN tumor suppressor gene, identified in about 85% of CS-associated and 10% of sporadic thyroid cancers. Germline variations in Krebs cycle enzyme complex succinate dehydrogenase (SDH) genes were first observed in patients with pheochromocytoma/paraganglioma and recently discovered in patients with CS. Moreover, CS patients with variants in succinate dehydrogenase subunit D (SDHD) gene either alone/in combination with PTEN mutation have increased thyroid and breast cancers risk compared to patients with PTEN-only mutations. Studies examining SDHD germline variations have identified the His50Arg (H50R) variation as associated more frequently with sporadic thyroid tumor cases in patients. Therefore, modeling this tumor progression in vivo is essential to better understand how such mutations in the human patient population lead to thyroid cancer. Previous studies of mouse models with thyroid-specific Sdhd deletion displayed larger thyroid volumes, decreased follicle size, and increased cell proliferation. Novel mouse models with thyroid-specific Sdhd deletion and/or global Sdhd H50R mutation were generated using CRISPR-Cas9 genome editing technology, yielding a heterozygous Sdhd H50R mutant founder mouse to generate experimental Sdhd H50R mouse models: SdhdH50R/Wt, SdhdH50R/H50R, and Tpo-SdhdH50R/- (heterozygous Sdhd H50R mutation with thyroid-specific deletion of wildtype allele). While SdhdH50R/H50R mice were viable and born in normal Mendelian ratios, early demise was observed in certain SdhdH50R/H50R mice, potentially indicating a mortality associated phenotype in mutant models. Survival analysis displayed lower trends of viability between SdhdH50R/H50R and SdhdH50R/Wt in comparison to the SdhdWt/Wt models at 12 months. To assess whether the demise could be related to physiological wasting, weights were recorded for the models, which revealed that the SdhdH50R/H50R had lower body weights compared to wildtypes. Ultrasound imaging of the thyroid glands revealed relatively little difference between wildtype and SdhdH50R/H50R thyroid glands. Pathology of the SdhdH50R/Wt founder mouse revealed that there were no tumors present within the thyroid glands. However, closer analysis of thyroid tissue revealed dysplasia of follicles and greater cellular proliferation compared to SdhdWt/Wt. In addition, histology of the adrenal glands in mutant models have displayed cellular alterations that were also indicative of cellular dysplasia, implying alterations in other endocrine glands. These effects may suggest putative initiation of tumor development and potential progression to cancer, lending support that the Sdhd H50R mutation may cause thyroid tumorigenesis due to a gain-of-function and/or dominant-negative effect instead of the postulated loss-of-function method. ItemStress-induced anxiety is dependent upon caspase-1 processing of active IL-1β in brain macrophages(The Ohio State University, 2015-05) Atcheson, Kyle; Godbout, JonathanThe murine repeated social defeat (RSD) model reveals that neuroimmune signaling is implicated in the development of stress-induced anxiety-like behavior. Work from our lab showed that the anxiety-like behavior exhibited following RSD is associated with microglial activation, increased recruitment of brain-macrophages, and increased IL-1b expression in the brain. Moreover, this neuroinflammatory process was dependent upon IL-1 receptor 1 in the CNS. To determine the source of cytokine production following stress, NanoString mRNA analysis was conducted on FACS-sorted microglia and brain-macrophages. This revealed that brain macrophages rather than resident microglia are the primary source of increased IL-1b mRNA production in the brain following repeated social defeat. To address the functional role of IL-1b in the development of anxiety-like behavior, caspase-1 knockout (KO) mice were used. Caspase-1 is required for the cleavage of pro-IL-1b into its active form. Results show that caspase-1 KO mice did not develop anxiety-like behavior following RSD. Furthermore, our data indicates that caspase-1 KO mice do not exhibit increased microglial activation as characterized by Iba-1 immunolabeling. However, caspase-1 KO mice still had increased peripheral macrophage trafficking to the brain and increased IL-1b mRNA expression in the brain. We interpret these results to indicate that the development of anxiety-like behavior is dependent on functional IL-1b released by brain macrophages that is cleaved by caspase-1. ItemThe effect of ball-handling on lower extremity mechanics in soccer(The Ohio State University, 2016-05) Pradarelli, Jared; Chaudhari, Ajit; Onate, JamesNearly 240,000 soccer injuries are estimated to have occurred in the United States in 2014 with a high number of them non-contact in nature and involving the lower extremities. These injuries result in time-loss from training or match play, potential psychological consequences, and financial burdens. Epidemiological research suggests that these non-contact injuries may occur more frequently while ball-handling compared to defending in soccer. However, no prior studies have investigated the biomechanical implications of controlling a soccer ball with the feet while running and cutting that may help explain this finding. The purpose of this study was to compare knee and ankle joint moments and angles implicated in non-contact soccer injury mechanisms demonstrated during run-to-cut maneuvers performed with and without dribbling a soccer ball. Our hypothesis was that the cutting maneuvers performed while dribbling a ball would have a detrimental effect on biomechanical parameters associated with non-contact ankle and knee injuries. Seventeen healthy male collegiate soccer players participated in the study. Subjects performed ball-handling and running maneuvers while running straight ahead and also at a 45° cutting angle. All data were collected using three-dimensional motion capture with force plates embedded in the floor. Ball-handling had a significant effect on peak ankle internal rotation angle (p=0.010) and knee abduction angle (p=0.024). Changes in other parameters of interest, including peak ankle inversion moment and peak knee abduction moment, did not reach significance (p>0.05). In conclusion, ball-handling in soccer can detrimentally alter lower extremity joint mechanics of dynamic movements. The results of this study support the need for coaches to consider the implications of an athlete’s sport-specific movements when creating training programs for teams and individuals. ItemInvestigating the Interactions Between Protocadherin-19 and Ryk and its Effect on Neurogenesis(The Ohio State University, 2016-05) Liebau, Brandon; Jontes, JamesProtocadherins are a large, diverse family of neural cell adhesion proteins, but their functions are not understood. Mutations or in several protocadherins have been associated with neurological disorders. For example, Protocadherin19 (PCDH19) causes epilepsy in females with mental retardation (EFMR). In this X chromosome-linked disease, girls randomly express one good allele or one mutant allele per cell due to a phenomenon known as X-linked inactivation. This mosaic expression leads to the symptoms classified as EFMR, but the effects on cellular pathways for the disease are not known. In zebrafish, the loss of Pcdh19 leads to the loss of columnar organization in the developing optic tectum through the loss of adhesion and an increase in neuronal proliferation and differentiation. The literature shows that Pcdh19 interacts with Ryk, a noncanonical Wnt receptor involved in neurogenesis. Based on previous data and the literature, we hypothesize that Pcdh19 binds to the extracellular domain of Ryk to inhibit binding of the Wnt3 ligand, preventing the intracellular domain of Ryk from being cleaved and translocated to the nucleus where it would initiate pathways for neuronal proliferation. Immunohistochemistry and co-immunoprecipitation were attempted to show if Pcdh19 affects cleavage of the Ryk intracellular domain, its nuclear translocation, and subsequent Wnt signaling. Transgenic and Ryk-knockout fish are being developed to perform experiments in vivo. ItemOncolytic HSV-mediated Regulation of the Host Hypoxia Response(The Ohio State University, 2014-05) Hurwitz, Brian; Kaur, BalveenGlioblastoma (GBM) is the most common and deadly primary brain tumor, accounting for over 10,000 new cancer diagnoses in the United States each year. The poor prognosis for GBM patients necessitates novel biological treatments. One such approach is the use of oncolytic herpes simplex virus 1 (oHSV). Like many novel treatments oHSV therapy causes off target effects that are not yet well understood. Our lab has demonstrated that oHSV treatment increases the vascularity of brain tumors. The goal of this study is to determine the mechanism by which oHSV treatment increases the vascularity of brain tumors. Preliminary data suggested the hypoxia inducible factor-1 alpha (HIF1α) may be activated in cells infected with oHSV, even in normal oxygen conditions. HIF1α is a transcription factor, which activates a variety of genes in response to a lack of oxygen. We hypothesized that HIF1α activation may be responsible for the increased vascularity of oHSV treated brain tumors. A screen of targetscan.org for the predicted target genes of herpes simplex virus 1 (HSV-1) revealed multiple miRNAs predicted to target a protein called, factor inhibiting HIF1α (FIH). This protein functionally inhibits HIF1α activation by preventing the binding of HIF1α to DNA. We believed that FIH would be negatively regulated in GBM cells infected with oHSV, thus allowing HIF1α activation. In this study, we demonstrate that HSV-1 expresses two miRNA molecules, which target and down regulate FIH. Transfection of miRNA inhibitors (antagomirs) was able to successfully abrogate the virus' ability to down regulate FIH as demonstrated by quantitative PCR and western blot. Moreover, transfection of HSV-1 miRNA mimics in the absence of virus was able to down regulate FIH protein levels (western blot) and activate the expression of a variety of HIF1α driven genes, including VEGF and IL-8 (quantitative PCR). However, after analyzing HIF1α promoter activity in a variety of cell lines, we determined that HIF1α was not activated during oHSV infection. The expression of VEGF and IL-8 was likely due to the activation of another transcription factor. ItemThe Role of GLI-1 in Endocrine Resistant Breast Cancer(The Ohio State University, 2014-05) Rudolph, Mark; Majumder, SarmilaEstrogen receptor positive (ER+) and estrogen receptor negative (ER-) are two major types of breast cancer. For women with ER+ positive breast cancer, patients are treated with the antiestrogenic compounds, tamoxifen or faslodex for five years, immediately after surgical resection of tumors. Unfortunately, 30-40% of these patients will develop resistance to endocrine therapy. Our recent study has shown that the Hedgehog (Hhg) signaling pathway plays a significant role in endocrine resistance and that the aberrantly activated transcription factor, GLI-1, is vital to the development of resistance. However, not much is known about the GLI-1 target genes that might contribute to endocrine resistance. Our goal is to determine novel target genes of GLI-1 and determine how these genes promote endocrine therapy resistance. ItemThe Role of Interleukin-4 in the Neuroprotective Benefits from Social Interaction(The Ohio State University, 2014-05) Fung, Christine J.; DeVries, A. CourtneyStroke is a leading cause of death in the US and results in over $73.7 billion in health care costs every year. Strong social support has been shown to reduce mortality in cardiovascular disease and stroke, yet little is known about the physiological mechanism underlying these health benefits. Extensive tissue damage following stroke results in part from activated microglia, the immune cells of the brain. Oxytocin, a hormone released during social interaction, increases expression of Cluster of Differentiation 200 (CD200), a neuronal marker known to suppress microglial activation. We hypothesize that Interleukin-4 (IL-4), an anti-inflammatory cytokine produced by T-cells, is involved in the oxytocin-mediated mechanism through which social interaction conveys neuroprotection by upregulating expression of CD200 and complement receptor 1-related gene/protein-y (Crry). Using an animal model of social interaction, we show that oxytocin, IL-4, CD200, and Crry are increased in the brains of pair housed mice relative to isolated mice and that exogenous administration of IL-4 in vitro upregulates neuronal CD200, Crry, and oxytocin receptor gene expression. However, IL-4 does not alter oxytocin gene expression. An absence of IL-4 also eliminates the increase in CD200R found in paired mice compared to isolated mice. These findings further elucidate the physiological mechanism by which social interaction is beneficial to one’s health, and may lead to new methods to reduce the risk for and improving recovery from multiple diseases such as stroke and cardiac arrest.