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  Astrocytic Influences in the Auditory Brainstem in Fragile X Syndrome

  Warner, Katharine D; School of Medicine

  Fragile X Syndrome is one of the leading genetic causes of autism spectrum disorder. Fragile X Syndrome is associated with auditory hypersensitivity and childhood audiogenic frontal-temporal lobe seizures, which may be the result of abnormalities in the auditory nuclei found in the brainstem. The purpose of this research is to expand on preliminary findings that dysfunction in the auditory brainstem contributes to sound sensitivity and subsequently to frontal-temporal lobe seizures. To test the role of the LSO, VCN, and MNTB we examined the astrocyte function in these areas by extracting the auditory brainstem from Fmr1 KO mice and wild type mice. We then co-cultured these astrocytes with neurons from either mouse genotype to determine differences in the neuronal size, morphology, and synaptogenic factors. Our research examined the levels of SPARC, hevin, and thrombospondin-1, which are synaptic proteins found in the synapses in the auditory brainstem. Our results showed that Fmr1 KO astrocytes altered the size of WT neurons to a size comparable to a standard Fmr1 KO neuron, and Fmr1 KO astrocytes secreted significantly less SPARC than WT astrocytes. Overall, abnormal astrocyte function among Fmr1 KO mice was found to cause morphological changes in auditory brainstem neurons and differences in the amount of the SPARC found in the auditory brainstem between wild type and Fmr1 KO mice.
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  WHAT DOES MIDDLE SCHOOL TEACH? THE CURRICULUM OF BEING/BECOMING MIDDLE SCHOOL TEACHERS

  Warren, Erica Adela; Tift College of Education

  This post-critical ethnographic study explored the curriculum of being/becoming a middle school teacher that 5 emergency-certified new-to-teaching teachers experienced through the quotidian interactions of their first fully in-person school year. The curriculum of being/becoming teachers is increasingly important as the percentage of teachers entering the profession through alternative and emergency certification pathways increases each year and school and district leaders inherit more of the responsibility to prepare and develop these teachers. The purpose of this study is to describe how participants developed a sense of students, content, and contexts through the curriculum of being/becoming teachers. Additionally, this study describes how the instructional coach/researcher co-developed and evolved a new teacher community of practice (NTCOP) that pushed participants toward becoming more sensitive, humane, and empathetic curriculum makers. Three questions guided this inquiry: (1) What are some of the core teachings in the curriculum of being/becoming teachers at this school site as evidenced by policies, practices, and relationships between teachers and students in place during their inaugural school year?; (2) In what ways does engagement in a learning group aimed at intervening in the curriculum of being/becoming teachers interact with new-to-teaching teachers' understanding of who they become, what they know, and how they interact with students and as middle school teachers?; and (3) What questions do new-to-teaching teachers’ experiences grappling with the curriculum of being/becoming teachers and emerging teacher identity raise about the ways districts and schools provide support? In middle schools, where nearly a third of adolescents experience academic and social challenges due to developmental and cultural mismatches between teachers and students, this study’s findings suggest that the curriculum of being/becoming teachers reinforced these mismatches in three ways: school and district leaders assigned teaching tasks as a mechanism of control, middle school policies and practices reinscribed deficit narratives about adolescents, and administrators and colleagues cultivated a hostile environment for new-teacher learning. The NTCOP provided a counter-space for participants to disrupt deficit narratives and to discuss and affirm placemaking practices. However, the participants did not adopt active advocacy stances toward adolescents despite the researcher’s efforts.
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  CEREBROVASCULAR COMPLICATIONS OF SARS-CoV-2 IN TYPE-2 DIABETES

  Burnett, Faith; School of Medicine

  Diabetics are more vulnerable to SARS-CoV-2 cerebrovascular complications, including brain fog, cognitive impairment, and strokes. This study aims to identify the molecular mechanisms of SARS-CoV-2-induced cerebrovascular dysfunction in diabetics. We hypothesize that SARS-CoV-2 exacerbates diabetes-induced cerebral oxidative stress and inflammation via activation of the destructive arm of the renin-angiotensin system (RAAS) and toll-like receptor (TLR) signaling. Methods: SARS-CoV-2 spike protein interacts with human ACE2 receptors but not murine Ace2. Therefore, type-two diabetes was induced in humanized ACE2 (hACE2) transgenic knock-in mice using low-dose streptozotocin followed by eight weeks of a high-fat diet. Recombinant SARS-CoV-2 spike protein was injected intravenously in control and diabetic mice. Cognitive functions were tested using Y-maze and Barnes maze. Cerebral blood flow was measured using laser speckle imaging. RAAS system and TLR signaling were assessed using RT-PCR and western blot analysis. The cerebrovascular architecture was measured using immunohistochemistry. Human brain microvascular endothelial cells were treated with hyperglycemia (25 mM glucose) to mimic diabetic conditions. Results: Spike protein exacerbated diabetes-induced cerebrovascular oxidative stress and inflammation as detected by increased (NOX1, NOX5) and (Il-6, Il-1β, and TNF-α) gene expression, respectively. Spike protein enhanced the destructive RAAS arm (angiotensin ll and AT1R) at the expense of the RAAS protective arm (ACE2 and AT2R) gene expression (P<0.05). In parallel, spike-protein exacerbated TLR signaling in diabetes as indicated by the increase in TLR-8 receptor and its ligands (HMGB1 and S100) and downstream adaptor proteins (MyD88, TRAF6, and NF-κB) expression (P<0.05). Spike-protein increased cerebrovascular rarefaction and decreased blood flow and cognitive functions in diabetes compared to control (P<0.05). Conclusion: SAR-CoV-2 spike protein intensified RAAS and TLR signaling in diabetes, increasing cerebrovascular damage and cognitive dysfunction. Targeting RAAS and TLR singling are possible therapeutic strategies to protect against SAR-COV-2-induced cerebrovascular dysfunction in diabetes.
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  Deciphering The Effect of Xenobiotic Exposure on The Immunobiology of Human Mesenchymal Stem Cells & Their Interaction with Dendritic Cells

  Uwazie, Crystal Chidinma; School of Medicine

  Mesenchymal Stromal Cells (MSCs) are nonhematopoietic multipotent stem cells that possess a myriad of immunomodulatory and regenerative functions and thus maintain immune physiology and tissue homeostasis in the body. MSCs primarily carry out these functions through intercellular immune interactions involving paracrine secretion of secretory factors such as cytokine, chemokines, and growth factors. Previous study has extensively shown that MSCs display immunosuppressive properties on immune cells of lymphoid lineage; however, MSC’s interactions with cells of myeloid lineage such as dendritic cells require further research. Furthermore, given the central role of dendritic cells in immunity as antigen-presenting cells and the bridge between the innate and adaptive immune systems, it is important to understand how disruptions in the interactions between MSCs and dendritic cells can have implications on immune physiology. This project seeks to address this lack of knowledge by 1) identifying the interactions between human bone marrow derived MSCs and two circulating dendritic cells subtypes (plasmacytoid and myeloid) and by 2) defining the functionality of human bone marrow derived MSCs upon exposure to atrazine. MSC and dendritic cell interactions were investigated by conducting MSC and peripheral blood mononuclear cell (PBMCs) co-cultures under TLR7 or TLR4 stimulation. The presence of MSCs and TLR7 stimulation showed no effects on the function of the plasmacytoid dendritic cell subset. Contrarily, the presence of MSCs and TLR7 stimulation led to a decrease in maturation in the myeloid dendritic cell subset, as measured by CD83 expression using flow cytometry. Interestingly, MSCs present in a co-culture and TLR4 stimulation led to an increase in maturation in the myeloid dendritic cell subset. Secretome analysis of stimulated MSCs using multiplex assays suggests that secretory factors may be the reason for the results seen. Disruptions to MSC immune interactions were investigated by exposing MSCs to the xenobiotic herbicide atrazine at a range of dosages and for various durations of time. Cells were analyzed using an assay matrix comprising of MTT assays, flow cytometry, and multiplex assays. Atrazine was shown to affect MSC metabolic viability, size and granularity in a dose and time dependent manner. Atrazine exposure also modulates certain immunomodulatory and angiogenic secretory factors. Furthermore, atrazine exposure attenuates MSC responsiveness to exogenous cues, namely IFNγ. These results provide the context necessary in understanding how xenobiotic disruptions from atrazine exposure can affect immune interactions between MSCs and circulating dendritic cells.
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  Application of Recurrent Neural Networks for Pharmacokinetic Modeling and Simulation

  Khusial, Richard Darien; College of Pharmacy

  Pharmacometrics and the utilization of population pharmacokinetics play an integral role in model informed drug discovery and development (MIDD). Recently, there has been a growth in the application of deep learning approaches to aid in areas within MIDD. In this work, we aim to explore the potential of deep learning approaches towards drug concentration prediction and simulation. A total of 1,527 olanzapine drug concentrations sparsely sampled from 523 individuals along with eleven patient-specific covariates provided by the CATIE studies were used in model development, validation, and simulation. LSTM and LSTM-ANN with multiple inputs were investigated towards olanzapine drug concentration predictions. The LSTM-ANN model captured the relationships within a pharmacokinetic dataset and generated olanzapine drug concentration predictions with a lower RMSE than the LSTM model. Bayesian optimization was implemented to tune the hyperparameters of the LSTM-ANN model. The LSTM-ANN model had a RMSE of 29.566 in the validation set. A population pharmacokinetic model using NONMEM model was constructed as a reference to compare the performance of the LSTM-ANN model. The RMSE of the NONMEM model was 31.129. Permutation importance revealed age, sex and smoking were highly influential covariates in the LSTM-ANN model. The LSTM-ANN model showed potential in drug concentration prediction as it performed comparably to the NONMEM model. Future studies investigating clinical studies with varying sample sizes and sampling strategies are required to further examine the potential of a LSTM-ANN model towards drug concentration prediction. For olanzapine drug concentration simulations, three RNN cells within an RNN-ANN model with multiple inputs were studied. The GRU-ANN model resulted in the optimal RNN-ANN model with the lowest RMSE in the simulation data. Bayesian optimization was implemented to optimize the hyperparameters of the GRU-ANN model. The optimized GRU-ANN model resulted in a simulation RMSE of 24.844. Visual inspection revealed the simulated olanzapine drug concentrations were lower than their respected observed olanzapine drug concentrations. Exploratory data analysis revealed the underperformance may have been a result of dosing levels between the CATIE studies having little overlap. A comprehensive clinical trial study is required to fully explore the potential of a GRU-ANN model towards drug concentration simulations.

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