Cell and Developmental Biology Commons^™

Evaluating The Sna1 Gene Using Crispr Mediated Knockdown In Ewing Sarcoma Cells, Rosanna Jees, Sonia Cerrillo, Terry Jo Shackleford

Mechanisms of Disease

Ewing sarcoma is a pediatric cancer with limited therapeutic options and poor long-term survival. CRISPR–Cas9–mediated gene editing provides a powerful tool for investigating tumor molecular behavior and identifying potential therapeutic targets. In this study, we worked with CRISPR–Cas9 in Ewing sarcoma ES8 cells to evaluate the roles of the transcription factors SNAI1 and SNAI2, both implicated in epithelial–mesenchymal transition and cancer progression. Using guide RNAs targeting each gene, we assessed proliferation, apoptosis, migration, and long-term survival through Incucyte live-cell imaging and colony-formation assays. Knockout of SNAI1 resulted in decreased cell proliferation and reduced migratory capacity compared with controls, suggesting a …

Effects Of Ptk2 Knockdown On Key Cellular Processes In Es8 Cells, Adrian Cruz, Emiliano Rueda, Dylan Andrews, Terry Jo Shackleford

Mechanisms of Disease

Ewing sarcoma is an aggressive form of pediatric cancer characterized by rapid growth and metastatic potential, with limited treatment options available. PTK2 is a key regulator of many pathways involved in tumor behavior, including integrin-mediated adhesion, survival, and migration. This research lab investigated how CRISPR-Cas9 knockdown of PTK2 can affect cellular processes in ES8 Ewing sarcoma cells. To assess the effectiveness of reducing PTK2 expression, we utilized live-cell imaging, wound-healing assays, measurements of caspase activity, colony formation, and qRT-PCR. Our results showed that incomplete knockdown decreased cell proliferation and colony formation, while also increasing apoptotic activity. Additionally, migratory ability was …

How Cxcr4 Knockdown Affects Tumorigenic Properties In Es8 Cells, Gabriella Galdeano, Damon James, Terry Jo Shackleford

Mechanisms of Disease

C-X-C chemokine receptor type 4 (CXCR4) is a seven-transmembrane G-protein–coupled receptor (GPCR). When activated by its ligand SDF-1 (CXCL12), CXCR4 triggers signaling pathways that promote cell survival, proliferation, angiogenesis, and chemotaxis. Many cancers exploit the CXCR4/SDF-1 axis to support tumor growth and metastasis. Ewing sarcoma (ES8) is an aggressive pediatric bone and soft-tissue cancer, and elevated CXCR4 signaling has been linked to increased migration and metastatic behavior. ES8 cells, a well-established ES8 cell line, provided a model to study how CXCR4contributes to tumor progression. CRISPR-Cas9 genome editing was used to knock down CXCR4 in ES8 cells, delivered through lipid-based transfection. …

Investigating The Role Of Twist1 In Cancer Progression Using Crispr Knockdown, Abby Guerrero, Sofia Perez, Terry J. Shackleford

Mechanisms of Disease

TWIST1 is a transcription factor that plays a critical role in epithelial-mesenchymal transition (EMT), a process that promotes cancer cell migration, invasion, and metastasis. This study investigates the functional role of TWIST1 in cancer progression by using CRISPR/Cas9-mediated knockdown to examine its effects on apoptosis, proliferation, migration, and invasion. TWIST1 was knocked down in cancer cell lines using CRISPR/Cas9 assay. Knockdown efficiency and changes in genes were confirmed by qRT-PCR. Functional assays included caspase assays for apoptosis, MTT assays for proliferation, wound-healing assays for migration, Matrigel invasion assays, and colony formation assays to assess long-term growth and invasive potential. Across …

Itgb1 Knockdown Alters Apoptosis And Proliferation In Ewing Sarcoma Cells, Aditi Kumar, Magdalena Saad, Terry J. Shackleford

Mechanisms of Disease

Integrin beta 1 (ITGB1) is an adhesion receptor that link cells to the extracellular matrix and regulates pathways involved in survival, proliferation, and migration. In order to understand its role in Ewing Sarcoma, we applied CRISPR-Cas9 with two guide RNAs to knock down ITGB1 in ES8 cells. We then assessed changes in cell growth, death, movement, and gene expression using Incucyte live-cell imaging, colony formation assays, wound healing assays, and qRT-PCR. qRT-PCR showed partial ITGB1 reduction, with ITGB1_gRNA2 producing the strongest decrease. ITGB1_gRNA2 also led to a small increase in caspase activity and a 20-30% increase in proliferation at the …

Elucidating The Mechanism Of Neprilysin-Regulated Triple-Negative Breast Cancer Invasion, Kai Lam, Ellie Griner

Student Scholar Symposium

Triple-negative breast cancer (TNBC) is a highly invasive and difficult-to-target form of cancer. It is characterized by a lack of estrogen receptors, progesterone receptors, and human epidermal growth factor 2 receptors, making current targeted therapies ineffective and leaving chemotherapy as the primary treatment option. Previous studies in this lab have linked reduced expression of neprilysin (NEP), a membrane-bound protease that cleaves and inactivates mitogenic peptides, to a highly invasive phenotype in TNBC. Our data indicates that while NEP promotes phosphoinositol 3-kinase (PI3K) signaling, it simultaneously inhibits cellular invasion. Here, we begin to explain a possible mechanism behind the anti-invasive role …

Δ-Valerobetaine As A Novel Inhibitor Of Breast Cancer Cell Migration: Hinders Cellular Migration Of Mcf-7 Cells Through Reducing Atp Concentrations And Fak Activation, Braxton Ivie, Lincoln Brown, Bryson Lovorn

Student Scholar Symposium

δ-valerobetaine as a Novel Inhibitor of Breast Cancer Cell Migration: Hinders Cellular Migration of MCF-7 Cells Through Reducing ATP Concentrations and FAK Activation

Lincoln Brown, Braxton Ivie, Bryson Lovorn, Joshua Owens

Based on female breast cancer diagnoses from 2014-2020, the CDC reports that the 5-year relative survival rate for local and regional breast cancers was 98.9% and 86.3%, respectively, while the survival rate for metastasized cancers was reportedly 32.4%. These discrepancies in survival highlight an increased mortality rate when cancer metastasizes. Interestingly, aggressive tumors have been shown to metabolize fats at higher rates than low-metastatic tumors. Recently, our group has …

Epidemiology, Presentation, And Diagnosis Of Type 2 Diabetes Mellitus In Children And Adolescents, Bisher Alsabbagh, Whard Alsabbagh

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The prevalence of type 2 diabetes mellitus (T2DM) in children and adolescents has increased substantially over recent decades, paralleling rising rates of pediatric obesity and metabolic risk. Youth-onset T2DM is characterized by significant insulin resistance accompanied by an inadequate compensatory insulin response, resulting in progressive hyperglycemia and early beta-cell dysfunction. Incidence varies widely among racial and ethnic groups, with the highest rates observed in non-Hispanic Black, Hispanic, Native American, Asian American, and Pacific Islander youth. Risk factors include obesity, family history, high-risk ethnicity, pubertal insulin resistance, prenatal metabolic exposures, and comorbid conditions such as polycystic ovary syndrome. Clinical presentation ranges …

Increased Intermembrane Space [Ca2+] Drives Mitochondrial Structural Damage In Cpvt, Shanna Hamilton, Radmila Terentyeva, Roland Veress, Fruzsina Perger, Zuzana Nichtova, Mark Bannister, Jinxi Wang, Sage Quiggle, Rachel Battershell, Matthew Gorr, Sandor Györke, Bum-Rak Choi, Christopher George, Andriy Belevych, György Csordás, Dmitry Terentyev

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: Mitochondrial dysfunction caused by abnormally high RyR2 (ryanodine receptor) activity is a common finding in cardiovascular diseases. Mechanisms linking RyR2 gain of function with mitochondrial remodeling remain elusive. We hypothesized that RyR2 hyperactivity in cardiac disease increases [Ca 2+ ] in the mitochondrial intermembrane space (IMS) and activates the Ca 2+ -sensitive protease calpain, driving remodeling of mitochondrial cristae architecture through cleavage of structural protein OPA1 (optic atrophy protein 1).

METHODS: We generated a highly arrhythmogenic rat model of catecholaminergic polymorphic ventricular tachycardia, induced by RyR2 gain-of-function mutation S2236L(Ser2336Leu)(+/-) . We created a new biosensor to measure IMS-[Ca2+ ] …

Protein-Protein Interaction–Interfering Peptide Rescues Dysregulated Nmda Receptor Signaling, Robert E. Featherstone, Hongbin Li, Ameet S. Sengar, Karin E. Borgmann-Winter, Olya Melnychenko, Lindsey M. Crown, Ray L. Gifford, Felix Amirfathi, Anamika Banerjee, Aivi Tran, Krishna Parekh, Margaret Heller, Wenyu Zhang, Robert J. Gallop, Adam D. Marc, Pragya Komal, Michael W. Salter, Steven J. Siegel, Chang-Gyu Hahn

Farber Institute for Neuroscience Faculty Papers

The complex and heterogeneous genetic architecture of neuropsychiatric illnesses compels us to look beyond individual risk genes for therapeutic strategies and target the interactive dynamics and convergence of their protein products. A mechanistic substrate for convergence of synaptic neuropsychiatric risk genes are protein-protein interactions (PPIs) in the N-methyl-D-aspartate receptor (NMDAR) complex. NMDAR hypofunction in schizophrenia is associated with hypoactivity of Src kinase, resulting from convergent alterations in PPIs of Src with its partners. Of these, the association of Src with PSD-95, which inhibits the activity of this kinase in the NMDAR complex, is known to be increased in schizophrenia. Here, …

A Persistent Homology Framework For Scrna-Seq: Assessing Clustering Robustness And Quantifying Preprocessing And Integration Effects On Topological Features., Jonah Daneshmand

Electronic Theses and Dissertations

As single-cell RNA sequencing (scRNA-seq) data expands, robust methods for integrating diverse datasets are critical. This dissertation applies Persistent Homology (PH), a technique from Topological Data Analysis (TDA), to a collection of scRNA-seq datasets spanning eight tissue types to quantify how data integration affects topological features and biological interpretability. We assessed global topological structure using Betti curves, Euler characteristics, and persistence landscapes across raw, normalized, and integrated data representations. Our analysis revealed a performance inversion: while conventional methods excelled on unintegrated data, high-granularity topological methods, particularly those sensitive to global data structure, became superior after integration. This suggests a synergy …

Further Characterization Of The Fungal Effector Mvlg-01732 In Planta And Heterologous Expression Of Fungal Effectors In Different Smut Fungi., Joseph Paul Ham

Electronic Theses and Dissertations

Host-pathogen interactions in smut fungi are characterized by effector proteins and their role in infection. The goal of this work is to better understand these interactions by further characterizing an effector from Microbotryum lychnidis-dioicae and heterologously expressing this and other effectors in heterologous species to see if the range of these effectors can play a role in pathogenicity when expressed in distantly and closely related fungal species. These effectors have been attuned over time to their host plant but have homologous interactors in other plant species. In this study, Microbotryum lychnidis-dioicae, Sporisorium reilianum, and Ustilago maydis, were all used …

Ontogenetic Analysis Of Parasaurolophus Braincases (Ornithischia, Hadrosauridae) From The Kaiparowits Formation (Upper Cretaceous) Of Utah, Usa: Comparing Juvenile And Adult Morphology, Amanda "Billie" C. Guerrero

Electronic Theses, Projects, and Dissertations

This study documents the ontogenetic development of the braincase in Parasaurolophus, a lambeosaurine hadrosaurid dinosaur known for its distinctive cranial crest. Whereas the crest has been extensively examined for its roles in communication and display, the braincase remains less understood, despite its structural and functional relationship with the crest. This research compares the braincase morphology of a juvenile specimen (RAM 14000, "Baby Joe"), a subadult specimen (RAM 17000), and an adult P. tubicen (NMMNH P-25100), to investigate how developmental changes influenced sensory systems, neurocranial architecture, and biomechanical adaptations across ontogeny.

Detailed morphological analysis reveals progressive changes from juvenile to …

Characterization Of A New Domestic Feline Macrophage Cell Line To Study Molecular Mechanisms Of Feline Coronavirus, Adam Espinoza

Electronic Theses, Projects, and Dissertations

Feline Coronavirus (FCoV) is a veterinary pathogen common in domestic cats. FCoV is transmitted via the fecal-oral route, results in mild enteric symptoms, and is considered feline enteric coronavirus (FECV). During infection the virus may transition from infecting host enteric epithelial cells to infecting host macrophages, resulting in a lethal systemic disease, and the virus is now considered feline infectious peritonitis virus (FIPV). FIPV arises in an infected individual, is not shed in the feces, and is not transmitted from cat to cat. There is limited research defining the host molecular mechanisms driving FCoV lethal outcomes, with few feline cell …

Mitochondria-Mediated Epigenetic Transfer Between Chondrosarcoma And Wild-Type Cells, Caleb C. J. Wyckoff

Biomedical Sciences Theses & Dissertations

Glioblastoma (GB), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), cholangiocarcinoma, and chondrosarcoma (CS) cancers all contain mutations in the gene isocitrate dehydrogenase 2 (IDH2). The mutant IDH2 enzyme exhibits transformation of alpha-ketoglutarate (αKG) into the oncometabolite D-2-hydroxyglutarate (D2HG) in the mitochondria of these cancers. Mitochondrial-mediated transfer between cancer cells and recipient cells is a significant event that impacts the physiology of the receiving cell, specifically the epigenetic landscape. Previous experiments indicating increased DNA methylation in mesenchymal stem cells exposed to IDH1 or IDH2 conditioned medium underscore the potential role of D2HG to alter methylation states. Additionally, the presence of …

The Genetic And Environmental Determinants Of Stress Resilience Across Model Systems, Carson Len Stacy

Graduate Theses and Dissertations

Survival in fluctuating environments demands that organisms effectively anticipate and respond to future challenges. This capacity is shaped by both genetic background and environmental context. In this dissertation, I apply a population aware lens to explore how genetic diversity and environmental conditions intersect to shape stress survival. Integrating functional genomics and transcriptomics in Saccharomyces cerevisiae (brewer’s yeast) and a vertebrate (common canary) system, I dissect gene-by-environment (GE) interactions that underpin resilience. First, I move beyond a single reference genome by using a pan-genomic CRISPR knockout screen to assess osmotic stress survival across diverse S. cerevisiae isolates. This analysis reveals a …

Characterizing The Ethanol Sensitivity Of The Planar Cell Polarity Pathway In Endoderm Morphogenesis And Jaw Development., Raéden Omneil C. C. Rolle Gray

Electronic Theses and Dissertations

Fetal Alcohol Spectrum Disorders (FASD) describes a large range of developmental defects due to prenatal alcohol exposure. It is characterized by neural and facial defects, including jaw hypoplasia. Many factors contribute to the susceptibility of ethanol-induced facial defects, including genetics, yet the genes involved, and the cell events they regulate, are poorly understood. Zebrafish have a large number of tools for genetic manipulation available, share 70% of genes with humans, are fertilized externally and are translucent during embryogenesis, making them great models to study FASD. Previous studies show that two members of the Planar Cell Polarity (PCP) pathway, zebrafish vangl2 …

Host-Intrinsic Factors Determine Anti-Tumor Efficacy Of Exercise In Pancreatic Ductal Adenocarcinoma, Sumedha Pareek

Dissertations and Theses (Open Access)

Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths with a low 5-year relative survival rate of approximately 13.3% for all stages combined. We have previously shown that exercise can improve quality of life and enhance functional capacity among patients with PDAC. Exercise induces a variety of changes in the tumor microenvironment with beneficial effects in several tumor types. However, our understanding of the clinical effects of exercise and the mechanisms that mediate anti-tumor effects are limited in pancreatic cancer.

In this project, using C57BL/6 mice from Taconic Biosciences (Tac) and Jackson Laboratories (Jax), we established a …

Neurogenesis And Functional Recovery Of Murine Retinal Ganglion Cells After Glaucoma Induction Following Treatment With Pnu-282987, Samantha Luzadre

Masters Theses

Experiments were performed to demonstrate electrical recovery in glaucoma-induced adult mice after neurogenesis of retinal ganglion cells (RGCs). Newly derived RGCs were induced following eye drop treatment with a selective alpha7 nicotinic acetylcholine receptor agonist, PNU-282987. Both sexes of transgenic (RlbpCre-ER (CRE⁺) and Rosa-tdTomato (tdTomato⁺)) adult mice were utilized to trace newly generated RGCs originating from Müller glia (MG). Experimental mice were injected with hypertonic saline into the episcleral vein of anesthetized mice to significantly increase intraocular pressure (IOP) and induce glaucoma-like conditions. One month after the injection and after significant death of RGCs occurred, mice …

Context Dependent Regulation Of Dicer Function During Oocyte-To-Embryo Transition, Nick A. Newkirk

Dissertations and Theses (Open Access)

Over two decades ago, Dicer was discovered as the primary enzyme responsible for the generation of small RNAs known as the microRNAs (miRNAs) and short interfering RNAs (siRNAs). Spatiotemporal regulation of Dicer-dependent small-RNA biogenesis affects many aspects of biology, including aging, disease progression, fertility, and cancer. Interestingly, a distinct population of Dicer-dependent small RNAs, known as the endogenous siRNAs (endo-siRNAs), are uniquely abundant in the germ cells and gametes, and are critical for normal fertility. Despite their function regulating new life, the mechanisms controlling their biogenesis and subsequent germ cell functions remain poorly defined. Work to parse these mechanisms in …