Medical Cell Biology Commons^™

Effect Of S100b Deletion On Membrane Properties And Localization Of Ncald And Hpca, Natasha Hesketh

Graduate School of Biomedical Sciences Theses and Dissertations

Calcium signaling is particularly important for neuronal function. Neurons utilize a wide range of calcium-binding proteins. Dysregulation of such proteins is linked to neurodegeneration. Neurocalcin delta (NCALD), hippocalcin (HPCA), and S100B are calcium sensors that are expressed in the hippocampus, a brain region essential to memory and severely damaged in Alzheimer’s disease (AD). Despite the potential importance of these proteins, we do not fully understand the physiological significance of their relationship. Because NCALD and HPCA are known to interact with S100B, we hypothesized that the loss of S100B affects NCALD and HPCA localization, and therefore electrical properties, of hippocampal neurons. …

Comparative Molecular Transporter Properties Of Cyclic Peptides Containing Tryptophan And Arginine Residues Formed Through Disulfide Cyclization, Eman H. M. Mohammed, Dindyal Mandal, Saghar Mozaffari, Magdy Abdel-Hamied Zahran, Amany Mostafa Osman, Rakesh Kumar Tiwari, Keykavous Parang

Pharmacy Faculty Articles and Research

We have previously reported cyclic cell-penetrating peptides [WR]₅ and [WR]₄ as molecular transporters. To optimize further the utility of our developed peptides for targeted therapy in cancer cells using the redox condition, we designed a new generation of peptides and evaluated their cytotoxicity as well as uptake behavior against different cancer cell lines. Thus, cyclic [C(WR)_xC] and linear counterparts (C(WR)_xC), where x = 4–5, were synthesized using Fmoc/tBu solid-phase peptide synthesis, purified, and characterized. The compounds did not show any significant cytotoxicity (at 25 µM) against ovarian (SK-OV-3), leukemia (CCRF-CEM), gastric adenocarcinoma (CRL-1739), breast …

The Anti-Proliferative Effects Of Methotrexate And Novel Ucp1162 On Acute Myeloid Leukemia Cell Lines, Jacqueline Klepinger, Charles Giardina, Didem Ozcan

Honors Scholar Theses

Cancer cells proliferate at rapid rates due to the aberrant activity of proteins involved in regulating the cell cycle. This characteristic allows mutated cancer cells to spread and metastasize, causing lesions to form throughout the body. Two treatment conditions, one classical antifolate methotrexate (MTX) and non-classical, novel antifolate UCP1162, were tested on a panel of acute myeloid leukemia (AML) cell lines to determine if UCP1162 has higher anti-proliferative activity. High dose MTX is used as a first line chemotherapy in common childhood malignancies such as acute lymphoid leukemia (ALL). Methotrexate is excluded from acute myeloid leukemia (AML) treatments based on …

Identifying Determinants Of Target Specificity In Two Related Bacterial Peptide Toxins, Andrew D. Holmes

Honors Thesis

Toxin-antitoxin (TA) systems were originally identified as two-component systems ensuring the stable inheritance of plasmids in bacterial populations. Recently, they have been identified on bacterial chromosomes where their functions remain mostly undefined. The par locus of E. faecalis plasmid pAD1 (par_pAD1) was the first TA system defined in a Gram-positive bacterium and a homolog encoded on the E. faecalis chromosome (par_EF0409) was later described. Related loci numbering in the hundreds have been identified throughout Gram-positive bacteria based on homology to the toxin of the system, Fst, and similarities in genetic organization and regulation. Despite …

Effects Of Nicotinamide Riboside And Beta-Hydroxybutyrate On C. Elegans Lifespan, Jeffery Peters

Undergraduate Honors Theses

The nicotinamide riboside (NR) form of vitamin B₃and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B₃are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD⁺decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD⁺levels. BHB is naturally produced by the body when individuals fast …

Cloning, Protein Expression, And Characterization Of Interleukin 1 Alpha, Musaab Habeeb Ali Al Ameer

Graduate Theses and Dissertations

Recombinant DNA technology and the ability to produce recombinant proteins have significantly changed the world of pharmaceutical market. Recombinant DNA technology using E. coli cells has successfully produced many therapeutic proteins. In this study, the designed tag Ark-RUBY-tag facilitates rapid, and reproducible purification of recombinant proteins expressed as inclusion bodies in E. coli cells. Purification of Ark-RUBY-fused recombinant protein(s) can be obtained by using imidazole fractions. Target protein can be easily removed from the Ark-RUBY-tag by enzymatic cleavage. Ark-RUBY-fused recombinant proteins can be quantitatively detected using polyclonal antibodies. Ark-RUBY-tag can be used to purify small peptides. By using Ark-RUBY-tag, purified …

Alternative Polyadenylation Modulates Expression Of Pro-Fibrotic Proteins And Contributes To Lung Fibrosis, Junsuk Ko

Dissertations and Theses (Open Access)

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease which affects about 5 to 8 million individuals in the world. Despite the high prevalence, there is currently no cure for IPF, and the cause of this disease is still unclear. Our laboratory and collaborators have shown that nudix hydrolase 21 (NUDT21, which is also known as cleavage factor 25, CFIm25) is a key regulator of alternative polyadenylation (APA). NUDT21 depletion causes 3’UTR shortening via APA leading to enhanced mRNA stability and protein translation. This NUDT21 reduction promotes tumor growth in glioblastoma by enhancing expression of oncogenes. Cancer and IPF share …

Prostate Field Cancerization And Exosomes: Association Between Cd9, Early Growth Response 1 And Fatty Acid Synthase, Farideh Amirrad, Philip A. Pytak, Neda Sadeghiani-Pelar, Julie P. T. Nguyen, Emily L. Cauble, Anna C. Jones

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Intracapsular and well‑defined adenocarcinomas of the prostate are often surrounded by tissue areas that harbor molecular aberrations, including those of genetic, epigenetic and biochemical nature. This is known as field cancerization, or a field effect and denotes a state of pre‑malignancy. Such alterations in histologically normal tumor‑adjacent prostatic tissues have been recognized as clinically important and are potentially exploitable as biomarkers of disease and/or targets for preventative/therapeutic intervention. The authors have previously identified and validated two protein markers of field cancerization: The expressional upregulation of the transcription factor early growth response 1 (EGR‑1) and the lipogenic enzyme fatty acid synthase …

Combination Of Investigational Cell-Based Therapy And Deep Brain Stimulation To Alter The Progression Of Parkinson’S Disease, Nader El Seblani

Theses and Dissertations--Pharmacy

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and the motor symptoms are caused by progressive loss of midbrain dopamine neurons. There is no current treatment that can slow or reverse PD. Our current “DBS-Plus” clinical trial (NCT02369003) features the implantation in vivo of autologous Schwann cells (SCs) derived from a patient’s sural nerve into the substantia nigra pars compacta (SNpc) in combination with Deep Brain Stimulation (DBS) therapy for treating patients with advanced PD.

The central hypothesis of our research is that transdifferentiated SCs within conditioned nerve tissue will deliver pro-regenerative factors to enhance the survival of …

Modulation Of Epileptogenesis: A Paradigm For The Integration Of Enzyme-Based Microelectrode Arrays And Optogenetics, Corwin R. Butler, Jeffery A. Boychuk, Francois Pomerleau, Ramona Alcala, Peter Huettl, Yi Ai, Johan Jakobsson, Sidney W. Whiteheart, Greg A. Gerhardt, Bret N. Smith, John T. Slevin

Physiology Faculty Publications

BACKGROUND: Genesis of acquired epilepsy includes transformations spanning genetic-to- network-level modifications, disrupting the regional excitatory/inhibitory balance. Methodology concurrently tracking changes at multiple levels is lacking. Here, viral vectors are used to differentially express two opsin proteins in neuronal populations within dentate gyrus (DG) of hippocampus. When activated, these opsins induced excitatory or inhibitory neural output that differentially affected neural networks and epileptogenesis. In vivo measures included behavioral observation coupled to real-time measures of regional glutamate flux using ceramic-based amperometric microelectrode arrays (MEAs).

RESULTS: Using MEA technology, phasic increases of extracellular glutamate were recorded immediately upon application of blue light/488 nm …

Residues And Residue Pairs Of Evolutionary Importance Differentially Direct Signaling Bias Of D2 Dopamine Receptors, María E Terrón-Díaz, Sara J Wright, Melina A Agosto, Olivier Lichtarge, Theodore G Wensel

Faculty, Staff and Students Publications

The D2 dopamine receptor and the serotonin 5-hydroxytryptamine 2A receptor (5-HT2A) are closely-related G-protein–coupled receptors (GPCRs) from the class A bioamine subfamily. Despite structural similarity, they respond to distinct ligands through distinct downstream pathways, whose dysregulation is linked to depression, bipolar disorder, addiction, and psychosis. They are important drug targets, and it is important to understand how their bias toward G-protein versus β-arrestin signaling pathways is regulated. Previously, evolution-based computational approaches, difference Evolutionary Trace and Evolutionary Trace–Mutual information (ET-Mip), revealed residues and residue pairs that, when switched in the D2 receptor to the corresponding residues from 5-HT2A, altered ligand potency …

Examining Multiple Cellular Pathways At Once Using Multiplex Hextuple Luciferase Assaying, Alejandro Sarrion-Perdigones, Lyra Chang, Yezabel Gonzalez, Tatiana Gallego-Flores, Damian W Young, Koen J T Venken

Faculty, Staff and Students Publications

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five …

Alzheimer's And Amyloid Beta: Amyloidogenicity And Tauopathy Via Dyshomeostatic Interactions Of Amyloid Beta, Jordan Tillinghast

Senior Honors Theses

This paper reviews functions of Amyloid-β (Aβ) in healthy individuals compared to the consequences of aberrant Aβ in Alzheimer’s disease (AD). As extraneuronal Aβ accumulation and plaque formation are characteristics of AD, it is reasonable to infer a pivotal role for Aβ in AD pathogenesis. Establishing progress of the disease as well as the mechanism of neurodegeneration from AD have proven difficult (Selkoe, 1994). This thesis provides evidence suggesting the pathogenesis of AD is due to dysfunctional neuronal processes involving Aβ’s synaptic malfunction, abnormal interaction with tau, and disruption of neuronal homeostasis. Significant evidence demonstrates that AD symptoms are partially …

Activation Of Lxrβ Inhibits Tumor Respiration And Is Synthetically Lethal With Bcl-Xl Inhibition, Trang Thi Thu Nguyen, Chiaki Tsuge Ishida, Enyuan Shang, Chang Shu, Consuelo Torrini, Yiru Zhang, Elena Bianchetti, Maria J. Sanchez-Quintero, Giulio Kleiner, Catarina M. Quinzii, Mike-Andrew Westhoff, Georg Karpel-Massler, Peter Canoll, Markus D. Siegelin

Publications and Research

Liver-X-receptor (LXR) agonists are known to bear anti-tumor activity. However, their efficacy is limited and additional insights regarding the underlying mechanism are necessary. By performing transcriptome analysis coupled with global polar metabolite screening, we show that LXR agonists, LXR623 and GW3965, enhance synergistically the anti-proliferative effect of BH3 mimetics in solid tumor malignancies, which is predominantly mediated by cell death with features of apoptosis and is rescued by exogenous cholesterol. Extracellular flux analysis and carbon tracing experiments (U-¹³C-glucose and U-¹³C-glutamine) reveal that within 5 h, activation of LXRβ results in reprogramming of tumor cell metabolism, leading …

Natural Autoantibodies: Origin, Function And Utility For Diagnosis Of Disease, Abhirup Sarkar

Graduate School of Biomedical Sciences Theses and Dissertations

Autoantibodies (aAbs) by the simplest definitions have been described as antibodies against self-antigens and were exclusively associated with autoimmune diseases. Eventually, studies demonstrated that they are abundant in the blood of all human sera, regardless of age, gender, or the presence or absence of disease, and were thus named as ‘natural autoantibodies’. The underlying reason for their ubiquity has remained elusive, but we have hypothesized that they are responsible for clearing blood-borne cell and tissue debris generated under conditions of health and disease. To test this, we chose to use two widely different disease model systems, namely neurodegenerative diseases and …

Cyclin C Regulated Oxidative Stress Responsive Transcriptome In Mus Musculus Embryonic Fibroblasts, David C Stieg, Kai-Ti Chang, Katrina F Cooper, Randy Strich

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The transcriptional changes that occur in response to oxidative stress help direct the decision to maintain cell viability or enter a cell death pathway. Cyclin C-Cdk8 is a conserved kinase that associates with the RNA polymerase II Mediator complex that stimulates or represses transcription depending on the locus. In response to oxidative stress, cyclin C, but not Cdk8, displays partial translocation into the cytoplasm. These findings open the possibility that cyclin C relocalization is a regulatory mechanism governing oxidative stress-induced transcriptional changes. In the present study, the cyclin C-dependent transcriptome was determined and compared to transcriptional changes occurring in oxidatively …

Real-Time Interactive Simulation Of Human Atrial Electrical Activity On Realistic 3d Atrial Structures Using Webgl, Abouzar Kaboudian, Yanyan Ji, Elizabeth Cherry, Flavio H. Fenton

Biology and Medicine Through Mathematics Conference

No abstract provided.

Development Of A Sonically Powered Biodegradable Nanogenerator For Bone Regeneration, Avi Patel

Honors Scholar Theses

Background: Reconstruction of bone fractures and defects remains a big challenge in orthopedic surgery. While regenerative engineering has advanced the field greatly using a combination of biomaterial scaffolds and stem cells, one matter of difficulty is inducing osteogenesis in these cells. Recent works have shown electricity’s ability to promote osteogenesis in stem cell lines when seeded in bone scaffolds; however, typical electrical stimulators are either (a) externally housed and require overcomplex percutaneous wires be connected to the implanted scaffold or (b) implanted non-degradable devices which contain toxic batteries and require invasive removal surgeries.

Objective: Here, we establish a biodegradable, piezoelectric …

The Role Of Ros In The Progression And Treatment Of Castration-Resistant Prostate Cancer, Dannah R. Miller

Theses & Dissertations

Prostate cancer is the second leading cause of cancer-related deaths in U.S. men, primarily due to the development of castration-resistant (CR) prostate cancer (PCa), of which there are no effective treatment options. Reactive oxygen species (ROS) plays a critical role in prostate carcinogenesis, including the progression of the CR PCa phenotype. ROS regulates both cell proliferation and apoptosis; a moderate increase in ROS can promote proliferation; however, a substantial rise in ROS levels will result in apoptosis. Oxidase p66Shc is elevated in clinical PCa cells and has been associated with a metastatic phenotype of CR PCa cells, promoting PCa cell …

Yeast Mitochondrial Protein Pet111p Binds Directly To Two Distinct Targets In Cox2 Mrna, Suggesting A Mechanism Of Translational Activation, Julia L Jones, Katharina B Hofmann, Andrew T Cowan, Dmitry Temiakov, Patrick Cramer, Michael Anikin

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in …

Evaluating The Potential Of Repurposing Commercially Available Drugs For The Treatment Of Viral Infections, Brennan Connor Mcewan

Undergraduate Honors Capstone Projects

Viral infections that are often overlooked as common seasonal illnesses such as influenza can rapidly become a public threat. They threaten society as new, more dangerous strains of these common viruses emerge and as strains develop resistance to current vaccines and antiviral treatments (Kochanek, Murphy, Xu, & Tejada-Vera, 2014). To combat this, the development of antiviral treatments with novel mechanisms of action is essential. Repurposing drugs instead of developing new drugs can save years of development time and hundreds of millions of dollars (DiMasi, Hansen, & Grabowski, 2003). To support the effort to discover drugs with unique mechanisms of action, …

Itch Nuclear Translocation And H1.2 Polyubiquitination Negatively Regulate The Dna Damage Response, Lufen Chang, Lei Shen, Hu Zhou, Jing Gao, Hangyi Pan, Li Zheng, Brian Armstrong, Yang Peng, Guang Peng, Binhua P. Zhou, Steven T. Rosen, Binghui Shen

Molecular and Cellular Biochemistry Faculty Publications

The downregulation of the DNA damage response (DDR) enables aggressive tumors to achieve uncontrolled proliferation against replication stress, but the mechanisms underlying this process in tumors are relatively complex. Here, we demonstrate a mechanism through which a distinct E3 ubiquitin ligase, ITCH, modulates DDR machinery in triple-negative breast cancer (TNBC). We found that expression of a nuclear form of ITCH was significantly increased in human TNBC cell lines and tumor specimens. Phosphorylation of ITCH at Ser257 by AKT led to the nuclear localization of ITCH and ubiquitination of H1.2. The ITCH-mediated polyubiquitination of H1.2 suppressed RNF8/RNF168-dependent formation of 53BP1 foci, …

Histology For Cytotechnologists I, Joanna Burkhardt

Library Impact Statements

No abstract provided.

Heat Shock Protein 40 And Immune Function In Altered Gravity, Amber M. Paul, Brooke D. Shepard, Sharmila Bhattacharya

Publications

In space, astronauts are more susceptible to pathogens, viral reactivation and immunosuppression, which poses limits to their health and the mission. Interestingly, during space flight, stress-inducible heat shock proteins (HSP) are robustly induced, and the overexpression of HSPs have been implicated in immune dysregulation, therefore HSPs may be critically involved in regulating immune homeostasis. HSP40/DNAJ1 plays a major role in proper protein translation and folding. Its loss of function has been implicated in susceptibility to microbial infection, while its overexpression has been implicated in autoimmunity, collectively suggesting its complicated, but necessary, role in maintaining immunological function. To determine the role …

Cysteine Residues Contribute To The Dimerization And Enzymatic Activity Of Human Nuclear Dutp Nucleotidohydrolase (Ndut)., Shawna M Rotoli, Julia L Jones, Salvatore J Caradonna

Rowan-Virtua School of Osteopathic Medicine Departmental Research

dUTPase is an enzyme found in all organisms that have thymine as a constituent of DNA. Through evolution, humans have two major isoforms of dUTPase: a mitochondrial (mDut) and a nuclear (nDut) isoform. The nuclear isoform of dUTPase is a 164-amino-acids-long protein containing three cysteine residues. nDut's starting methionine is post-translationally cleaved, leaving four unique amino acids on its amino-terminus including one cysteine residue (C3). These are not present in the mitochondrial isoform (mDut). Using mass spectrometry analyses of recombinant dUTPase constructs, we have discovered an intermolecular disulfide bridge between cysteine-3 of each nDut monomer. We have found that these …

Editorial: Ion Channel Trafficking And Cardiac Arrhythmias, Marcel A. G. Van Der Heyden, Brian P. Delisle, Hugues Abriel

Physiology Faculty Publications

No abstract provided.

Myocyte [Na+]I Dysregulation In Heart Failure And Diabetic Cardiomyopathy, Sanda Despa

Pharmacology and Nutritional Sciences Faculty Publications

By controlling the function of various sarcolemmal and mitochondrial ion transporters, intracellular Na⁺ concentration ([Na⁺]_i) regulates Ca²⁺ cycling, electrical activity, the matching of energy supply and demand, and oxidative stress in cardiac myocytes. Thus, maintenance of myocyte Na⁺ homeostasis is vital for preserving the electrical and contractile activity of the heart. [Na⁺]_i is set by the balance between the passive Na⁺ entry through numerous pathways and the pumping of Na⁺ out of the cell by the Na⁺/K⁺-ATPase. This equilibrium is perturbed in heart failure, …

Escherichia Coli Itat Is A Type Ii Toxin That Inhibits Translation By Acetylating Isoleucyl-Trnaile, Brendan Wilcox, Ilya Osterman, Marina Serebryakova, Dmitry Lukyanov, Ekaterina Komarova, Bridget Gollan, Natalia Morozova, Yuri I Wolf, Kira S Makarova, Sophie Helaine, Petr Sergiev, Svetlana Dubiley, Sergei Borukhov, Konstantin Severinov

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Prokaryotic toxin-antitoxin (TA) modules are highly abundant and are involved in stress response and drug tolerance. The most common type II TA modules consist of two interacting proteins. The type II toxins are diverse enzymes targeting various essential intracellular targets. The antitoxin binds to cognate toxin and inhibits its function. Recently, TA modules whose toxins are GNAT-family acetyltransferases were described. For two such systems, the target of acetylation was shown to be aminoacyl-tRNA: the TacT toxin targets aminoacylated elongator tRNAs, while AtaT targets the amino acid moiety of initiating tRNAMet. We show that the itaRT gene pair from Escherichia coli …

N-Terminal Domain Of Human Uracil Dna Glycosylase (Hung2) Promotes Targeting To Uracil Sites Adjacent To Ssdna-Dsdna Junctions, Brian P Weiser, Gaddiel Rodriguez, Philip A Cole, James T Stivers

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias …

Uplc-Ms/Ms Analysis Of Dextromethorphan-O-Demethylation Kinetics In Rat Brain Microsomes, Barent N. Dubois, Reza Mehvar

Pharmacy Faculty Articles and Research

Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. …