Identification Of A Β-Arrestin-Biased Negative Allosteric Modulator For The Β2-Adrenergic Receptor, Michael Ippolito, Francesco De Pascali, Nathan Hopfinger, Konstantin E. Komolov, Daniela Laurinavichyute, Poli Adi Narayana Reddy, Leon A. Sakkal, Kyle Z. Rajkowski, Ajay P. Nayak, Justin Lee, Jordan Lee, Gaoyuan Cao, Preston S. Donover, Melvin Reichman, Stevens. An, Joseph M. Salvino, Raymond B. Penn, Roger S S. Armen, Charles P. Scott, Jeffrey L. Benovic

Department of Biochemistry and Molecular Biology Faculty Papers

Catecholamine-stimulated β2-adrenergic receptor (β2AR) signaling via the canonical Gs–adenylyl cyclase–cAMP–PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous β-agonists in the treatment of airway disease. β2AR signaling is tightly regulated by GRKs and β-arrestins, which together promote β2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias β2AR signaling toward the Gs pathway while avoiding β-arrestin-mediated effects may provide a strategy to improve the functional consequences of β2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the β2AR have been largely unsuccessful, here we …

Genomic Features Underlie The Co-Option Of Sva Transposons As Cis-Regulatory Elements In Human Pluripotent Stem Cells, Samantha M Barnada, Andrew Isopi, Daniela Tejada-Martinez, Clément Goubert, Sruti Patoori, Luca Pagliaroli, Mason Tracewell, Marco Trizzino

Department of Biochemistry and Molecular Biology Faculty Papers

Domestication of transposable elements (TEs) into functional cis-regulatory elements is a widespread phenomenon. However, the mechanisms behind why some TEs are co-opted as functional enhancers while others are not are underappreciated. SINE-VNTR-Alus (SVAs) are the youngest group of transposons in the human genome, where ~3,700 copies are annotated, nearly half of which are human-specific. Many studies indicate that SVAs are among the most frequently co-opted TEs in human gene regulation, but the mechanisms underlying such processes have not yet been thoroughly investigated. Here, we leveraged CRISPR-interference (CRISPRi), computational and functional genomics to elucidate the genomic features that underlie SVA domestication …

Lysosomal Zn 2+ Release Triggers Rapid, Mitochondria-Mediated, Non-Apoptotic Cell Death In Metastatic Melanoma, Wanlu Du, Mingxue Gu, Meiqin Hu, Timothy Nold, Prateeksunder Pinchi, Wei Chen, Michael Ryan, Ahmed Bannaga, Haoxing Xu

Medical Student Research Symposium

During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca2+ and Zn2+ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn2+-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in …

In-Silico Determination Of Phytochemicals Against Spike Protein Of Covid-19, Muhammad Irfan, Muhammad Adil, Areej Fatima, Arooj Fatima, Ammara Khalid, Muhammad Bilal

Journal of Bioresource Management

Spike protein is present on the exterior of SARS-CoV-2 that mediates the binding of virus with human ACE2 receptor. S-protein has the ability to mutate in a short span of time. Using S-protein as a therapeutic target, Covid-19 infection can be prevented. Many plant-derived phytochemicals are found effective to treat viral infections. In this study, we selected top 10 phytochemicals following the Lipinski’s rule of five from total 82 candidate phytochemicals. The binding energies were determined through molecular docking of the phytochemical ligands. Top three compounds having maximal interactions and lowest binding energies were visualized. We suggested Dictamnine, Deoxypodophyllotoxin and …

Evaluation Of Hippocampal Allostatic Load-Associated Factors In Animal Models Of Post-Traumatic Stress Disorder: Relevance To Human Ptsd, Dennis Parker Kelley

LSU Doctoral Dissertations

Post-traumatic stress disorder (PTSD) is associated with elevated allostatic load, nearly double the risk for metabolic syndrome, reduced hippocampal volume, and contextual memory processing deficits. Emerging evidence suggests that these stress effects may predispose individuals to the development of PTSD, and there is a known relationship between chronic stress and metabolic dysfunction. In this work, we utilized two rat models of PTSD to explore these connections. We used an acute predator odor stressor to investigate the relationship between PTSD-like behaviors and mitochondrial dysfunction in the hippocampus of rats, and we observed that conditioned place avoidance was associated with reduced mitochondrial …

Creating Tools To Study The Signaling And Function Of The Adhesion Family Of Gpcrs, Victor M. Mirka

Electronic Thesis and Dissertation Repository

Adhesion GPCRs (aGPCRs) are difficult to study because they are activated by mechanical force. aGPCRs are autoproteolytically cleaved into N-terminal and C-terminal fragments. Mechanical force removes the N-terminal fragment revealing a tethered ligand activating the receptor. Proteinase Activated Receptors (PARs) are N-terminally cleaved by proteinases revealing a tethered ligand activating the receptor. We hypothesized the tethered ligand of aGPCRs could be revealed by replacing the N-terminal fragment with a PAR N-terminus. We fused the PAR2 N-terminus to the C-terminal fragments of four aGPCRs: CD97, EMR2, GPR56, and BAI1. PAR2-aGPCR chimeric receptors dose dependently recruited G-proteins and β-arrestins, supporting our hypothesis. …

Hiv-1 Drug Resistance To Integrase Strand Transfer Inhibitors In Hiv-1 Non-B Subtypes, Emmanuel Ndashimye

Electronic Thesis and Dissertation Repository

Human immunodeficiency syndrome (HIV-1) has infected over 75 million people and over 35 million have succumbed to virus related illnesses. Despite access to a variety of antiretroviral therapy (ART) options, ART programs have been disproportionally spread in the world with low-and middle-income countries (LMICs) facing challenges to access the most potent ART options. With less potent ART remaining in use in LMICs, HIV-1 drug resistance (HIVDR) presents a growing challenge in LMICs. Since approval of the first-generation integrase strand transfer inhibitor (INSTIs), Raltegravir (RAL) in 2007, INSTIs remain the best choice as a backbone of ART. Access to second generation …

Generation Of In-Frame Gene Deletion Mutants In Pseudomonas Aeruginosa And Testing For Virulence Attenuation In A Simple Mouse Model Of Infection, Meagan E. Valentine, Brandon D. Kirby, Hongwei D. Yu

Biomedical Sciences

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas eruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative …

The Development Of Novel Apurinic/Aprymidinic Endonuclease/Redox-Factor 1 Inhibitors For The Treatment Of Human Melanoma, Bella Sharifi

Pharmaceutical Sciences (MS) Theses

Apurinic/apyrimidinic DNA repair endonuclease-1 (APE1), first recognized as an important DNA excision repair enzyme, is also known as Redox Factor-1 (Ref-1) involved in the activation of many nuclear transcription factors in both redox-dependent and independent manner. It has been well-documented that the overexpression of APE/Ref-1 contributes to the development of chemo-resistance and is associated with tumor progression in many human malignancies [1].

Our previous study in melanoma demonstrated that the development of novel inhibitors targeting the redox regulation domain of APE/Ref-1 is a promising strategy for melanoma treatment. To date, limited successes have been reported in developing novel …

Investigating Trail Sensitivity In Platinum-Resistant Ovarian Cancer, Nicholas Pathoulas

All College Thesis Program, 2016-2019

Ovarian cancer is the deadliest gynecologic malignancy in the United States. While these tumors may have a promising initial response to platinum-based chemotherapies, the patient’s prognosis is commonly hindered by the development of platinum resistant cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been implicated as a potential treatment for many cancers based on its tumor selective nature. Combination therapies with TRAIL and platinum drugs have been shown to increase apoptosis and have been used in a variety of clinical trials, which have thus far failed to pass phase II.1 Researchers have not yet elucidated the complex mechanism(s) of TRAIL …

Pediatric Leukemia: Diagnosis To Treatment–A Review, Samantha C. Bernard, Ehab H. Abdelsamad, Paisley A. Johnson, Daniel L. Chapman, Madhukiran Parvathaneni

Faculty Works

Leukemia is cancer of the blood and bone marrow, it is the most common cancer found in children and is found to be more than one fourth of pediatric cancers. It causes white blood cells to become abnormal and the body to become weak. This deficiency in the immune system reduces the body's ability to fight infection or simple airborne illnesses, causing extensive treatment of common pathogens and cancer treatment. The present review covers all topics, from diagnosis to treatment of pediatric leukemia, as well as the stages of growth and physiological changes throughout the process. As leukemia has a …

The Role Of Pxr And Ikkβ Signaling In Cardiometabolic Disease, Robert N. Helsley

Theses and Dissertations--Pharmacology and Nutritional Sciences

Cardiovascular disease (CVD) is the leading cause of death worldwide and is partially attributed to perturbations in lipid metabolism. Xenobiotics, such as pharmaceutical drugs and environmental chemicals, have been associated with increased risk of CVD in multiple large-scale human population studies, but the underlying mechanisms remain poorly defined. We and others have identified several xenobiotics as potent agonists for the pregnane X receptor (PXR), a nuclear receptor that can be activated by numerous drugs as well as environmental and dietary chemicals. However, the role of PXR in mediating the pathophysiological effects of xenobiotic exposure in humans and animals remains elusive. …

Dual Pi3k/Mtor Inhibition With Bez235 Augments The Therapeutic Efficacy Of Doxorubicin In Cancer Without Influencing Cardiac Function, David E. Durrant

Theses and Dissertations

Cancer continues to be a leading cause death in the United States despite improved treatments. Cancerous lesions form after acquiring oncogenic driver mutations or losing tumor suppressor function in normal cells. Traditional therapies have included use of genotoxic substances that take advantage of the increased growth rate and loss of tumor suppressor function to cause cell death. One such drug is the anthracycline antibiotic doxorubicin (DOX). DOX interchelates into DNA and disrupts transcriptional machinery while also poisoning topoisomerase II. This results in single and double stranded DNA breaks, which if severe enough leads to either necrotic or apoptotic cell death. …

Regulation Of The High-Affinity Choline Transporter Activity And Trafficking In Alzheimer’S Disease-Related Pathological Conditions, Leah K. Cuddy

Electronic Thesis and Dissertation Repository

Cholinergic neurons play a key role in cognitive processes through the action of the neurotransmitter acetylcholine (ACh). Dysfunction of these neurons occurs in several neurodegenerative disorders, including Alzheimer’s disease (AD). The high-affinity choline transporter CHT recycles choline back into synaptic terminals, which is the rate-limiting step to ACh production. CHT proteins traffic between the cell surface and subcellular organelles in a constitutive manner, which maintains plasma membrane transporter levels, thereby regulating CHT activity and maintaining cholinergic transmission. Pathological conditions associated with AD may alter CHT function in a manner that reduces choline uptake activity and impairs cholinergic neurotransmission. Thus, my …

Fty720 (Fingolimod) Provides Insight Into The Molecular Mechanisms Of Multiple Sclerosis, Madelyn Elizabeth Crawford

Pursuit - The Journal of Undergraduate Research at The University of Tennessee

Multiple sclerosis (MS) is a neurodegenerative disorder caused by a prolonged immune- mediated inflammatory response that targets myelin. Nearly all of the drugs approved for the treatment of MS are general immunosuppressants or only function in symptom management. The oral medication fingolimod, however, is reported to have direct therapeutic effects on cells of the central nervous system in addition to immunomodulatory functions. Fingolimod is known to interact with sphingosine-1-phosphate (S1P) receptors, and the most widely- accepted theory for its mechanism of action is functional antagonism of the receptor. This review examines significant neuromodulatory effects achieved by functional antagonism of the …

Lrh1 As A Driving Factor For Cancer Development, Alissa M. Margraf

Senior Honors Projects

LRH1 as a driving factor for cancer development

Alissa Margraf, Qi Tang, Qiushi Lin, Xiaoqun Dong

Department of Biomedical and Pharmaceutical Science, College of Pharmacy, The University of Rhode Island, Pharmacy Building, 7 Greenhouse Road, Kingston, RI 02881 USA

Cancer is a major public health problem worldwide. Colon cancer ranks as the third most common causes of cancer mortality in the United States, with an estimated 96,830 new cases and 50,310 deaths in 2014. Colon cancer develops in the digestive tract where benign growths called polyps transform into malignant tumors. Colon cancer cells invade and destroy nearby tissue and can …

Identification And Functional Analysis Of Alternatively Spliced Isoforms Of Soluble Adenylyl Cyclase In Human Bronchial Epithelial Cells, Xi Chen

Xi Chen

No abstract provided.

Constitutive Mu-Opioid Receptor Activity Leads To Long-Term Endogenous Analgesia And Dependence, Gregory Corder, Suzanne Doolen, Renee R. Donahue, Michele K. Winter, Brandon L. Jutras, Y He, X Hu, Joseph S. Wieskopf, Jeffrey S. Mogil, Daniel R. Storm, Z J. Wang, Kenneth E. Mccarson, Bradley K. Taylor

Renee R. Donahue

Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced m-opioid receptor (MOR) constitutive activity (MORCA) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3′,5′-monophosphate overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1. Thus, MORCA initiates both analgesic signaling and a compensatory opponent …

Supplemental Data For Science 2013 Corder Et Al. Constitutive Mu-Opioid Receptor Activity Leads To Long-Term Endogenous Analgesia And Dependence, Renee R. Donahue

Renee R. Donahue

Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced m-opioid receptor (MOR) constitutive activity (MORCA) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3′,5′-monophosphate overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1. Thus, MORCA initiates both analgesic signaling and a compensatory opponent …

Pparg Activation Blocks Development And Reduces Established Neuropathic Pain In Rats, Jenny Morgenweck, Ryan B. Griggs, Renee R. Donahue, James E. Zadina, Bradley K. Taylor

Renee R. Donahue

Peroxisomeproliferator-activated receptor gamma (PPARg) isemerging as a newpharmacotherapeutic target for chronic pain.When oral (3e30 mg/kg/day in chowfor 7 wk) or twice-daily intraperitoneal (1e10 mg/kg/ day for 2 wk) administration began before spared nerve injury (SNI), pioglitazone, a PPARg agonist, dosedependently prevented multiple behavioral signs of somatosensory hypersensitivity. The highest dose of intraperitoneal pioglitazone did not produce ataxia or reductions in transient mechanical and heat nociception, indicating that inhibitory effects on hypersensitivity were not secondary to adverse drug-induced behaviors or antinociception. Inhibitory effects on hypersensitivity persisted at least one week beyond cessation of pioglitazone administration, suggestive of long-lasting effects on gene …

The Use Of Cerium Oxide And Curcumin Nanoparticles As Therapeutic Agents For The Treatment Of Ventricular Hypertrophy Following Pulmonary Arterial Hypertension, Madhukar Babu Kolli

Theses, Dissertations and Capstones

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by inflammation, increased pulmonary vascular resistance, right ventricular failure and premature death. Monocrotaline (MCT) has been used to induce PAH in laboratory rats. Previous in vitro and in vivo work suggested that cerium oxide (CeO2)-and curcumin nanoparticles exhibit anti-inflammatory activity; however, it is unknown if these materials are effective for the treatment of PAH induced cardiac hypertrophy. To determine the efficacy of CeO2 nanoparticle treatment in preventing MCT-induced RV hypertrophy, male Sprague Dawley rats were divided into one of three groups (control, MCT, or MCT + CeO2 nanoparticle, n=6/group). …

Mitochondrial Lysyl-Trna Synthetase Independent Import Of Trna Lysine Into Yeast Mitochondria., Naresh Babu V Sepuri, Madhavi Gorla, Michael P King

Department of Biochemistry and Molecular Biology Faculty Papers

Aminoacyl tRNA synthetases play a central role in protein synthesis by charging tRNAs with amino acids. Yeast mitochondrial lysyl tRNA synthetase (Msk1), in addition to the aminoacylation of mitochondrial tRNA, also functions as a chaperone to facilitate the import of cytosolic lysyl tRNA. In this report, we show that human mitochondrial Kars (lysyl tRNA synthetase) can complement the growth defect associated with the loss of yeast Msk1 and can additionally facilitate the in vitro import of tRNA into mitochondria. Surprisingly, the import of lysyl tRNA can occur independent of Msk1 in vivo. This suggests that an alternative mechanism is present …

Substrate Envelope And Drug Resistance: Crystal Structure Of Ro1 In Complex With Wild-Type Human Immunodeficiency Virus Type 1 Protease, Moses Prabu-Jeyabalan, Nancy M. King, Ellen A. Nalivaika, Gabrielle Heilek-Snyder, Nick Cammack, Celia A. Schiffer

Celia A. Schiffer

In our previous crystallographic studies of human immunodeficiency virus type 1 (HIV-1) protease-substrate complexes, we described a conserved "envelope" that appears to be important for substrate recognition and the selection of drug-resistant mutations. In this study, the complex of HIV-1 protease with the inhibitor RO1 was determined and comparison with the substrate envelope provides a rationale for mutational patterns.

Desulfovibrio Desulfuricans G20 Tetraheme Cytochrome Structure At 1.5 A˚ And Cytochrome Interaction With Metal Complexes, Mrunalini Pattarkine, J J. Tanner, C A. Bottoms, Y H. Lee, Judy D. Wall

Faculty Works

The structure of the type I tetraheme cytochrome c3 from Desulfovibrio desulfuricans G20 was determined to 1.5 A˚ by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated …

Studies On The Formation Of Dna-Cationic Lipid Composite Films And Dna Hybridization In The Composites, Murali Sastry, Vidya Ramakrishnan, Mrunalini Pattarkine, Krishna N. Ganesh

Faculty Works

The formation of composite films of double-stranded DNA and cationic lipid molecules (octadecylamine, ODA) and the hybridization of complementary single-stranded DNA molecules in such composite films are demonstrated. The immobilization of DNA is accomplished by simple immersion of a thermally evaporated ODA film in the DNA solution at close to physiological pH. The entrapment of the DNA molecules in the cationic lipid film is dominated by attractive electrostatic interaction between the negatively charged phosphate backbone of the DNA molecules and the protonated amine molecules in the thermally evaporated film and has been quantified using quartz crystal microgravimetry (QCM). Fluorescence studies …

Cationic Surfactant Mediated Hybridization And Hydrophobization Of Dna Molecules At The Liquid/Liquid Interface And Their Phase Transfer, Murali Sastry, Ashavani Kumar, Mrunalini Pattarkine, Vidya Ramakrishnan, Krishna N. Ganesh

Faculty Works

Hybridization of complementary oligonucleotides mediated by a cationic surfactant at the water/hexane interface leads to hydrophobic, double-helical DNA which may be readily phase transferred to the organic phase and cast into thin films on solid substrates.

Hybridization Of Dna By Sequential Immobilization Of Oligonucleotides At The Air-Water Interface, Murali Sastry, Vidya Ramakrishnan, Mrunalini Pattarkine, Anand Gole, K. N. Ganesh

Faculty Works

The hybridization of DNA by sequential electrostatic and hydrogen-bonding immobilization of single-stranded complementary oligonucleotides at the air-water interface with cationic Langmuir monolayers is demonstrated. The complexation of the single-stranded DNA molecules with octadecylamine (ODA) Langmuir monolayers was followed in time by monitoring the pressure-area isotherms. A large (and slow) expansion of the ODA monolayer was observed during each stage of complexation in the following sequence: primary single-stranded DNA followed by complementary single-stranded DNA followed by the intercalator, ethidium bromide. Langmuir-Blodgett (LB) films of the ODA-DNA complex were formed on different substrates and characterized using quartz-crystal microgravimetry (QCM), Fourier transform infrared …

Anion Induced Blue To Purple Transition In Bacteriorhodopsin, Mrunalini Pattarkine, Anil K. Singh

Faculty Works

Purple membrane (PM, λ" role="presentation">λmax" role="presentation">max 570 nm) of H. halobium on treatment with sulphuric acid changes its colour to blue (λ" role="presentation">λmax" role="presentation">max 608 nm). The purple chromophore can be regenerated from the blue chromophore by exogeneous addition of anions such as CI−" role="presentation">− and HPO42−" role="presentation">2−4. Chloride ion is found to be more effective than the dibasic phosphate ion in regenerating the purple chromophore. Nevertheless, one thing common to the anion regeneration is that both CI−" role="presentation">− and HPO42−" role="presentation">2−4 show marked pH effect. At pH 1.0 the efficiency of …