Molecular Biology Commons^™

Zinc Chloride Enhanced Chondrogenesis Is Vegf Dependent, Gilbert M. Sharp Iv

Seton Hall University Dissertations and Theses (ETDs)

Researchers have begun investigating whether insulin mimetics such as ZnCl2 could promote bone healing in both non-diabetic and diabetic fracture healing similarly to insulin. Our research focused on understanding the mechanism by which ZnCl2 affects chondrogenesis, an important component of bone fracture healing. The increases in proteoglycan deposition and cell proliferation seen in our data may be a result of ZnCl2 induction of the IGF-1 pathway. When the VEGF pathway was inhibited in ZnCl2- or insulin-treated cells significant decreases in proteoglycan deposition occurred on day 7 and 14 (P=0.007 for ZnCl2, P=0.028 for insulin) when compared to controls. This data …

Development Of A Lectin-Fc Fusion Protein With Antiviral And Anti-Cancer Activity., Matthew William Dent

Electronic Theses and Dissertations

This thesis describes the development of a novel lectin-Fc fusion protein and its antiviral and anti-cancer activity. The molecule, Avaren-Fc (AvFc), is a fusion of a variant of the actinomycete lectin actinohivin (Avaren) and the Fc region of human IgG1, and is selective for the terminal α1,2-mannose residues found at the ends of high-mannose-type glycans that can be found on the surface of certain heavily glycosylated viruses and cancer cells. Here, AvFc was found to be able to neutralize simian immunodeficiency virus as well as Hepatitis C virus with nanomolar IC₅₀ values. Furthermore, AvFc recognizes a number of cell …

Ck2 Negatively Regulates 5-Ht4 Receptor Signaling In The Prefrontal Cortex And Mediates Depression-Like Behaviors, Julia Castello Saval

Dissertations, Theses, and Capstone Projects

The serotonergic system has been the major candidate in the pathophysiology of mood related disorders such as anxiety and major depressive disorder (MDD). Unfortunately, current antidepressant drugs are ineffective in 50% of the population and require chronic administration for a period of 3-6 weeks before the onset of therapeutic response. 5-HT4 receptor (5-HT₄R) agonists have emerged as potential candidates for fast antidepressant action, since an antidepressant response can be achieved after 3 days of pharmacological administration in rodents.

This dissertation aims to investigate the role of casein kinase 2 (CK2) as a regulator of 5-HT₄R expression …

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, …

Toward An Enzyme-Coupled, Bioorthogonal Platform For Methyltransferases: Probing The Specificity Of Methionine Adenosyltransferases, Tyler D. Huber

Theses and Dissertations--Pharmacy

Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to …

Eralpha Isoforms Modulate The Tumorigenicity Of 24r,25(Oh)2d3 In Estrogen-Responsive Cancer, Anjali Verma

Theses and Dissertations

Over 200,000 cases of breast cancer are diagnosed every year. Nearly 20% of these patients supplement their diets with some form of vitamin D. This high frequency of vitamin D supplement use may be due in part to research suggesting that cancer patients with higher serum vitamin D₃ levels have better prognoses than patients with low serum vitamin D_3. However, double-blind clinical trials on the efficacy of vitamin D₃ supplementation in breast cancer have been inconclusive. A recent meta-analysis showed evidence of reduced cancer recurrence in patients taking vitamin D₃ supplements who had ‘estrogen receptor positive’ …

The First In Vivo Human Methionine Sulfide Proteome And The Impact Of Smoking, Abdullah Qassab

Graduate Theses and Dissertations

Reactive oxygen species are naturally generated within the human body and they are known to modulate signaling pathway and mediate other physiological activities. However, excessive generation of ROS and the inability of body defense system in detoxifying them results in the so called “oxidative stress”. Methionine has powerful antioxidant properties due to the presence of electronegative sulfur in its structure. Therefore, Met is readily oxidized, and methionine sulfoxide has been linked to several pathological conditions.

The urinary proteome is an attractive candidate for the discovery of biomarkers to diagnose and classify health conditions because of the non-invasive collection procedure. However, …

Development Of A Pd-L1 Pet Imaging Biomarker, Caleb Jack Bridgwater

Posters-at-the-Capitol

Immunotherapy strategies are very promising treatments for cancer patients. Specifically, Immune checkpoint inhibitor therapy focusing on the PD-1/PD-L1 pathway shows long-lasting positive results in many cancer patients. Unfortunately, not all the patients can benefit from this highly effective treatment. Hence, there is a great need for predictive biomarkers. Immunohistochemical (IHC) staining has been used as a way of predicting patient response, yet shows many problems. For example, IHC utilizes an invasive biopsy and sample fixing, which creates an incomplete and delayed picture of the patient’s biochemistry and the tumor microenvironment, consequently ignoring metastases.

The purpose of this study is to …

Direct Quantification Of Deubiquitinating Enzyme Activity In Single Intact Cells, Nora Safabakhsh

LSU Doctoral Dissertations

Challenges in drug efficacy occur during the treatment of most types of cancer due to the heterogeneity of the tumor microenvironment. This has led to the development of personalized medicine. Due to the clinical success of the proteasome inhibitors Bortezomib and Carfilzomib in treatment of multiple myeloma, interest has shifted towards molecularly-targeted chemotherapeutics for ubiquitin-proteasome system (UPS). Deubiquitinating enzymes (DUBs) are an essential part of this pathway which have been found to promote Bortezomib resistance in multiple myeloma patients. Unfortunately, there is a lack of specific, high throughput biochemical assays to characterize DUB activity in patient samples before and after …

Fars2 Mutations Presenting With Pure Spastic Paraplegia And Lesions Of The Dentate Nuclei, Supreet K. Sahai, Rebecca E. Steiner, Margaret G. Au, John M. Graham, Norikio Salamon, Michael Ibba, Tyler M. Pierson

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Mutations in FARS2, the gene encoding the mitochondrial phenylalanine‐tRNA synthetase (mtPheRS), have been linked to a range of phenotypes including epileptic encephalopathy, developmental delay, and motor dysfunction. We report a 9‐year‐old boy with novel compound heterozygous variants of FARS2, presenting with a pure spastic paraplegia syndrome associated with bilateral signal abnormalities in the dentate nuclei. Exome sequencing identified a paternal nonsense variant (Q216X) lacking the catalytic core and anticodon‐binding regions, and a maternal missense variant (P136H) possessing partial enzymatic activity. This case confirms and expands the phenotype related to FARS mutations with regards to clinical presentation and neuroimaging findings.

Inhibition Of Ribosome Biogenesis Through Genetic And Chemical Approaches, Leonid Anikin

Graduate School of Biomedical Sciences Theses and Dissertations

In order to maintain the ability to generate proteins, proliferating cells must continuously generate ribosomes, designating up to 80% of their energy to ribosome biogenesis (RBG). RBG involves transcription of rDNA by RNA polymerases I (Pol I) and III (Pol III), expression of approximately 80 ribosomal proteins, and assembly of these components in a process referred to as ribosome maturation. During maturation, the Pol I transcribed 47S pre-rRNA undergoes a number of processing events, while simultaneously interacting with processing factors and ribosomal proteins that drive pre-ribosome assembly. Inhibition of RBG has become one of the pursued targets for cancer therapy …

The N-Terminal Methyltransferase Homologs Nrmt1 And Nrmt2 Exhibit Novel Regulation Of Activity Through Heterotrimer Formation., Jon David Faughn

Electronic Theses and Dissertations

Protein, DNA, and RNA methyltransferases have an ever-expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered through formation of complexes with close homologs. This work involves the N-terminal methyltransferase homologs NRMT1 and NRMT2. NRMT1 is a ubiquitously expressed distributive trimethylase. NRMT2 is a monomethylase expressed at low levels and in a tissue-specific manner. They are both nuclear methyltransferases with overlapping target consensus sequences …

Transcriptomics Of Learning, Pablo Iturralde

Theses

Learning is a basic and important component of behavior yet we have very little empirical information about the interaction between mechanisms of learning and evolution. In our work, we are testing hypotheses about the neurogenetic mechanisms through which animal learning abilities evolve. We are able to test this directly by using experimentally evolved populations of flies, which differ in learning ability. These populations were previously evolved within the lab by creating worlds with different patterns of change following theoretically predicted effects on which enhanced learning will evolve. How has evolution acted to modulate genes and gene expression in the brain …

Codon Usage Revisited: Lack Of Correlation Between Codon Usage And The Number Of Trna Genes In Enterobacteria, Joaquín Rojas, Gabriel Castillo, Lorenzo Eugenio Leiva, Sara Elgamal, Omar Orellana, Michael Ibba, Assaf Katz

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

It is widely believed that if a high number of genes are found for any tRNA in a rapidly replicating bacteria, then the cytoplasmic levels of that tRNA will be high and an open reading frame containing a higher frequency of the complementary codon will be translated faster. This idea is based on correlations between the number of tRNA genes, tRNA concentration and the frequency of codon usage observed in a limited number of strains as well as from the fact that artificially changing the number of tRNA genes alters translation efficiency and consequently the amount of properly folded protein …

Abcg5 And Abcg8: More Than A Defense Against Xenosterols, Shailendra B. Patel, Gregory A. Graf, Ryan E. Temel

Pharmaceutical Sciences Faculty Publications

The elucidation of the molecular basis of the rare disease, sitosterolemia, has revolutionized our mechanistic understanding of how dietary sterols are excreted and how cholesterol is eliminated from the body. Two proteins, ABCG5 and ABCG8, encoded by the sitosterolemia locus, work as obligate dimers to pump sterols out of hepatocytes and enterocytes. ABCG5/ABCG8 are key in regulating whole-body sterol trafficking, by eliminating sterols via the biliary tree as well as the intestinal tract. Importantly, these transporters keep xenosterols from accumulating in the body. The sitosterolemia locus has been genetically associated with lipid levels and downstream atherosclerotic disease, as well as …

The Regulation Of Dna Methylation In Mammalian Development And Cancer, Nicolas Veland

Dissertations and Theses (Open Access)

DNA methylation is an essential epigenetic modification in mammals, as it plays important regulatory roles in multiple biological processes, such as gene transcription, maintenance of chromosomal structure and genomic stability, genomic imprinting, retrotransposon silencing, and X-chromosome inactivation. Dysregulation of DNA methylation is associated with various human diseases. For example, cancer cells usually show global hypomethylation and regional hypermenthylation, which have been implicated in genomic instability and tumor suppressor silencing, respectively. Although great progress has been made in elucidating the biological functions of DNA methylation over the last several decades, how DNA methylation patterns and levels are regulated and dysregulated is …

Role Of Protein Charge Density On Hepatitis B Virus Capsid Formation, Xinyu Sun, Dong Li, Zhaoshuai Wang, Panchao Yin, Rundong Hu, Rundong Hu, Hui Li, Qiao Liu, Yunyi Gao, Baiping Ren, Jie Zheng, Yinan Wei, Tianbo Liu

Chemistry Faculty Publications

The role of electrostatic interactions in the viral capsid assembly process was studied by comparing the assembly process of a truncated hepatitis B virus capsid protein Cp149 with its mutant protein D2N/D4N, which has the same conformational structure but four fewer charges per dimer. The capsid protein self-assembly was investigated under a wide range of protein surface charge densities by changing the protein concentration, buffer pH, and solution ionic strength. Lowering the protein charge density favored the capsid formation. However, lowering charge beyond a certain point resulted in capsid aggregation and precipitation. Interestingly, both the wild-type and D2N/D4N mutant displayed …

Ef-P Post-Translational Modification Has Variable Impact On Polyproline Translation In Bacillus Subtilis, Anne Witzky, Katherine R. Hummels, Rodney Tollerson Ii, Andrei Rajkovic, Lisa A. Jones, Daniel B. Kearns, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is a ubiquitous translation factor that facilitates translation of polyproline motifs. In order to perform this function, EF-P generally requires posttranslational modification (PTM) on a conserved residue. Although the position of the modification is highly conserved, the structure can vary widely between organisms. In Bacillus subtilis, EF-P is modified at Lys³² with a 5-aminopentanol moiety. Here, we use a forward genetic screen to identify genes involved in 5-aminopentanolylation. Tandem mass spectrometry analysis of the PTM mutant strains indicated that ynbB, gsaB, and ymfI are required for modification and that yaaO, yfkA, and …

Increased Liver Tumor Formation In Neutral Sphingomyelinase-2-Deficient Mice, Liansheng Zhong, Ji Na Kong, Michael B. Dinkins, Silvia Leanhart, Zhihui Zhu, Stefka D. Spassieva, Haiyan Qin, Hsuan-Pei Lin, Ahmed Elsherbini, Rebecca Wang, Xue Jiang, Mariana N. Nikolova‑Karakashian, Guanghu Wang, Erhard Bieberich

Physiology Faculty Publications

Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C₁₆-ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, …

In Vitro Genetic Code Expansion And Selected Applications, Emil S. Iqbal

Theses and Dissertations

The ability of incorporation non-canonical amino acids (ncAAs) using translation offers researchers the ability of extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the three projects where the addition of ncAAs to in vitro translation systems creates useful chemical biology techniques. In the first, a fluorinated histidine derivative is used to create a novel affinity tag that allows for the selective purification of peptides from a complex mixture of proteins. In the second, the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P) …

9-Aminoacridine Inhibits Ribosome Biogenesis And Synergizes With Cytotoxic Drugs To Induce Selective Killing Of P53-Deficient Cells, Leonid Anikin, Dimitri G Pestov

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Common cancer treatments target rapidly dividing cells and do not discriminate between cancer and normal host cells. One approach to mitigating negative side‐effects of cancer treatment is to temporarily arrest cell cycle progression and thus protect normal cells during cytotoxic treatments, a concept called cyclotherapy. We recently proposed that transient inhibition of post‐transcriptional steps of ribosome biogenesis (RBG) can be used to selectively arrest p53‐positive host cells and not p53‐null cancer cells. In this study, we investigated whether cytoprotective RBG inhibition can be achieved through small molecule treatment.

Structural Basis For Earp-Mediated Arginine Glycosylation Of Translation Elongation Factor Ef-P, Ralph Krafczyk, Jakub Macošek, Pravin Kumar Ankush Jagtap, Daniel Gast, Swetlana Wunder, Prithiba Mitra, Amit Kumar Jha, Jürgen Rohr, Anja Hoffmann-Röder, Kirsten Jung, Janosch Hennig, Jürgen Lassak

Pharmaceutical Sciences Faculty Publications

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-β-L-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for …

Translational Fidelity, Mistranslation, And The Cellular Responses To Stress, Kyle Mohler, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Faithful translation of mRNA into the corresponding polypeptide is a complex multistep process, requiring accurate amino acid selection, transfer RNA (tRNA) charging and mRNA decoding on the ribosome. Key players in this process are aminoacyl-tRNA synthetases (aaRSs), which not only catalyse the attachment of cognate amino acids to their respective tRNAs, but also selectively hydrolyse incorrectly activated non-cognate amino acids and/or misaminoacylated tRNAs. This aaRS proofreading provides quality control checkpoints that exclude non-cognate amino acids during translation, and in so doing helps to prevent the formation of an aberrant proteome. However, despite the intrinsic need for high accuracy during translation, …

Carbonyl Reduction By Ymfi Completes The Modification Of Ef-P In Bacillus Subtilis To Prevent Accumulation Of An Inhibitory Modification State, Katherine R. Hummels, Anne Witzky, Andrei Rajkovic, Rodney Tollerson Ii, Lisa A. Jones, Michael Ibba, Daniel B. Kearns

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Translation elongation factor P (EF‐P) in Bacillus subtilis is required for a form of surface migration called swarming motility. Furthermore, B. subtilis EF‐P is post‐translationally modified with a 5‐aminopentanol group but the pathway necessary for the synthesis and ligation of the modification is unknown. Here we determine that the protein YmfI catalyzes the reduction of EF‐P‐5 aminopentanone to EF‐P‐5 aminopentanol. In the absence of YmfI, accumulation of 5‐aminopentanonated EF‐P is inhibitory to swarming motility. Suppressor mutations that enhanced swarming in the absence of YmfI were found at two positions on EF‐P, including one that changed the conserved modification site (Lys …

Fret Biosensors: Engineering Fluorescent Proteins As Biological Tools For Studying Parkinson’S Disease, Nathan J. Leroy, Jacob R. Norley, Saranya Radhakrishnan, Mathew Tantama

The Summer Undergraduate Research Fellowship (SURF) Symposium

Parkinson’s Disease (PD) is a common neurodegenerative disease with over 200,000 new cases each year. In general, the cause of the disease is unknown, but oxidative stress inside of neurons has been associated with the disease’s pathology for some time. Currently, techniques to study the onset of PD inside of neurons are limited. This makes treatments and causes difficult to discover. One solution to this has been fluorescent protein biosensors. In short, these proteins can be engineered to glow when a certain state is achieved inside a cell. The present research discusses the engineering of a genetically-encoded fluorescent protein (FP) …

Elongation Factor P Interactions With The Ribosome Are Independent Of Pausing, Rodney Tollerson Ii, Anne Witzky, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Bacterial elongation factor P (EF-P) plays a pivotal role in the translation of polyproline motifs. To stimulate peptide bond formation, EF-P must enter the ribosome via an empty E-site. Using fluorescence-based single-molecule tracking, Mohapatra et al. (S. Mohapatra, H. Choi, X. Ge, S. Sanyal, and J. C. Weisshaar, mBio 8:e00300-17, 2017, https://doi.org/10.1128/mBio.00300-17 ) monitored the cellular distribution of EF-P and quantified the frequency of association between EF-P and the ribosome under various conditions. Findings from the study showed that EF-P has a localization pattern that is strikingly similar to that of ribosomes. Intriguingly, EF-P was seen to bind ribosomes more …

Gcn5 Impacts Fgf Signaling At Multiple Levels And Activates C-Myc Target Genes During Early Differentiation Of Embryoid Bodies, Li Wang

Dissertations and Theses (Open Access)

Precise control of gene expression during development is orchestrated by transcription factors, signaling pathways and co-regulators, with complex cross-regulatory events often occurring. Growing evidence has identified chromatin modifiers as important regulators for development as well, yet how particular chromatin modifying enzymes affect specific developmental processes remains largely unclear. Embryonic stem cells (ESCs) are self-renewing, pluripotent, and have the abilities to generate almost all cell types in adult tissues. The dual capacity of ESCs to self-renew and differentiate offers unlimited potential for studying gene regulation events at specific developmental stages in vitro that parallel developmental events during embryogenesis in vivo. …

Quaternary Interactions And Supercoiling Modulate The Cooperative Dna Binding Of Agt, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ω_ave. We found …

B7h6: A Cancer Biomarker For The Development Of Novel Immunotherapy Approaches, Mariana Phillips

Seton Hall University Dissertations and Theses (ETDs)

Cancer-based immunotherapy has led the evolution of biologics that can stimulate immune responses towards tumor eradication. The synthesis of small to intermediate size molecules with the targeting and effector functions of mAb may represent a novel class of immunotherapeutics that may overcome the limitations of their biological counterparts.Towards this objective, B7H6 has been identified as a protein ligand localized on the cell surface of transformed tumor cells. B7H6 binds specifically to the activating receptor NKp30, constitutively expressed on all resting and active NK cells. Upon ligand:receptor binding, B7H6 triggers NK cell activation and release of chemokines and pro-inflammatory cytokines such …

Lymphoid Hematopoiesis And The Role Of B-Cells In Transgenic Mouse Model Of Sickle Cell Disease, Christina Cotte

University Scholar Projects

Sickle cell disease (SCD) has been shown to be associated with decreased baseline immunity and thus increased susceptibility to infection. I sought to discern possible causes of this by looking into the correlations between SCD and hematopoiesis, the immune system and the neuroendocrine system, and ultimately by conducting experiments surrounding the impaired immune system of SCD. These experiments focused on the potential causes and effects of the diminution of B-1a cells in the SCD spleen. Adoptive transfers, infections with Streptococcus pneumoniae, and histologic imaging were conducted to establish if the diminution of the B-1a cells in the SCD spleen …