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Effect Of Salt Concentration On Electrochemical Detection Of Dna, Ziming Dong, Eddie Madrigal, Ryan West Dr. 2019 University of San Francisco

Effect Of Salt Concentration On Electrochemical Detection Of Dna, Ziming Dong, Eddie Madrigal, Ryan West Dr.

Creative Activity and Research Day - CARD

Electrochemical approaches for biological sensing offer the potential advantages of facile sample preparation, fast response times, ease of parallel and multiplexed measurements, and the possibility of miniaturization (of sample sizes, electrodes, and associated electronics). All of these factors contribute towards the feasibility of electrochemical methods in biological sensing and analysis. This potential has already been achieved with the commercialization of blood glucose meters, which often rely on an electrochemical transduction mechanism. We have previously demonstrated the ability to electrochemically detect and differentiate complementary and mismatched DNA using our method of melting DNA duplexes at electrified gold surfaces, i.e. e-melting. Recently, …


Saturated Fatty Acid Activates T Cell Inflammation Through A Nicotinamide Nucleotide Transhydrogenase (Nnt)-Dependent Mechanism, Grace McCambridge, Madhur Agrawal, Alanna Keady, Philip A. Kern, Hatice Hasturk, Barbara S. Nikolajczyk, Leena P. Bharath 2019 Merrimack College

Saturated Fatty Acid Activates T Cell Inflammation Through A Nicotinamide Nucleotide Transhydrogenase (Nnt)-Dependent Mechanism, Grace Mccambridge, Madhur Agrawal, Alanna Keady, Philip A. Kern, Hatice Hasturk, Barbara S. Nikolajczyk, Leena P. Bharath

Honors Senior Capstone Projects

Circulating fatty acids (FAs) increase with obesity and can drive mitochondrial damage and inflammation. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that positively regulates nicotinamide adenine dinucleotide phosphate (NADPH), a key mediator of energy transduction and redox homeostasis. The role that NNT-regulated bioenergetics play in the inflammatory response of immune cells in obesity is untested. Our objective was to determine how free fatty acids (FFAs) regulate inflammation through impacts on mitochondria and redox homeostasis of peripheral blood mononuclear cells (PBMCs). PBMCs from lean subjects were activated with a T cell-specific stimulus in the presence or absence of generally pro-inflammatory …


The Atpase Mechanism Of Uvra2 Reveals The Distinct Roles Of Proximal And Distal Atpase Sites In Nucleotide Excision Repair, Brandon C. Case, Silas Hartley, Memie Osuga, David Jeruzalmi, Manju M. Hingorani 2019 Wesleyan University

The Atpase Mechanism Of Uvra2 Reveals The Distinct Roles Of Proximal And Distal Atpase Sites In Nucleotide Excision Repair, Brandon C. Case, Silas Hartley, Memie Osuga, David Jeruzalmi, Manju M. Hingorani

Publications and Research

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the highaffinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition …


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 2019 Beckman Research Institute

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 2019 University of Kentucky

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 …


Characterization Of The Microbial Phosphonate-Activating Pntc Enzymes, Kyle Rice 2019 Wilfrid Laurier University

Characterization Of The Microbial Phosphonate-Activating Pntc Enzymes, Kyle Rice

Theses and Dissertations (Comprehensive)

New strategies are urgently needed to combat infectious diseases in an era of rising antibiotic resistance. Furthermore, an emerging appreciation for the human microbiome’s role in maintaining health motivates discovery of species-specific antibiotics that minimally disrupt our native bacterial communities. Small molecule modifications to bacterial cell surfaces represent a potentially rich source of new targets for next generation antibiotics, as these molecules mediate virulence and evasion of the host immune response. Phosphocholine (PCho) is a rare cell surface modification that contributes to virulence, and modifications with phosphonates like 2-aminoethylphosphonate (AEP) are even more unusual and therefore provide opportunities for species- …


Isolation Of Rna From A Mixture And Its Detection By Utilizing A Microgel-Based Optical Device, Molla R. Islam, Shakiba Azimi, Faranak Teimoory, Glen Loppnow, Michael J. Serpe 2018 Chapman University

Isolation Of Rna From A Mixture And Its Detection By Utilizing A Microgel-Based Optical Device, Molla R. Islam, Shakiba Azimi, Faranak Teimoory, Glen Loppnow, Michael J. Serpe

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

In this investigation, we show that RNA can be separated from a solution containing DNA and RNA and the isolated RNA can be detected using poly (N-isopropylacrylamide-co-N-(3-aminopropyl) methacrylamide hydrochloride) microgel-based optical devices (etalons). The isolation of RNA was accomplished by using hairpin-functionalized magnetic beads (MMPDNA) and differential melting, based on the fact that the DNA–RNA hybrid duplex is stronger (i.e., high melting temperature) than the DNA–DNA duplex (i.e., low melting temperature). By performing concurrent etalon sensing and fluorescent studies, we found that the MMPDNA combined with differential melting was capable of selectively separating RNA from DNA. This selective separation and …


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 2018 Cedars-Sinai Medical Center

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.


Fluorescently Labeled Sirnas And Their Theranostic Applications In Cancer Gene Therapy, Stephen David Kozuch 2018 Seton Hall University

Fluorescently Labeled Sirnas And Their Theranostic Applications In Cancer Gene Therapy, Stephen David Kozuch

Seton Hall University Dissertations and Theses (ETDs)

Gene therapy has emerged as a promising precision nano-medicine strategy in the treatment of numerous diseases including cancer. At the forefront of its utility are the applications of short-interfering RNA (siRNA), that silence oncogenic mRNA expression leading to cancer cell death through the RNA interference (RNAi) pathway. Despite the therapeutic potential, siRNAs are limited by poor pharmacological properties, which has hindered their translation into the clinic. Recent studies, however, have highlighted the applications of modified siRNAs, including the use of fluorescent probes and siRNA nanostructures in cancer detection and treatment. The siRNAs reported in this thesis are designed to target …


Azido- And Triazolyl-Modified Nucleoside/Tide Analogues: Chemistry, Fluorescent Properties, And Anticancer Activities, Zhiwei Wen 2018 Florida International University

Azido- And Triazolyl-Modified Nucleoside/Tide Analogues: Chemistry, Fluorescent Properties, And Anticancer Activities, Zhiwei Wen

FIU Electronic Theses and Dissertations

Two classes of C5 azido-modified pyrimidine nucleosides were synthesized and explored as radiosensitizers. The 5-azidomethyl-2'-deoxyuridine (AmdU) was prepared from thymidine and converted to its cytosine counterpart (AmdC). The 5-(1-azidovinyl) modified 2'-deoxyuridine (AvdU) and 2'-deoxycytidine (AvdC) were prepared employing regioselective Ag-catalyzed hydroazidation of 5-ethynyl pyrimidine substrates with TMSN3. AmdU and AmdC were converted to 5'-triphosphates AmdUTP and AmdCTP, and incorporated into DNA-fragments via polymerase-catalyzed reaction during DNA replication and base excision repair. Radiation-mediated prehydrated electrons formed in homogeneous aqueous glassy (7.5 M LiCl) systems in the absence of oxygen at 77 K led to site-specific formation of π-type aminyl …


Optimization Of The Split-Spinach Aptamer For Monitoring Contiguous Rna Nanoparticle Assembly, Jack M. O'Hara 2018 Seattle Pacific University

Optimization Of The Split-Spinach Aptamer For Monitoring Contiguous Rna Nanoparticle Assembly, Jack M. O'Hara

Honors Projects

The emerging field of RNA nanotechnology takes advantage of the RNA’s ability to self-assemble into exquisite structures. As nanoparticle design continues to advance and move into increasingly complex biological systems, tools to monitor their assembly and location will be of great importance. Here, a split-aptamer system is used to monitor assembly of a six-membered nanoring based on fluorescence feedback of a fluorophore. First, the split-aptamer is designed into two of the six pieces of the ring. Through mutation and deletion, we optimize the fluorescence feedback established when a six membered nanoparticle assembles, compared to partial assembly. We demonstrate that with …


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 2018 Universidad de Chile

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 …


Impact Of Single Nucleotide Polymorphisms On Hpa Axis Functionality In Depression, Claire Kelly 2018 Union College

Impact Of Single Nucleotide Polymorphisms On Hpa Axis Functionality In Depression, Claire Kelly

Honors Theses

The hypothalamic-pituitary-adrenal (HPA) axis plays a primary role in stress response through the regulated secretion of the glucocorticoid hormone cortisol. Diseases of cortisol dysregulation such as Cushing’s syndrome (hypercortisolemia) and Addison’s Disease (hypocortisolemia) are both associated with depression. Based on this we, and others, have hypothesized that mutations in the genes for the glucocorticoid receptor (GR), the closely related mineralocorticoid receptor (MR), and regulatory proteins associated with cortisol or GR function may contribute to depression in the absence of hyper- or hypo-cortisolemia. Our study investigated the genotypic frequency in the clinical population of several single nucleotide polymorphisms (SNPs) that affect …


Development Of Lc-Ms For The Identification And Characterization Of Non-Adjacent Dna Photoproduct Formation In G-Quadruplex Forming Sequences, Claudia Posadas 2018 Washington University in St. Louis

Development Of Lc-Ms For The Identification And Characterization Of Non-Adjacent Dna Photoproduct Formation In G-Quadruplex Forming Sequences, Claudia Posadas

Arts & Sciences Electronic Theses and Dissertations

Ultraviolet light is well known to induce cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts in duplex DNA, which interfere with DNA replication and transcription. Recently, a new class of DNA photoproducts known as anti cyclobutanepyrimidine dimers have been discovered, which form in G-quadruplex forming sequences in solution. G-quadruplex structures have been proposed to form in human DNA telomeres and certain promoters in vivo but evidence for their existence has been lacking. Since anti-cyclobutante pyrimidine dimers have been shown to form in G-quadruplex forming sequences, their formation in irradiated human cells could be used to confirm the existence …


The Regulation Of Dna Methylation In Mammalian Development And Cancer, Nicolas Veland 2018 The University of Texas M D Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences

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

The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences 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 …


Catalyzed And Uncatalyzed Modifications Of Nucleosides, Synthesis Of Hippadine, And Deuterated 1,2,3-Triazoles, Hari K. Akula 2018 The Graduate Center, City University of New York

Catalyzed And Uncatalyzed Modifications Of Nucleosides, Synthesis Of Hippadine, And Deuterated 1,2,3-Triazoles, Hari K. Akula

Dissertations, Theses, and Capstone Projects

The C4 amide carbonyl of O-t-butyldimethylsilyl-protected thymidine, 2’-deoxyuridine, and 3’-azidothymidine (AZT) was activated by reaction with (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate (BOP) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in THF as solvent. This led to the formation of corresponding O4-(benzotriazol-1-yl) derivatives, which are reactive intermediates. Substitution at the C4 position was then carried out by reactions with alkyl and aryl amines, and thiols. Typically, reactions were conducted as a two-step, one-pot transformation, and also as a one-step conversion. After examining the reactions, the formation of 1-(4-pyrimidinyl)-1H-benzotriazole-3-oxide derivatives from the pyrimidine nucleosides was identified. However, these too underwent conversion to …


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 2018 The Ohio State University

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 …


Towards The Development Of A Novel Dna Binding Fluorescent Cell Stain; An Analysis Of Common Dna Dyes And Their Applications, Lauren Kapteyn 2018 Liberty University

Towards The Development Of A Novel Dna Binding Fluorescent Cell Stain; An Analysis Of Common Dna Dyes And Their Applications, Lauren Kapteyn

Senior Honors Theses

Fluorescent deoxyribonucleic acid (DNA) stains that permeate cells are used to observe cellular processes in vivo, making them valuable tools. For the proposal of a new stain, ethidium bromide, cyanine dyes, Hoechst stains, macarpine, DAPI and DRAQ5 are first examined. Features contributing to DNA binding mechanism, toxicity, and cell membrane transport are analyzed for these compounds. The mechanism of DNA binding contributes to the toxicity of the compound. For dyes with well-understood transport mechanisms, multi-drug protein transporters are vital players, though this remains an area for ongoing research as many mechanisms are not well studied. A novel anthraquinone-based compound …


A Chimeric Nucleobase - Phenylazo Derivative As An Intrinsic Nucleobase Quencher, Gyeongsu Park, Timothy Martin-Chan, Amer El Samm, Robert H.E. Hudson 2018 Western University

A Chimeric Nucleobase - Phenylazo Derivative As An Intrinsic Nucleobase Quencher, Gyeongsu Park, Timothy Martin-Chan, Amer El Samm, Robert H.E. Hudson

Western Research Forum

Molecular beacons are important bioanalytical probes which are most often

constructed from a single-stranded oligonucleotide which has been labeled at

opposite termini with a fluorophore and a quencher. When the fluorophore and

quencher are in close proximity, no fluorescence is observed due to FRET

(Fluorescence Resonance Energy Transfer). DABCYL (4-dimethylaminoazobenzene-

4'-carboxylic acid) has been used as a quencher in the molecular beacon to absorbs

excitation energy from a fluorophore and to dissipate the energy as heat. However,

DABCYL is unable to form a base-pair and is conventionally placed as an overhanging

residue. This produces a derivative wherein the chromophore has …


Microrna 1207-3p In Prostate Cancer, Dibash Das 2018 The Graduate Center, City University of New York

Microrna 1207-3p In Prostate Cancer, Dibash Das

Dissertations, Theses, and Capstone Projects

Prostate cancer (PCa) is the most commonly diagnosed male cancer and the second leading cause of cancer-related death for men in the United States. Understanding the molecular mechanisms involved in progression from the asymptomatic androgen-dependent PCa to the lethal castration resistant prostate cancer (CRPC) is a major challenge. MicroRNAs (miRNAs), are known to be dysregulated in PCa. MicroRNA-1207-3p (miR-1207-3p) is encoded by the non-protein coding gene locus PVT1 on the 8q24 human chromosomal region, an established PCa susceptibility locus. However, the role of miR-1207-3p in PCa is unclear. We have discovered that miR-1207-3p is significantly underexpressed in PCa cell lines …


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