VIRTUAL STUDENT SCHOLARS SYMPOSIUM 2020 PRESENTATIONS

Cell Biology and Biochemistry - Poster Presentations

High School Division

Effects of β-Tubulin Mutations on Microtubules: First steps in targeted cancer treatment
Maya J Hunter, Cherry Creek High School

Microtubules, which form by stacking alpha- and beta-tubulin dimers on top of each
other, are involved in dividing the cell’s chromosomes during cell division, transporting proteins
and vesicles across the cell, and maintaining cell structural support. These proteins are coded at the beta-tubulin gene in the cell’s DNA. Microtubules are highly dynamic, being a highly
non-equilibrium system (Desai). This project is designed to study the effects of beta-tubulin
mutations on the behavior of microtubules in stressed environments, such as under
microtubule-targeting cancer drugs that affect the dynamic instability of microtubules. Yeast cells were mutated at the beta-tubulin gene and two mutations were found that grow well under Benomyl. These mutations were measured under two different microtubulin-targeting drugs to determine the mutations’ effect on microtubule dynamics, and visualized under a microscope. Results indicate that C354V grows better than wild type on Benomyl, and grows similarly to the wild type in Nocodazole and EpoA. The mutation G126L grows well in Benomyl, is resistant to Nocodazole, and its doubling time was much higher at higher concentrations of EpoA than wild type. Mutations were unaffected by temperature. Overall, mutations in the beta-tubulin gene affected the growth rates of yeast cells. Cancers with G126L/possibly similar mutations have more stable microtubules, making them responsive to low levels of microtubule stabilizing drugs, specifically EpoA. Cancers with C354V or G126L/possibly similar mutations are resistant to Benomyl, precluding it as an effective cancer treatment. Possible implications for targeted cancer treatments are discussed.

Epigallocatechin Gallate Blunts Inflammation Stimulated by Tumor Necrosis Factor-α in Liver Sinusoidal Endothelial Cells Under Varying Oxygen States
Emi Miyazaki, Blacksburg High School

Liver diseases result in over 2 million deaths annually around the world. Among such diseases, non-alcoholic fatty liver disease involves liver inflammation and fat buildup in liver cells. Nutraceutical products, such as green tea, have been researched extensively for health benefits to reduce such diseases. Epigallocatechin gallate (EGCG) is a catechin found in green tea leaves, and previous studies have shown EGCG to have potential anti-inflammatory effects. However, the effects of EGCG on inflamed cells in a low oxygen (hypoxic) state, which acts as an added stressor, are not fully understood. This experiment modeled the effects of EGCG on inflamed liver sinusoidal endothelial cells (LSECs) in both normoxic and hypoxic conditions using an in vitro technique. LSECs were put into a state of inflammation using tumor necrosis factor alpha (TNF-α). The effects of EGCG were analyzed in an adhesion assay to measure cell adherence and qPCR to measure VCAM-1 (an adhesion molecule upregulated by TNF-α) expression. EGCG decreased cell adherence and VCAM-1 expression in both normoxic and hypoxic conditions, but hypoxia was not found to have an effect on inflammation or the success of EGCG. While these results suggest the potential for EGCG to be an anti-inflammatory agent, future work should further research the hypoxia condition by analyzing a variety of adhesion genes, pretreating the LSECs with EGCG, and assessing the effect of the duration LSECs spent in hypoxia.

The Microenvironment of the Subventricular Zone Enhances the Malignancy of Glioblastoma Multiforme
Raha Riazati, Stanton College Preparatory School

Purpose: GBM (glioblastoma multiforme), a primary brain tumor, exhibits more invasive and malignant behavior when developed proximal to the subventricular zone (SVZ) of the brain. The SVZ is unique in that it is home to a large population of neural progenitor cells (NPCs), which are believed to be a possible cause of this increased malignancy in the SVZ.

Methods: GBM cells were cultured in a transwell system in a variety of conditions, either exposed to the conditioned media or actual cells of GBM cells or NPCs. The migration of the GBM cells across the semi-porous membrane was quantified via staining with DAPI and then compared across the different conditions to ascertain which condition most enhanced the migration of GBM.

Results: The GBM cells consistently migrated to a greater extent when in contact with actual cells versus just the conditioned media of the cells, whether the cells be GBM cells or NPCs. Also, the GBM cells appeared to migrate more when in contact with certain neural progenitor cell lines (F50) than when in contact with GBM cells.

Conclusion: Since the actual cells and the conditioned media of the cells produced markedly different effects on the behavior of GBM cells, some sort of cell-to-cell communication is most likely occurring between the cells in the transwell system that is not simply accounted for with the secretome of the cells. Also, it appears that neural progenitor cells do play a role in enhancing the migration and thus aggressiveness of GBM cells in vitro.

Bio Enzymatic Sensors: Environmental Health Monitoring System
Dev R Udata, Jesuit High School

In recent years several pollution monitoring programs in USA and Europe determined that environmental risk assessment cannot be based solely upon chemical analysis of environmental samples and bioassays. This approach does not provide any indication of deleterious effects of contaminants on the biota. Therefore, the measurement of the biological effects of pollutants at molecular and cellular level has become of major importance for the assessment of the quality of the environment. Bio enzymatic sensors are as measurable indicators at molecular and cellular level and can be used as sensitive “early warning” tools for biological effect measurement.
The commercially important giant prawn, Macrobrachium rosenbergii, spends its premature stages in brackish water where they are subjected to numerous environmental fluctuations. Therefore, the larval stages are extremely sensitive to pollutants and environmental factors. Due to that fact, this type of post larvae was selected for this study.
ATPases play important roles in intracellular functions for all types of physiological activities. The membrane bound Na⁺,K⁺-ATPase is ubiquitous in nature. This Na⁺, K⁺-ATPase machines moves Na, K ions across membranes which is crucial for cell function.
All the tested metals Cu, Cd, Cr, Pb, Zn and mixture of these metals affected Na⁺, K⁺-ATPase activity of the Macrobrachium rosenbergii post larvae. Na⁺, K⁺-ATPase activity of post larvae decreased with increasing metal concentrations and exposure time. The decrease in ATPase activity plateaued after certain concertation metals. Hanes-Woolf plot studies indicate that Cd and Cr inhibits Na⁺, K⁺-ATPase activity competitively. whereas Cu, Zn and Pb inhibits non-competitively in post larvae of Macrobrachium rosenbergii.
A prominent inhibition of Na⁺,K⁺-ATPase activity was along with good correlation between in vitro and in vivo studies indicates that the mechanism of inhibition is common in both conditions and this enzyme system is sensitive to heavy metals, it can be used as biosensor to monitor the environment.

The Effect of DCLK1 Inhibition on Cancer Stem Cell Properties in Early Pancreatic Cancer Development
Aditi Verma, Stanton College Preparatory High School

Pancreatic Cancer is a major cancer with a 5-year survival rate of ~9%. Current treatments for patients with Pancreatic Cancer are largely ineffective due to dense fibrotic tumor microenvironments that prevent drugs and therapies from reaching tumor cells. A form of PDAC development begins with oncogenic mutations in the KRAS gene that contribute to inflammation and ADM, which progress to PanIN formation, and eventually contribute to PDAC. The SM3 cell-line used is derived from murine PanIN lesions which develop from a KrasG12D mutation. Increased levels of DCLK1 were identified in this precancerous cell-line and reduced utilizing inhibitors: XMD8-92 and LRRK2-IN-1. Identifying molecular targets, such as DCLK1, in precancerous development, enhancing drug delivery systems against these novel targets, and understanding whether DCLK1 is sufficient to induce stemness is necessary to improve therapeutic results against drug-resistant PDAC. This study involves utilizing inhibitors to prevent DCLK1’s kinase activities to understand the protein’s functions. I analyzed the role of DCLK1 in the SM3 cell-line in vitro . The effect of DCLK1 inhibitors XMD8-92 and LRRK2-IN-1 was investigated in a cytotoxic assay, analyses of treatment in culture, immunofluorescence, western blot, and qPCR. A western blot was utilized to identify proteins and determine downregulation, and qPCR was used to analyze the effect of the inhibitors on mRNA levels of cancer stem cell markers using the genes of interest: DCLK1, Hes1, Prom1, Cd44, and Pou2f3. The results provide evidence that DCLK1 inhibitors affect both transcriptional and protein levels. We determine that DCLK1 inhibition induces pancreatic cancer cell death.

Modeling Distant Metastasis-Free Survival: Applications to Hazard Prediction and Pairwise Gene Interaction Discovery
Russell Yang, The Harker School

Models of time until metastasis (metastasis-free survival) based on microarray gene expression can be powerful tools for clinicians in quantifying risk, genetic research, and palliative assessment/care.

This project focused on modeling distant metastasis-free survival. We used 1 of 3 dimensionality reduction methods (PCA, Univariate Cox Regression, and Diffusion Mapping) to transform a set of 20,000+ genes to a smaller dataset. For each of 4 survival models (the Cox Proportional Hazards Model, a Ridge-Regularized Cox Model, Random Survival Forests, and a Cox Proportional Hazards Deep Neural Network), we evaluated a combination of dimensionality reduction and survival model using a nonparametric bootstrapping approach (B=100). We also tested model assumptions using Schoenfeld, Deviance, and Martingale residuals.
Using the survival models that we developed, we devised an algorithm to automatically identify pairwise (epistatic) gene interactions between MYC (a proto-oncogene) and other genes in the dataset. We performed a Kolmogorov-Smirnov test and found a significant difference between gene z-score density before/after MYC was added (p=0.008062).

We conducted gene set enrichment analysis (using Hallmark and c1 positional reference datasets) and pathway analysis.

The novelty of this project is that many cancer-related bioinformatics projects focus on pre-diagnosis problems, like detecting cancer in images. However, there’s a lot of potential for ML in post-diagnosis settings. The models developed in this study have critical applications in post-diagnosis settings: they can be used to predict risk of distant metastasis for any individual at any time after diagnosis and can help doctors realistically estimate time until end of life.

Undergraduate Division

Global Gene Expression Analysis in the Absence of a Non-Receptor Tyrosine Kinase During Post-Embryonic Development of the Nematode C. elegans
Tu N Hoang, Whitworth University

Fer-related kinase -1 (FRK-1) is a non-receptor tyrosine kinase that regulates cell proliferation and differentiation during embryonic development of the nematode Caenorhabditis elegans. During the embryogenesis, the movement of hypodermal cells allows for enclosure of the embryo. Furthermore, FRK-1 limits asymmetric Wnt signaling dependent for specification of a subset of hypodermal cells called seam cells and for endoderm proliferation.

A deletion of genomic frk-1, allele (ok760), results in larvae that are severely uncoordinated and fail to progress developmentally, arresting in the first larval stage. Our previous studies have shown that deletion of the frk-1 gene results in an excess number of seam cells, changes in cell morphology and alters expression of heterochronic regulators. Due to the severity of the frk-1(ok760) mutant phenotype, we sought to investigate FRK-1 influenced signaling pathways by measuring global gene expression levels.

Using RNA Seq, the relevance of genes that are differentially expressed in the absence of FRK-1 were analyzed using Functional Enrichment Analysis (FEA). FEA identified twenty- one gene clusters of significantly affected genes of which seven were chosen for further analysis. Once identified, we confirmed gene expression data in vivo, examined associated interactions of differentially expressed genes, and began examining novel gene signaling in association with FRK-1 activity. Here we present our RNA Seq analysis and results that show FRK-1 is required as a master regulator for proper expression in specific pathways associated with developmental growth, extra-cellular matrix cuticle formation, cell motility, cell adhesion and cellular localization.

The Effect of Lovastatin on Processing of Activating Transcription Factor 6
Aida Husain, California State University, Long Beach

The Endoplasmic Reticulum (ER) mediates integral cellular processes such as protein synthesis, folding, and trafficking. Perturbation of these processes leads to a type of cellular stress known as ER stress, which activates the Unfolded Protein Response (UPR), that works to restore ER homeostasis. The UPR is regulated by three ER transmembrane proteins, including Activating Transcription factor 6 (ATF6). In response to ER stress, AFT6 is translocated to the Golgi where it undergoes proteolytic cleavage to release its N-terminal fragment. It is known that Sterol Regulatory Element Binding Proteins (SREBPs) are cleaved in a manner similar to ATF6 in order to regulate lipid homeostasis. Previous studies have shown that cleavage of SREBPs is promoted by Lovastatin, a drug prescribed to lower blood cholesterol levels. We hypothesized that Lovastatin should induce cleavage of ATF6 in a manner similar to SREBPs, thus triggering the UPR. To test our hypothesis, we treated HeLa cells, a cervical cancer cell line, with either Lovastatin or induced ER stress. The cells were lysed and the levels of known UPR proteins were tested by immunoblotting. Preliminary data suggest that Lovastatin treatment induces ER stress, as determined by enhanced level of a key chaperone known to be upregulated under ER stress conditions. Our results also demonstrate that HeLa cells upregulate the cytoprotective Akt signaling when treated with Lovastatin. Collectively, our results suggest that Lovastatin indeed induces ER stress-like conditions, triggering the UPR. Our current and future experiments are focused on determining the effect of lovastatin on cell viability.

Effects of DNA-Modifying Reagents on Splicing Regulation of the BRCA2 Tumor Suppressor Gene
Talia Ishfaq, Benedictine University

BRCA1 and BRCA2 are tumor suppressor genes associated with hereditary breast/ovarian cancer (HBOC) syndrome. Patients from affected families will often have their BRCA1 and BRCA2 genes sequence to identify inherited mutations in BRCA1, BRCA2, or other genes. Some pathogenic mutations with modest penetrance in these genes affect mRNA splicing patterns, yet literature suggests that DNA damage and DNA methylation states can affect splicing fidelity. We reasoned that commonly used breast/ovarian cancer therapies that promote DNA damage or demethylation may exacerbate the risk associated with some of these mutations. We hypothesize first that BRCA2 alternate splicing patterns can be affected by changing the methylation status of intron-exon boundaries. To test this, we treated a breast cancer cell line (MCF7) and a non-cancer breast cell line (MCF-10A) with 5-aza 2’-deoxycytidine (5 aza 2’dC), an analog of cytidine that cannot be methylated. We find that demethylating drugs reduced levels of alternate splicing of BRCA2 exon 3 in MCF7 but not MCF 10A. Thus, demethylation therapies could compromise tumor suppressor function by altering the balance of alternate splice variants for certain genes. We hypothesize next that DNA damaging agents could affect alternate splicing rates. Surprisingly, we find that In MCF7, the DNA damaging agent doxorubicin increased the level of alternate BRCA2 exon 3 splicing, while bleomycin decreased it. This may indicate that DNA damage that directly cause backbone breakage is qualitatively different from damage that accumulates from topoisomerase inhibition with respect to splicing regulation.

Mutations of Ubiquitin Proteasome System Genes Protect Cells from the Effects of Anisomycin
Julia Niekamp, Ball State University

The rates of protein synthesis and degradation are controlled to maintain protein molecules at the appropriate abundance and in the appropriate conformations. The conserved ubiquitin proteasome system (UPS) maintains protein homeostasis. This balance can be disrupted; for example, the antibiotic anisomycin, produced by the bacterium Streptomyces griseolus, interferes with translation by inhibiting polypeptide elongation. Anisomycin has been found to impair new memory formation in animal studies and has been proposed as a therapeutic for disorders associated with traumatic memory formation (such as post-traumatic stress disorder). Previous work revealed that Saccharomyces cerevisiae strains lacking specific protein quality control genes exhibit growth advantages in the presence of anisomycin. This was surprising, since anisomycin is expected to promote formation of aberrant, truncated protein molecules. Yeast strains lacking a limited panel of genes involved in protein quality control and regulated protein degradation were also tested for resistance to anisomycin; no clear pattern to which mutants exhibited a growth advantage was observed. Ongoing efforts are aimed at comprehensively evaluating the roles of UPS genes in anisomycin resistance. Toward this end, yeast lacking each of 95 genes involved in protein degradation are being cultured in the presence and absence of anisomycin. Following growth assays, data will be analyzed to identify commonalities in genes conferring similar responses to anisomycin. Since the UPS is overwhelmingly conserved between yeast and humans, this work may contribute to an understanding of the cellular response to anisomycin and its potential therapeutic role for memory-associated disorders.

Detection of Cervical Cancer from Pap-Smear Cell Images Using Texture Features
Donnell Perkins, New York Institute of Technology

Cervical cancer is the fourth most frequent cancer in women representing 6.6% of all female cancers. Early detection through a pap smear increases the chance of treating it before it reaches critical stages. The collected cells from a pap test are analyzed under a microscope to detect nuclear abnormalities or dysplasia. This method is subjective to human error and relies on qualitative analysis. This is why an automated system that is able to detect the presence of pre-cancerous cells in a pap smear is necessary. An automated system would increase the efficiency and accuracy of detection and provide a quantitative method of classification. We examined abnormal cell detection based on texture-based features. Using the benchmark dataset of Herlev pap-smear images, our algorithm utilizes a mask for image segmentation defined by a set threshold values and includes gray-level co-occurrence matrix (GLCM) function to quantify the texture features of the nuclei in a simulated group setting. The group setting consists of mixed images of normal and abnormal cells in one combined panel. The texture features are defined by Contrast, Energy, Homogeneity and Correlation. The experimental analysis reveals that a group of cells with one or more abnormal cells have an Energy value of 0.88 or below. The Energy value is a more discriminative feature as compared to other GLCM features and the algorithm may be used reliably to detect abnormal cells in the data set. In future, the classification can be expanded to differentiate between mild and severe dysplasia in pap smears.

Structural Characterization of the Covalent Binding of KEAP1 and Thiol-containing Small Molecules
Gwendolyn Pyeatt, Boston University

Oxidative stress is involved in many diseases, as high levels of reactive oxygen species (ROS) can damage DNA and other vital biomolecules. Kelch-like ECH-associated protein 1 (KEAP1) regulates the body’s response to oxidative stress. In the absence of oxidative stress, a KEAP1 homodimer binds transcription factor nuclear factor-like 2 (Nrf2), causing Nrf2 degradation. Oxidative stress causes KEAP1 to release Nrf2, allowing it to activate the production of antioxidant enzymes. Blocking the protein-protein interaction (PPI) between KEAP1 and Nrf2 could have great therapeutic value. However, PPIs frequently take place between large, flat interfaces, making these interactions difficult to block. A previous study with the Arkin Lab at UCSF identified 43 thiol-containing compounds (monophores) that can be covalently tethered to KEAP1 at a cysteine near the protein interface. The goal of this project is to characterize interactions between KEAP1 and the monophores using X-ray crystallography to obtain structures of the protein-monophore complexes, or adducts. Structural data will allow us to select monophores suitable for scaffold optimization, a technique in which a smaller compound is elaborated to improve its potency and selectivity. To identify adduct crystallization conditions, the DTNB assay, which measures reactive cysteine groups in a protein, was used to screen buffer conditions without dithiothreitol (DTT). A high-throughput version of this assay was developed and utilized, along with differential scanning fluorimetry (DSF), to identify stabilizing conditions for adduct formation. This assay was used to identify a new buffer condition (50 mM EPPS, 50 mM NaCl, pH 8.0) for adduct crystallization attempts.

Determining the Effects of Impaired Muscle Fatty Acid Oxidation on Liver Metabolism
Arvind Rajan, East Carolina University

Carnitine palmitoyltransferase II (CPT2) is an irreplaceable enzyme for oxidation of long-chain fatty acids, converting acylcarnitines into acyl-CoAs within the mitochondrial matrix. Defects in CPT2 impair mitochondrial fatty acid oxidation (FAO) and result in the buildup of acylcarnitines within the skeletal muscle which are predicted to impair glucose metabolism. To assess the specific effects of Cpt2-deficiency on muscle and whole-body physiology, we generated a skeletal muscle specific CPT2-knockout mouse model (Cpt2^Sk-/-). Compared to controls, the muscles of Cpt2^Sk-/- mice have an 80% decrease in fatty acid oxidation and accumulate up to 200- fold more acylcarnitines. To study systemic glucose homeostasis, control and Cpt2^Sk-/- mice were fed low- and high-fat diets. Because glucose homeostasis is highly regulated by liver metabolism, we analyzed gene expression in the liver to reveal upregulated genes related to fatty acid oxidation, ketogenesis and gluconeogenesis in Cpt2^Sk-/- mice fed either diet. Next, we supplemented free carnitine to aid the metabolic flux of long-chain acylcarnitines and thus attenuate accumulation. In response to carnitine supplementation, Cpt2^Sk-/- mice fed either low- or high-fat diets did not increase expression of genes related to fatty acid oxidation or ketogenesis. These data suggest that carnitine supplementation suppressed the transcriptional response of fatty acid oxidation genes in the liver of a skeletal-muscle specific FAO deficient mouse model. These data demonstrate the role of multi-organ physiological cross talk as a regulator of metabolic control.

Heterogeneity in Water Dynamics in a Sodium pumping Rhodopsin
Aniruddha Seal, National Institute of Science Education and Research (NISER)

The conversion of light energy into ion gradients across biological membranes is one
of the most fundamental reactions in primary biological energy transduction.. Driven by light,
rhodopsins facilitate the translocation of ions across the cell membrane. By introducing such
photosensitive transporters into the cell, we can then use light pulses to manipulate the potential of the neuron cell membrane, thereby controlling its activity. Recently discovered[1], the first light activated Na+ pump Krokinobacter eikastus rhodopsin 2 (KR2), which allows ion translocation without any change in pH of the surrounding environment making it revolutionary for optogenetics. The mechanism of ion conduction is fundamental both for understanding its functioning and for modeling new optogenetic tools. A previous study[2] showed a change in hydration pattern of the internal cavities of the protein while sodium moves across the channel, suggesting a possible crucial role of water. To explore the role of water, we performed extensive classical and quantum molecular dynamics simulations of the early photocycle states. We have identified putative internal hydration sites along the channel and have shown heterogeneity in water dynamics across different sites, which was compared between the closed and the open states of the channel. We have further demonstrated very rare hopping transitions of water from site to site gated by protein motions in the closed state.

Suppression of Zika Virus Infection in Mice by p53 Inhibitors
Winnie Wong, Rutgers University

Zika virus (ZIKV), a mosquito-transmitted flavivirus with a (+)ss RNA genome, infection prompts cell-cycle arrest and apoptosis in proliferating neural progenitors. The tumor suppressor p53 plays a major role in apoptosis and inflammation in the central nervous system during viral infection. Previous in vitro experiments in our laboratory demonstrated that activation of p53 has a proviral effect upon ZIKV infection, as inhibition of p53 decreased viral replication compared to controls. Our current study focuses on elucidating roles for p53 signaling in neuroinflammation and host defense in vivo using mouse models of ZIKV encephalitis. Two trials were conducted: (1) to use isolated mice brain tissue for RNA gene expression and viral infectivity analyses; (2) and to monitor for survival. In each trial, wild-type mice, males and females, were divided into two arms, uninfected or infected with ZIKV (MR766), and each arm has three groups given a certain drug treatment: DMSO (control), pifithrin-α, or pifithrin-μ. Pifithrin-α inhibits all p53-dependent activities, including gene transcription, whereas pifithrin-μ only inhibits p53 signaling to mitochondria. Inhibition of p53 signaling following ZIKV infection in vivo decreased mortality compared to control animals. Plaque assay analysis confirmed that both p53 inhibitors decreased brain viral burden; however, expression of p53 transcriptional targets in mouse brain tissue following ZIKV infection was not impacted by either pifithrin-α or pifithrin-μ. These results support our previous work finding a proviral function for p53. Given the lack of expression of p53 transcriptional targets upon ZIKV infection, future work will explore mechanisms involving the mitochondria-dependent p53 apoptotic pathway.

Graduate Division

ATRX Loss Of Function Impairs Self-Renewal In Glioma Stem Cells By Sensitization To Proton Radiation Therapy
Ángel A. Garcés, MD Anderson Cancer Center

Glioblastoma Multiforme (GBM) is the most prevalent and aggressive form of malignant glioma in the United States with a five-year survival rate of only 6.8%.1 Glioma stem cells (GSCs), a subpopulation of undifferentiated, self-renewable, stem-like cells in GBM tumors, have been recently implicated in promoting GBM chemoradioresistance, metastasis, and tumorigenesis.2 Interestingly, 70-80% of low-grade glioma (LGG) and secondary GBM patients carry the IDH1^R132H mutation, which has been shown to inhibit GSC aggression by impairing migration and invasion along with promoting GSC differentiation.3 However, the effects of alpha-thalassemia/mental retardation syndrome X-linked gene inactivating mutations (ATRX^Loss), which often co-present with IDH1^R132H in younger LGG patients, on GSC response to chemoradiotherapy are not well understood. Proton radiation therapy has been recognized as an effective treatment modality in LGG and GBM due to its superior ability to target tumors and induce more complex DNA damage than conventional X-ray therapy (XRT).4 Based on previous studies that associate ATRX^Loss with XRT sensitivity,5 we hypothesize that ATRX^Loss promotes diminished survival compared to ATRX^WT in GSCs treated with proton radiation.

To test this hypothesis, we treated TS543-wtATRX and puromycin activated TS543-shATRX isogenic GSCs with 1-4 Gy proton radiation. GSC self-renewal was quantified using extreme limiting dilution analysis (ELDA).6 Our results demonstrate that ATRX^Loss significantly impairs GSC self-renewal compared to ATRX^WT in response to proton radiation based on neurosphere formation frequency. In the future, we will elucidate changes in the DNA damage repair of ATRX^Loss isogenic GSCs to further the development of optimal treatment regiments for IDH1^R132H/ATRX^Loss glioma patients.

Investigating Aberrant Neurite Outgrowth in an SBMA Cell Model
Jordyn S Karliner, Thomas Jefferson University

Spinal and bulbar muscular atrophy (SBMA) is an X-linked neurodegenerative disease characterized by the loss of brainstem and spinal cord lower motor neurons and muscle atrophy. It is caused by a CAG repeat expansion in exon 1 of the androgen receptor (AR) gene, which encodes a mutant protein with an expanded polyglutamine (polyQ) tract. Upon binding to androgens, mutant AR misfolds and aggregates, forming intranuclear inclusions, a molecular hallmark of disease. An ill-studied cellular phenomenon in SBMA cell models is aberrant neurite outgrowth, and cell cycle re-entry was revealed as a potential mechanism, involving reduced function of the ubiquitin ligase and cell cycle regulator complex, APC/C-Cdh1. This study aims to further characterize aberrant neurite outgrowth over time, and investigate the role of Cdh1 in the phenotypes observed, using an SBMA PC12 cell model. Through live cell imaging, we quantified the differentiation capabilities of wild type and SBMA model cells over time, and quantified Cdh1 expression through western blot analysis. Preliminary data reveal that SBMA model cells have an enhanced differentiation capacity at various timepoints, and reduced Cdh1 expression, in the absence of androgens. In addition to replicating these experiments, future work will incorporate SBMA patient-derived, iPSC-derived motor neurons to explore neurite outgrowth competency in a more disease-relevant model. We will also investigate regulators and downstream effectors of Cdh1 to further reveal the mechanisms underlying APC/C-Cdh1 loss of function and aberrant neurite outgrowth. Overall, this research can provide insight into lower motor neuron degeneration and muscle reinnervation in SBMA.

VIRTUAL STUDENT SCHOLARS SYMPOSIUM 2020 PRESENTATIONS

Cell Biology and Biochemistry - Poster Presentations

High School Division

Undergraduate Division

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