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Mechanoactivation of VEGFR-2 Promotes Angiogenesis in the Tumor Microenvironment
Get PDFCancer progression relies on blood vessel growth; without vasculature, tumors would be limited to less than 200µm in diameter. With this reasoning, several cancer therapies have been developed that target the process of angiogenesis, or the growth of vessels off pre-existing vasculature. These treatments target the major promoters of angiogenesis, vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2); however, anti-angiogenic therapies are not effective by themselves. This is likely due to mechanoactivation of VEGFR-2 through physical alterations in the tumor microenvironment (TME). One prevalent mechanical change is increased strain on surrounding cells and matrix due to highly contractile cancer-associated fibroblasts (CAFs) present in the TME. In this project, we applied strain to endothelial cells (ECs) through a 2D Flexcell system or in 3D through CAFs or magnetic beads. This allowed us to determine the molecular and functional effects of strain on ECs in the context of angiogenesis in the TME. For molecular studies, we focused on VEGFR-2 expression and phosphorylation at Y1054/Y1059 and Y1214. We observed that the application of 2D strain caused increased phosphorylation of both sites, indicating activation of the VEGFR-2 pathway. To further study the roles of these residues, we developed EC CRISPR/Cas9 mutants with Y1054 or Y1214 being changed to phenylalanine (Y1054F, Y1214F), which is not phosphorylatable. We discovered that in 3D microtissue studies, these mutant ECs developed significantly less vasculature compared to control lines. Our functional studies utilized three-chamber microfluidic devices to analyze angiogenesis, and we determined that cell-free strain through embedded magnetic beds promoted angiogenesis, which result was lost in Y1054F and Y1214F lines. Through this project, we demonstrate that strain can cause VEGFR-2 phosphorylation, which is likely the cause of increased angiogenesis. Y1054 and Y1214 are both required for normal vessel growth, suggesting potential targets of future cancer treatments. Tumor mechanics should be taken into consideration when developing novel anti-angiogenic therapies due to mechanoactivation of the VEGFR-2 pathway which may overcome common treatments used today
Evaluation of in vitro cardiac models for characterizing the role of autophagy in Streptococcus pneumoniae infection
Get PDFStreptococcus pneumoniae (Spn) is a gram-positive bacterium that causes community-acquired pneumonia, the most prevalent type of pneumonia and a predominant infectious disease. There is evidence linking Spn infection to cardiovascular damage through the ability of Spn to invade and replicate in the myocardium, inducing cardiomyocyte death and disrupting cardiac function. Spn infects cardiomyocytes intracellularly through clathrin-mediated endocytosis, with demonstrated evidence of inhibitory mechanisms including clathrin-mediated endocytosis inhibitors and antioxidants. As a result, autophagy is being investigated as a potential pathway for cardiomyocyte self-defense against Spn, as it may protect cardiomyocytes intracellularly. The primary objective of this study was to evaluate methods for characterizing the role of autophagy in cardiomyocyte protection following Spn infection in vitro in two- and three-dimensional cell architecture. Experiments utilized normal human-induced pluripotent stem cell-derived cardiomyocytes treated with an autophagy inhibitor and promoter, Bafilomycin A and Tat-Beclin1, respectively. Analyses of bacterial mNeonGreen fluorescence, lactate dehydrogenase, and cytokine results concluded that autophagy promotion leads to lower cell cytotoxicity and bacterial infection compared to autophagy inhibition in Spn infected cells. A primary advantage of three-dimensional cell architecture was its ability to form bacterium-filled microlesions characteristic of Spn infection in the heart, rendering it superior to two-dimensional. Characterization of a polydimethylsiloxane membrane cardiac tissue chip was also performed, as the cardiac tissue chip enables in vitro modulation of stretch and pressure. The study was intended to advance the understanding of cardiac response to Spn infection and lead to enhanced approaches for the study of cardiac injury
An Investigation Into The Effects Of Preservatives On ß-Hydroxybutyrate Concentrations Measured By Commercial Ketone Meter And Gas Chromatography-Mass Spectrometry
No full textKetoacidosis occurs when fatty acids are metabolized into ketone bodies in the liver and released into the bloodstream. This condition, which can be fatal, is commonly observed in diabetics, alcoholics, and individuals who experienced starvation or hypothermia. Although the ketone ß-hydroxybutyrate (BHB) is an accepted biomarker of ketoacidosis, the effects of preservatives on BHB concentrations in postmortem blood are unaddressed. The first aim of this research was to determine whether preservatives affect ketone levels as measured by a point-of-care device. Paired preserved and unpreserved blood samples were obtained from the Jefferson County morgue: preserved samples in grey top tubes and unpreserved samples in red top tubes. BHB was measured using an Abbott ketone meter with ß-ketone test strips. Due to variation observed between the ketone meter readings from the morgue and the red top tubes it was concluded that a factor other than preservatives was influencing the ketone meter readings. The second aim of this research was to determine whether the ketone meter was a viable point-of-care device for measuring BHB in blood samples during autopsy. Concentrations of BHB in previously tested paired postmortem blood samples were quantified using a GC-MS method validated according to ANSI/ASB 036 guidelines. Additionally, matrix stability and processed sample stability were assessed to ensure the accuracy and reliability of analysis in variable conditions. Although the ketone meter results may not be accurate, both the ketone meter and GC-MS were able to indicate if a significant BHB concentration was present. Further, unextracted BHB was stable at -20°C, 4°C, and 20°C for 90 days and processed BHB was stable for 72 hours at room temperature. The GC-MS method developed in this research was used to evaluate a case study for a decedent with no known medical history. The results revealed a clear case of ketoacidosis characterized by elevated glucose, acetone, and isopropyl alcohol levels, as well as a ketone meter reading greater than 2.5 mmol/L. The BHB levels were confirmed using the validated GC-MS method in triplicate to demonstrate the value of BHB analysis in determining the cause of death
Environmental Exposure To Metals In Children And Their Effects On Autism Spectrum Disorder
Get PDFABSTRACT As scientific understanding of environmental determinants of health advances, growing attention has turned to the role of toxic metal exposures in exacerbating neurodevelopmental outcomes, particularly in vulnerable populations such as children with autism spectrum disorder (ASD). This dissertation investigates the intersection of environmental metal exposure, biomonitoring innovation, and social factors shaping ASD severity in Alabama. Using a multidisciplinary framework, four interrelated studies explore biological, environmental, and participatory dimensions of ASD research and public health. The first study evaluates the utility of Volumetric Absorptive Microsampling (VAMS) for measuring exposure to cadmium (Cd), chromium (Cr), lead (Pb), manganese (Mn), and mercury (Hg) in children diagnosed with ASD. VAMS offers a minimally invasive and accessible alternative to venous blood collection, especially beneficial for pediatric populations. However, technical challenges such as contamination and limited sample volume highlight the need for refinement before widespread adoption in environmental epidemiology. The second study investigates the correlation between census-tract-level airborne metal concentrations estimated via environmental screening tools and ASD severity across Alabama. Findings reveal that children residing in areas with elevated airborne metal pollution, particularly in industrialized zones, tend to exhibit more severe ASD symptoms, underscoring the spatial inequalities in exposure risk. The third study analyzes the association between blood metal concentrations and ASD severity, identifying significant positive correlations between cadmium and lead and increased symptom severity. These findings reinforce concerns about the neurotoxic potential of environmental metals and their disproportionate burden on children with developmental vulnerabilities. The fourth study assesses parental awareness and participation in environmental ASD research. It reveals notable gaps in environmental health literacy and highlights how previous research engagement influences communication preferences and study participation. Barriers such as mistrust, time constraints, and perceived lack of benefit reduce participation among caregivers, particularly in underserved communities. Together, these studies provide a comprehensive understanding of how environmental exposures, spatial disparities, and community engagement intersect to influence ASD outcomes in Alabama. They collectively underscore the urgent need for targeted public health interventions, refined exposure assessment techniques, and inclusive research practices
Uncovering The Role Of Heparanase-Armed Extracellular Vesicles In Sepsis-Associated Vascular Endotheliopathy
Get PDFSepsis remains one of the most frequent causes of mortality and life-altering morbidity in humans of all ages globally. Though sepsis pathobiology is incompletely understood, it is clear that a unifying feature of all sepsis phenotypes is systemic vascular endotheliopathy and damage to the luminal glycocalyx of endothelial cells, or eGC, that leads to organ injury. While prior work has established that heparan sulfate (HS) is an especially important eGC constituent that helps regulate a host of endothelial cell (EC) functions, its role in promoting endotheliopathy during sepsis has not been fully characterized. Additionally, previous studies in the context of multiple myeloma have shown that small extracellular vesicles (EV) secreted from myeloma cells harbor heparanase (HPSE), the only known mammalian enzyme with activity on HS. However, HPSE+ EVs have not been characterized outside of cancer biology. As EVs are known to home select enzymes to specific tissues for targeted remodeling, HPSE+ EVs may have a role in causing HS damage in the eGC during sepsis. Thus, the objectives of our work were to (1) determine the impact of HS cleavage from the eGC on homeostatic EC signaling and gene expression and (2) characterize HPSE+ EVs within the plasma of humans with and without sepsis. We discovered that damage to HS in the eGC reduces homeostatic adenosine 5’-monophosphate-activated protein kinase signaling, which promotes an increase in angiopoietin-2 expression, a cytokine that promotes endothelial monolayer destabilization. We also uncovered that HS cleavage from the eGC disrupts homeostatic shear stress-responsive gene expression and reduces expression of genes that promote EC junctional integrity and cell polarity. Finally, we found that HPSE+ EVs circulate in healthy humans, constituting the bulk of total HPSE observed in human plasma, and that circulating levels of HPSE+ EVs decline during sepsis, coincident with reduced total levels of HPSE and increased levels of HS and angiopoietin-2 in the plasma of sepsis patients. Though our findings require more robust characterization and validation, they suggest a novel paradigm by which EVs are key drivers of sepsis-associated endotheliopathy. In the process of clarifying the mechanisms by which EVs contribute to endotheliopathy in sepsis, we hope to discover novel therapeutic targets for this disease
Modeling Wieacker-Wolff Syndrome Using CRISPR/Cas9 Gene Editing to Study Motor Abnormalities
Get PDF2025 Spring Expo Poster Presentation Works in Progress - class projecthttps://digitalcommons.library.uab.edu/sp-expo/1046/thumbnail.jp
Exploring the Role of BIG1 and BIG2 in Protein Trafficking and Sorting at the Trans-Golgi Network and Endosome
Get PDF2023 Spring Expo Poster Presentation Biological & Life Scienceshttps://digitalcommons.library.uab.edu/sp-expo/1060/thumbnail.jp
Exploring Entitativity: Understanding Social Movements and Ethnic Identity
Get PDF2025 Spring Expo Oral Presentation Arts & HumanitiesSocial & Behavioral Scienceshttps://digitalcommons.library.uab.edu/sp-expo/1064/thumbnail.jp
Voices in Between: Navigating Linguistic and Cultural Identity as Bilingual Puerto Rican High School Students in Miami
Get PDF2025 Spring Expo Online Poster Presentation Arts & Humanitieshttps://digitalcommons.library.uab.edu/sp-expo/1076/thumbnail.jp
