Biochemistry, Biophysics, and Structural Biology Commons^™

A Homogeneous Time-Resolved Fluorescence Screen To Identify Sirt2 Deacetylase And Defatty-Acylase Inhibitors, Jie Yang, Joel Cassel, Brian C Boyle, Daniel Oppong, Young-Hoon Ahn, Brian P Weiser

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Human sirtuin-2 (SIRT2) has emerged as an attractive drug target for a variety of diseases. The enzyme is a deacylase that can remove chemically different acyl modifications from protein lysine residues. Here, we developed a high-throughput screen based on a homogeneous time-resolved fluorescence (HTRF) binding assay to identify inhibitors of SIRT2's demyristoylase activity, which is uncommon among many ligands that only affect its deacetylase activity. From a test screen of 9600 compounds, we identified a small molecule that inhibited SIRT2's deacetylase activity (IC50 = 7 μM) as well as its demyristoylase activity (IC50 = 37 μM). The inhibitor was composed …

Rewiring The Sex-Determination Pathway During The Evolution Of Self-Fertility., Yongquan Shen, Shin-Yi Lin, Jonathan Harbin, Richa Amin, Allison Vassalotti, Joseph Romanowski, Emily Schmidt, Alexis Tierney, Ronald E Ellis

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Although evolution is driven by changes in how regulatory pathways control development, we know little about the molecular details underlying these transitions. The TRA-2 domain that mediates contact with TRA-1 is conserved in Caenorhabditis. By comparing the interaction of these proteins in two species, we identified a striking change in how sexual development is controlled. Identical mutations in this domain promote oogenesis in Caenorhabditis elegans but promote spermatogenesis in Caenorhabditis briggsae. Furthermore, the effects of these mutations involve the male-promoting gene fem-3 in C. elegans but are independent of fem-3 in C. briggsae. Finally, reciprocal mutations in these genes show …

Omani Camels From A Cultural And Genomics Perspective, Al Muatasim Al Zadjali

Electronic Theses and Dissertations

The Dromedarian camel, Camelus dromedarius, is native to the Arabian Peninsula, including the Sultanate of Oman. These camels are used for food, milk, as well as show and racing competitions. Despite their economic and cultural importance research on camels in Oman is limited. The goal of this study was to examine their genomic variation, relationship with camels in other parts of the Arabian Peninsula, and to determine if selective breeding has led to the establishment of distinct breeds in Oman. Information was compiled from multiple sources to produce a comprehensive review on the breeding, management, economic and cultural use, …

Interactions Between Black Soldier Fly Larvae (Hermetia Illucens) And Pathogenic Bacteria In Culled Potato Waste, Matthew A. Moyet

Electronic Theses and Dissertations

The focus of this dissertation is to investigate the feasibility and safety of rearing black soldier fly larvae (Hermetia illucens) on culled potato waste in the presence of pathogens. World food and feed supplies remain insufficient while the demand for alternative protein sources is steadily increasing. Recycling organic waste into ingredients of animal feeds using black soldier larvae, which are omnivorous saprotrophs native to North America, is a rapidly emerging technology that shows great promise for creating circular agricultural systems. To optimize this technology on an industrial scale, assessment of rearing conditions on different feeding substrates and the …

Characterizing The Neuronal Signaling Required For Muscle-Specific And Whole Body Enhanced Proteostasis Effects Of Low Mrna Translation, Marissa N. Ruzga

Electronic Theses and Dissertations

In the study of aging, there is a common paradigm that organisms allocate energy between surviving stressful conditions and growth/reproduction (Masoro, 2000). Driving mechanisms that favor the former extend lifespan and protect against cellular stress in different animal systems, including C. elegans. Dietary restriction is one such environmental stressor, which leads to downregulation of the energy-intensive process of mRNA translation (Karol, 2009; Tavernarakis, 2008). When knocked down in adulthood, IFG-1, a component of translation regulation, also protects against cellular stress. Protective effects of low translation appear to be orchestrated by certain tissues in C. elegans, namely neurons and …

The Role Of Med13 In Proteaphagy, John Sauer, Brittany Friedson, Katrina Cooper

Rowan-Virtua Research Day

Regulation of proteasomes is important for adaptation to cellular stress. Previous studies have shown that following starvation stress, proteasomes are targeted for destruction by autophagy. However, how cells control proteasomes in response to nitrogen starvation remains unclear. This study delves into the intricate interplay between Med13, proteaphagy, and stress response regulation, aiming to elucidate their roles in cellular survival mechanisms. It focused on the highly conserved Cdk8 kinase module (CKM) of the Mediator complex a that plays a pivotal involvement in cellular signaling and gene regulation under stress conditions. During the investigation, we asked if the degradation of specific proteasome …

The Purification And Thermal Stability Of The Peroxidase Enzyme In Cucurbita Moschata, Garen Hamner

Senior Honors Theses

Peroxidases are enzymes that catalyze the reduction of hydrogen peroxide to water while oxidizing organic substrates and are valuable in spheres like industrial and medical applications and histochemistry. Limitations still exist in the use of the well-studied horseradish peroxidase for certain activities due to limitations like poor thermal stability, thus the search for novel peroxidases that can overcome these limitations is an active area of research. Butternut squash peroxidase (Cucurbita moschata) (BSP) shows promise due to significant activity being found in the skin and apparent enhanced thermal stability, but an efficient purification scheme for it is lacking, as well as …

Using Edna To Assess Impacts Of Oyster Restoration On Ecosystem Biodiversity At A Heavily Impacted Coastal Lagoon, Dominique Di Domenico, Emily Bonacchi, Elizabeth Suter

Molloy Multidisciplinary Undergraduate Research Conference

The Great South Bay (GSB), a bar-built, temperate, coastal lagoon on the south shore of Long Island, New York, USA, is a highly modified estuary due to heavy urbanization and suburbanization in the last century. GSB historically provided up to 50% of the nation’s hard clam (Mercenaria mercenaria) harvest, and is the legal home of the “Blue Point” oyster (eastern oyster; Crassostrea virginica). However, due to overexploitation as well as persistent stress from ongoing nitrogen pollution and harmful algal blooms, shellfish populations are fractions of their original abundances. Restoration efforts in GSB, driven both by community efforts …

Trna Anticodon Cleavage By Target-Activated Crispr-Cas13a Effector, Ishita Jain, Matvey Kolesnik, Konstantin Kuznedelov, Leonid Minakhin, Natalia Morozova, Anna Shiriaeva, Alexandr Kirillov, Sofia Medvedeva, Alexei Livenskyi, Laura Kazieva, Kira S Makarova, Eugene V Koonin, Sergei Borukhov, Konstantin Severinov, Ekaterina Semenova

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA–guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus …

In Silico Identification Of Small Molecule Agonist Binding Sites On Kcc2, Kenyon Mitchell, Alfred Amendolara, Ruth Hunter, Jaden Miner, Andrew Payne

Annual Research Symposium

Purpose: Potassium-Chloride Cotransporter 2 (KCC2) is a neuronal membrane protein specific to the central nervous system. It is responsible for removing Cl- ions from the intracellular space, maintaining a normal Cl- gradient essential for proper function at inhibitory synapses. Dysregulation causes an upward shift in the Cl- reversal potential resulting in a hyperexcitable state of the postsynaptic neuron. Existing literature indicates that KCC2 may be involved in the addiction pathway of a variety of drugs of abuse, including opioids and alcohol. This makes KCC2 an attractive potential drug target when treating substance use disorders. A novel direct KCC2 agonist, VU0500469, …

Remodeling Anaplastic Thyroid Cancer's Aggressive Profile And Metabolic Signature By Natural Alkaloid Berberine, Tara Elizabeth Jarboe

NYMC Student Theses and Dissertations

Anaplastic thyroid cancer is a rare, fatal cancer with a five-year survival of 4%. Universally diagnosed at stage IV, anaplastic thyroid cancer is characterized by its lack of differentiation, rapid proliferative rate, highly inflammatory tumor microenvironment, and metabolic dysregulation. Refractory to all established therapies, anaplastic thyroid cancer requires a novel therapeutic approach that targets all of these drivers of anaplastic thyroid cancer carcinogenesis. We propose natural alkaloid berberine as a therapeutic with multitarget efficacy to alter mitochondrial metabolism and reprogram anaplastic thyroid cancer’s aggressive phenotype. Our in vitro model uses monocyte cell line U937, anaplastic thyroid cancer cell lines T238 …

Ksp1 Is An Autophagic Receptor Protein For The Snx4-Assisted Autophagy Of Ssn2/Med13, Sara E Hanley, Stephen D Willis, Steven J Doyle, Randy Strich, Katrina F Cooper

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Ksp1 is a casein II-like kinase whose activity prevents aberrant macroautophagy/autophagy induction in nutrient-rich conditions in yeast. Here, we describe a kinase-independent role of Ksp1 as a novel autophagic receptor protein for Ssn2/Med13, a known cargo of Snx4-assisted autophagy of transcription factors. In this pathway, a subset of conserved transcriptional regulators, Ssn2/Med13, Rim15, and Msn2, are selectively targeted for vacuolar proteolysis following nitrogen starvation, assisted by the sorting nexin heterodimer Snx4-Atg20. Here we show that phagophores also engulf Ksp1 alongside its cargo for vacuolar proteolysis. Ksp1 directly associates with Atg8 following nitrogen starvation at the interface of an Atg8-family interacting …

The Biobehavioral Effects Of Embryonic Exposure To Neural Inflammation And Oxidative Stress In Zebrafish, Dalton J. Anderson

Honors College Theses

The purpose of this research is to improve understanding of the neurodevelopmental effects of embryonic exposure to elevated inflammation and oxidative stress induced by the antipyretic drug acetaminophen (APAP). Our study was the first to examine the interactive effects of APAP and inflammation in zebrafish embryos and how the treatments affect brain development and larval behavior. Experimental groups of zebrafish larvae were exposed to lipopolysaccharide (LPS) to induce inflammation, APAP, or LPS + APAP and larval behavior was analyzed using Ethovision automated behavioral tracking software. We also measured changes in whole-brain Glycogen Synthase Kinase 3 Beta (GSK3B) and GSK3B phosphorylation, …

The Effects Of Vaping Coil Temperature On The Viability Of Veillonella Parvula And Streptococcus Mutans, Marvelous O. Aluko

Harrisburg University Research Symposium: Highlighting Research, Innovation, & Creativity

No abstract provided.

Irbp In Diabetic Retinopathy – Cell Culture & Ophthalmic Pathology Studies, Laura Valdez

Research Colloquium

Background: Interphotoreceptor-Retinoid-Binding-Protein (IRBP) is restricted to the subretinal space by the external limiting membrane and RPE zonulae occludens. Its expression is reduced in diabetic retinopathy (DR) and increased expression is protective. Hypoxia decreases viable conelike photoreceptors (661W) but increased VEGF, suggesting a role of photoreceptors in proliferative DR (PDR) (Rodriguez et al, Arch Clin Exp. Ophthal 3:23,2021). Here, we investigated the effects of high glucose and VEGF on IRBP expression by 661W and rod-like Y79 cells and distribution of IRBP and albumin in human globes with PDR and NPDR.

Methods:  661W and Y79 cells were cultured in DMEM or RPMI …

Effect Of Igf-1 On Post-Translational Modifications (Ptms) On A Model Of Diabetes-Induced Cardiac Hypertrophy In H9c2 Cardiomyoblast Cell Line, Andres Medina, Lizbeth Trevino, Alejandra Salinas, Yadira Chaires, Erick Rodriguez

Research Symposium

Background: Diabetic cardiomyocytes alter their post-translational modification levels, especially in OGlcNAcylation and Phosphorylation. Insulin-like growth factor 1 (IGF-1) is a peptide known to induce favorable cardiovascular effects in patients with heart failure. Here, we focus on the downstream effects of IGF-1 as a potential DCM treatment.

Methods: H9c2 cells were cultured in DMEM-10% FBS at 80% of confluence. As a cellular model of cardiac hypertrophy, we used a high-glucose medium (30 mM glucose) in the presence or absence of 10 µmol/L of IGF-1 (HG and HG+IGF-1). As control groups, we used cells cultured in low-glucose DMEM (glucose 5mM) in the …

Profiling And Verifying The Substrates Of E3 Ubiquitin Ligase Rsp5 In Yeast Cells, Shuai Fang, Geng Chen, Yiyang Wang, Rakhee Ganti, Tatiana A Chernova, Li Zhou, Savannah E Jacobs, Duc Duong, Hiroaki Kiyokawa, Yury O Chernoff, Ming Li, Natalia Shcherbik, Bo Zhao, Jun Yin

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.

Modeling Biphasic, Non-Sigmoidal Dose-Response Relationships: Comparison Of Brain- Cousens And Cedergreen Models For A Biochemical Dataset, Venkat D. Abbaraju, Tamaraty L. Robinson, Brian P. Weiser

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Biphasic, non-sigmoidal dose-response relationships are frequently observed in biochemistry and pharmacology, but they are not always analyzed with appropriate statistical methods. Here, we examine curve fitting methods for “hormetic” dose-response relationships where low and high doses of an effector produce opposite responses. We provide the full dataset used for modeling, and we provide the code for analyzing the dataset in SAS using two established mathematical models of hormesis, the Brain-Cousens model and the Cedergreen model. We show how to obtain and interpret curve parameters such as the ED50 that arise from modeling, and we discuss how curve parameters might change …

Utilizing Crispr Cas9 To Visualize Dopamine Receptors In Caenorhabditis Elegans, Lauren Michelle Velasquez

Electronic Theses, Projects, and Dissertations

Dopamine (DA) is a neurotransmitter with imperative implications in many functions including movement, reward, and cognition. Studying the pathways of dopaminergic neurons at multiple levels allows us to understand the ways in which these systems can go wrong. We study dopamine in a model system such as the worm Caenorhabditis elegans because of its relatively simple and well-characterized nervous system. DA is involved in regulating chemosensory behaviors in worms. The purpose of this research project is to definitively answer the following question: Are the dopamine receptors DOP-1 and DOP-4 expressed in chemosensory neurons? Previous reporter assays show that neither of …

Invited Review: Adrenocortical Function In Avian And Non-Avian Reptiles: Insights From Dispersed Adrenocortical Cells., Rocco V. Carsia, Patrick J. Mcilroy, Henry B John-Alder

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Herein we review our work involving dispersed adrenocortical cells from several lizard species: the Eastern Fence Lizard (Sceloporus undulatus), Yarrow's Spiny Lizard (Sceloporus jarrovii), Striped Plateau Lizard (Sceloporus virgatus) and the Yucatán Banded Gecko (Coleonyx elegans). Early work demonstrated changes in steroidogenic function of adrenocortical cells derived from adult S. undulatus associated with seasonal interactions with sex. However, new information suggests that both sexes operate within the same steroidogenic budget over season. The observed sex effect was further explored in orchiectomized and ovariectomized lizards, some supported with exogenous testosterone. Overall, a suppressive effect of testosterone was evident, especially in cells …

Role Of Chronic Stress-Induced Neuroinflammation In Rodent Locus Coeruleus Physiology And Anxiety-Like Behaviors, Arthur Anthony Alfonso Reyes

Graduate School of Biomedical Sciences Theses and Dissertations

The locus coeruleus (LC), the primary site of brain norepinephrine (NE), is a key anatomical brain region implicated in the stress response. Stress is a neuroendocrine physiologic response to a stressor that promotes organism survival through adaptive change and restoration of homeostasis. The central stress response, which drives behavioral and physiological change, is primarily mediated by activating the hypothalamic-pituitary-adrenal (HPA) axis. While advantageous in the short term, chronic stress exposure can lead to HPA axis and LC dysregulation, which are thought to contribute to the etiology of anxiety disorders. Previous studies demonstrate the effects of acute stress in increasing LC …

Migratory Material: Epigenetics & Weaving At The Us-Mexico Border, Valerie Navarrete

Masters Theses

Discourse often sutures the body shut, disallowing representations of identity to outgrow sociopolitical interests. This issue may originate from borders, but also from the unnamable pathology that generational colonial trauma transmits to the mind, body, and environment. Without a direct form of translatability, this thesis proposes a new materialism that deviates from any object-oriented ontology. Untethered and intra-active, epigenetics and weaving represent objects that transform typical ways of knowing and seeing. Their sensitivity to the environment, in addition to their mobility across generations of time, broaden the spatiotemporal loci of the body and its embodiment. Proposing new materials that expand …

Identifying The Temporal N-Linked Glycosylation Changes During Liver Disease Progression: From Liver Injury To End-Stage Liver Disease, Shaaron Ochoa-Rios

MUSC Theses and Dissertations

The high mortality rates of liver diseases and primary liver cancers can be attributed to the lack of screening and diagnostic strategies currently available for early detection. Non-alcoholic fatty liver disease (NAFLD) is an early stage of liver disease known to progress to a variety of pre-malignant and malignant conditions, like advanced fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and cholangiocarcinoma (CCA). Based on the wide variety of diseases that NAFLD can progress to, strategies to understand and detect the progression of NAFLD are of great value. Core fucosylation of N-linked glycans has been demonstrated to be useful for the clinical diagnosis …

Identifying Co-Factors That Drive Tra-1 Activator Function, Jibran Imtiaz, Youngquan Shen, Ronald Ellis

Rowan-Virtua Research Day

Gli proteins are involved in cell fate determination, proliferation, and patterning in many species and are major effectors of Hedgehog (Hh) signaling. There are three Gli proteins in humans, and mutations or errors in their regulation lead to a variety of developmental disorders or cancer. However, the mechanisms by which they interact with co-factors are poorly understood. We are analyzing co-factors of Gli proteins using TRA-1 in Caenorhabditis nematodes. The TRA-1 zinc fingers are structurally like those of other Gli proteins, and TRA-1 can be cleaved like other Gli proteins to form a repressor. However, its function has changed during …

The Involvement Of Ubiquitin In Med13 Cyclin C Degradation Following Cellular Stress, Ayesha Gurnani, Brittany Friedson, Katrina Cooper

Rowan-Virtua Research Day

The Cdk8 Kinase Module is a dissociable regulator of cellular stress response genes, with degradation of its components Med13 and cyclin C eventually determining cell fate decisions such as engaging cell survival or cell death mechanisms. We aimed to explore the roles of ubiquitin in degradation of the Cdk8 Kinase Module following nitrogen starvation, with respect to the potential involvement of deubiquitinating enzyme Doa4, lysine linkage at position K63, and E2 ubiquitin conjugating enzymes Ubc4 and Ubc5. We utilized Western blot analysis to observe nitrogen starvation-induced degradation of Med13-HA in wild-type, doa4 mutant, and K63R yeast strains; degradation of cyclin …

Immunomodulatory Effects Of Resolvin D2 In A Model Of Infection, Prem Yugandhar Kadiyam Sundarasivarao

Graduate School of Biomedical Sciences Theses and Dissertations

Dysregulated hyperinflammatory host immune response to underlying bacterial infections is a characteristic of sepsis. In sepsis, bacteria often trigger abnormal hyperinflammatory responses which can cause multiple organ failure and if sustained can lead to an immunosuppressive phase where the host is susceptible to secondary infections caused by opportunistic bacteria like Pseudomonas aeruginosa (P. aeruginosa). In our studies, we used a 2-hit model of cecal ligation and puncture (CLP) followed by P. aeruginosa secondary lung infection to investigate cellular and molecular mechanisms in the beneficial action of resolvin D2 (RvD2). Resolvins of the D-series are a group of fatty acids known …

Targeting Metabolic Alterations Associated With Smooth Muscle Α-Actin Pathogenic Variant Attenuates Moyamoya-Like Cerebrovascular Disease, Anita Kaw

Dissertations & Theses (Open Access)

Heterozygous pathogenic variants in ACTA2, encoding smooth muscle α-actin (α-SMA), predispose to thoracic aortic aneurysms and dissections. De novo missense variants disrupting ACTA2 arginine 179 (p.Arg179) cause a multisystemic disease termed smooth muscle dysfunction syndrome (SMDS), which is characterized by early onset thoracic aortic disease and moyamoya disease-like (MMD) cerebrovascular disease. The MMD-like cerebrovascular disease in SMDS patients is marked by bilateral steno-occlusive lesions in the distal internal carotid arteries (ICAs) and their branches. To study the molecular mechanisms that underlie the ACTA2 p.Arg179 variants, a smooth muscle-specific Cre-lox knock-in mouse model of the heterozygous Acta2 R179C variant, termed …

Determination Of Cadmium Uptake In Crassostrea Virginica Shell Under Controlled Conditions, Joseph John Pavelites Ii

<strong> Theses and Dissertations </strong>

The objective of this thesis was to meet growing demand for the development of environmental biomonitors that protect ecosystems and public health. To do this, I determined the potential of oyster shell as a bioindicator of cadmium (Cd) in the environment by determining the mode of Cd uptake and relationships between Cd concentrations in the environment, shell, and soft tissues of juvenile eastern oysters (Crassostrea virginica Gmelin). I performed a review of the literature on the ability of oyster shell to retain metal contaminants and the factors that could affect this process (Chapter 2). I then reared C. virginica …

The Role Of Cerium(Iii) In Bacterial Growth And The Microbial Transformation Of Aromatic Hydrocarbons, Shruti Sathish

Honors Theses

Biofilms are communities of surface-attached bacterial cells encased in an exopolymeric matrix. In this state, they are more resistant to antimicrobial treatment and can have adverse effects in medical, agricultural, and industrial settings. Whereas, as biocatalysts, biofilms from nonpathogenic bacteria enhance their performance and stability in catalysis. Unfortunately, there are several challenges when using bacteria in organic transformations due to their complex cellular chemistry. Trivalent lanthanide metals were discovered to serve regulatory roles in some bacterial catalytic processes, including those of Pseudomonas putida KT2440 (P. putida), a non-infectious Gram-negative bacterium. The main goal of our research is to use cerium(III) …

Med13 Degradation Defines A New Receptor-Mediated Autophagy Pathway Activated By Nutrient Deprivation, Sara E. Hanley

Graduate School of Biomedical Sciences Theses and Dissertations

Cells are exposed to an enormous amount of diverse extracellular cues but have a limited arsenal of weapons for protecting and maintaining homeostasis. To overcome these restrictions, nature has engineered proteins that have multiple functions. The pleiotropy of using one protein to carry out a variety of functions allows cells to rapidly execute tailored responses to a diverse set of signals. The Cdk8 kinase module (CKM) is a conserved detachable unit of the Mediator complex predominantly known for its role in transcriptional regulation. The CKM is composed of four proteins, the scaffolding proteins Med13 and Med12, as well as the …