Biochemistry Commons^™

Molecular Biochemical Study Of The Cataract-Associated Mutants In Human Gammad-Crystallin, Cindy Yeung

Legacy Theses & Dissertations (2009 - 2024)

Cataract is a vision impairment disease that leads to light scattering and opacity of the eye lens. It is recognized as the most prevalent cause of blindness. Most cataracts occur due to aging, but those that occur in childhood or in young adults are typically of genetic origin. In the young (developing) eye, post–surgical complications such as glaucoma are common after cataract surgery. Hence there is a great need to develop non-surgical intervention strategies for the treatment of cataract. Here, two cataract-associated mutants of human γD-crystallin (HGD): i) Arg76 to Ser (R76S) and ii) a truncation mutation at Trp156 (W156X …

Structural Biology Of The Receptor For Advanced Glycation End Products (Rage), Jing Xue

Legacy Theses & Dissertations (2009 - 2024)

The receptor for advanced glycated end products (RAGE), due to its location in the major histocompatibility complex class III (MHC III) region, suggests its involvement in immune responses. Because RAGE has been linked to complications of diabetes and chronic inflammation, the severity of Alzheimer's disease and cancer, it's significant to understanding the biological mechanism of signal transduction of RAGE.

New Tools To Study Amyloid Fibrils And Intrinsically Disordered Proteins In Vitro And In Vivo, Jacqueline D. Washington

Legacy Theses & Dissertations (2009 - 2024)

Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman and Nuclear Magnetic Resonance spectroscopy combined with hydrogen-deuterium exchange. Our new approach of combining NMR and Raman spectroscopy with molecular dynamic simulations for characterizing amyloid fibrils provided exclusive knowledge about fibril structure at amino acid residue resolution.

Y-Family Dna Polymerases : Mechanism Of Single-Base Deletion And Effect Of Pcna Interaction, Yifeng Wu

Legacy Theses & Dissertations (2009 - 2024)

Translesional synthesis (TLS) by specialized Y-family DNA polymerases is a DNA damage tolerance pathway to bypass DNA lesions that have not been repaired by other DNA repair mechanisms. Despite their valuable lesion bypass ability, the Y-family DNA polymerases display a much lower fidelity upon replicating undamaged DNA. Their activity therefore needs to be well regulated in the cell. In our first project, we examined how an archaeal Y-family DNA polymerase, Dpo4, makes single-base deletions during replication. Dpo4 belongs to the DinB subfamily which is known to make single-base deletions. Although previous studies suggested that it uses a dNTP-stabilized misalignment mechanism, …

The Sxrna Platform : Biophysical Characterization Of Trans-Acting Rna Switches For Gene Expression Control, Christine Bazinet

Legacy Theses & Dissertations (2009 - 2024)

In the past decade, microRNAs (miRNAs) have become a hot topic in biochemical research. These very short ~ 22 nucleotide strands of RNA have been recognized as a functional RNA group and found to play a key role in mediating gene regulation along with RNA-binding proteins (RBPs). For example, by binding to the histone stem-loop (HSL) region of the target mRNA, normally in the 3'- untranslated region (UTR), gene expression can either be activated or deactivated for up or down regulation of the gene product. The majority of miRNAs have been found to down regulate gene expression upon binding to …

2'-O-Methyl Substitutions In The Yeast Telomerase Pseudoknot And Their Effects On Telomerase Activity, Katelyn Mae Jasper

Legacy Theses & Dissertations (2009 - 2024)

Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomeric repeats at the ends of linear chromosomes to form the DNA–protein complexes known as telomeres. Telomeres protect the ends of chromosomes from degradation during replication due to the end-replication problem. When replication occurs, gaps are created at the beginning of the lagging and leading strands that result in the loss of a small amount of DNA at every replication cycle. By adding thousands of copies of telomeric repeats, the repeats are lost in the replication process and not precious genetic information. The telomerase RNA varies drastically among different species both in …

(S)-Trifluoroselenomethionine : A New Non-Natural Amino Acid With Enhanced Methioninase-Induced Cytotoxicity Toward Human Colon Cancer Cells, Stephene Nathele Lodge

Legacy Theses & Dissertations (2009 - 2024)

Abstract

Voltage-Controlled Enzyme-Catalyzed Glucose–Gluconolactone Conversion Using A Field-Effect Enzymatic Detector, Siu Tung Yau, Yan Xu, Yang Song, Ye Feng, Jiapeng Wang

Chemistry Faculty Publications

The field-effect enzymatic detection (FEED) technique was used to control the kinetics of the enzymatic conversion of glucose to gluconolactone. The glucose–gluconolactone conversion occurring at an enzyme-immobilized electrode, a well-studied process, was confirmed using mass spectrometry. Electrochemical studies showed that the glucose oxidation current depends on the gating voltage VG and the ion concentration of the sample solution. Additionally, the depletion of glucose in the sample also showed a dependence on VG. FEED was used to detect H2O2 on the zepto-molar level in order to show the ultrasensitive detection capability of the technique. These results, while providing evidence for the …

Molecular Considerations In The Design Of Novel Alpha/Beta Hydrolase Inhibitors, Shana Victoria Stoddard

Electronic Theses and Dissertations

Alpha/beta hydrolases (ABHs) are a superfamily of hydrolytic enzymes that process a wide variety of substrates. A subfamily of ABHs called carboxylesterases (CEs) are important enzymes that catalyze biological detoxification, hydrolysis of certain pesticides, and metabolism of many esterified drugs. The chemotherapy drug irinotecan used for treatment of colorectal cancer is metabolized to SN-38, the active drug metabolite, by two CE isozymes CES1 (localized in the liver) and CES2 (localized in the small intestines). CES2's ability to activate irinotecan at a faster rate than CES1 creates a localization of activated SN-38 in the gut epithelium, resulting in the dose limiting …

Spliceosomal Prp24 Unwinds A Minimal U2/U6 Complex From Yeast, Chandani Manoja Warnasooriya

Wayne State University Theses

Splicing plays a major role in eukaryotic gene expression by processing pre-mRNA to form mature mRNA. Pre-mRNAs undergo splicing to remove introns, non–protein coding regions, and religate exons, protein coding regions. This process is catalyzed by the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5 and U6) and numerous protein factors. Proper assembly of spliceosomal components is critical for function, and thus, defects in assembly can be lethal. Several spliceosomal proteins facilitate structural rearrangements important for spliceosomal assembly and function. Prp24 is an essential factor in U6 snRNP assembly, and it has been proposed …

Investigation Of Bacterial Rna-Directed Dna Methylation Via Dcm And Hfq, Dandan Li

Wayne State University Theses

Bacterial small RNAs and the RNA chaperone Hfq play crucial roles in post-transcriptional gene regulation, often as parts of stress-response pathways, but little is known about their roles in regulation of gene transcription. A recent report showed that changes in methylation patterns caused by DNA cytosine methyltransferase (Dcm) were linked to gene regulation occurring during the transition to stationary phase. Here, we show that Dcm involves in the stress responses under nutrient starvation and cold stress. Dcm and Hfq together mediate gene expression under cold stress. Hfq promotes Dcm-catalyzed cytosine methylation at specific sites near the rpoS promoter, which is …

Lc3i And Lc3ii As Autophagy Markers For The Development And Improvement Of Products And Techniques Used In Research, Caitlin Jane Williams

Wayne State University Theses

Abstract

LC3I and LC3II as Autphagy Markers for the Development and Improvement of Products and Techniques used in Research

By

Caitlin J. Williams

May 2013

Advisor: Dr. Robert Akins

Major: Biochemistry & Molecular Biology

Degree: Master of Science

Autophagy is an intracellular process that functions to maintain homeostasis in the cell by degrading misfolded proteins, old or nonfunctioning organelles, and outside invaders such as bacteria or viruses. This process can be split into three different types, microautophagy, chaperone mediated autophagy and macroautophagy. Macroautophagy is the most commonly studied form and is believed to be regulated by Atg proteins, as well …

Cysteine Desulfurase And Isd11: A Drosophila Model, Rahul Ravindran Nair

Wayne State University Theses

ABSTRACT

Cysteine desulfurase and Isd11: A Drosophila model

Iron-sulfur clusters are cofactors with evolutionary origins that date back to the pre-biotic world. Ever since life originated, these cofactors have intermingled with proteins to play vital roles in sustaining life. My research focuses on one such protein, the cysteine desulfurase (Nfs) that has the PLP cofactor incorporated in its active site and avails of the catalytic property of PLP to provide sulphur for Iron-sulfur cluster biogenesis and assembly in a cell. Interestingly, in a eukaryotic cell, despite the versatility of PLP, cysteine desulfurase's role as a "sulphur-extractor" is incomplete without another …

Optimization Of Labeling Techniques; Determination Of Best Parameter For Olfactory Mucosal Progenitor Cell Delivery And Study Of Effects Of Methylene Blue And Polyethylene Glycol In An Animal Model Of Spinal Cord Injury, Kiran Kumar Rokkappanavar

Wayne State University Theses

In the United States, the incidence of TSCI is about 40 per million persons per year, with approximately 250,000 living survivors of TSCI in the United States in July 2005. A number of human clinical trials (85) are ongoing using stem cells to evaluate the methods to reduce the injury following TBI. Among the stem cells, olfactory mucosal progenitor cells have several advantages with respect to ease of obtaining, fate of cells, etc. Our study was intended to optimize the labeling technique and to determine the best parameter to deliver the stem cells to the site of injury. We found …

Characterizing Cyclin J By Identifying Conserved Protein-Protein Interactions, Phillip Jacob Selman

Wayne State University Theses

Cyclins are proteins that bind to Cyclin-dependent kinases, or Cdks, through a conserved domain called the Cyclin Box. Many Cyclins regulate the cell cycle. A few Cyclins impact cellular processes outside of the cell cycle. Also, a few Cyclins have poorly understood functions.

Cyclin J is a member of the Cyclin superfamily of proteins. Cyclin J is conserved among all metazoans, but is presently not well understood. All the research done on Cyclin J has been done in Drosophila.

Its mRNA is present in the early embryo, then disappears, only to reappear in adult females. When probing protein extracts with …

Spectroscopic Analysis Of Polymerization And Exonuclease Proofreading By A High-Fidelity Dna Polymerase During Translesion Dna Synthesis, Babho Devadoss, Irene Lee, Anthony J. Berdis

Chemistry Faculty Publications

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient …

Towards Elucidation Of A Viral Dna Packaging Motor, Chad T. Schwartz

Theses and Dissertations--Pharmacy

Previously, gp16, the ATPase protein of phi29 DNA packaging motor, was an enigma due to its tendency to form multiple oligomeric states. Recently we employed new methodologies to decipher both its stoichiometry and also the mechanism in which the protein functions to hydrolyze ATP and provide the driving force for DNA packaging. The oligomeric states were determined by biochemical and biophysical approaches. Contrary to many reported intriguing models of viral DNA packaging, it was found that phi29 DNA packaging motor permits the translocation of DNA unidirectionally and driven cooperatively by three rings of defined shape. The mechanism for the generation …

Amalgamation Of Nucleosides And Amino Acids In Antibiotic Biosynthesis, Sandra H. Barnard

Theses and Dissertations--Pharmacy

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading …

Regulation Of 7-Dehydrocholesterol Reductase By Vitamin D3, Ling Zou

Theses and Dissertations--Pharmacy

7-Dehydrocholesterol (7-DHC) is the substrate of 7-dehydrocholesterol reductase (DHCR7) in the cholesterol synthesis pathway. Keratinocytes in human skin possess the enzymes necessary for cholesterol synthesis but are also responsible for vitamin D₃ synthesis from 7-DHC by exposure to UVB irradiation. It has been well established that DHCR7 is regulated by the SREBP pathway in the regulation of cholesterol synthesis, but little is known about the regulation of DHCR7 by the vitamin D pathway. In this study, the regulation of DHCR7 activity by vitamin D was explored. Treatment of adult human epidermal keratinocyte (HEKa) cells with vitamin D₃ resulted …

Epigenetic Regulation Of Oxysterol Formation, Steve Meaney

Articles

Oxysterols are oxygenated derivatives of cholesterol that may be formed by either enzymatic or non-enzymatic mechanisms. Expression of the genes responsible oxysterol synthesis (GROS) is known to be restricted across different tissues and cell types. Regulation of the transcription of GROS and the activity of their enzyme transcripts has been the subject of intense activity for many years. Recent studies have sought to decipher the mechanism(s) that underpin the restricted expression of the GROS. Available data indicates that epigenetic mechanisms have an important role to play in the control of the expression of GROS. In the current review we summarize …

Osteogenic And Antimicrobial Nanoparticulate Calcium Phosphate And Poly-(D, L-Lactide-Co-Glycolide) Powders For The Treatment Of Osteomyelitis, Vuk Uskoković, Charles Hoover, Marija Vukomanović, Dragan P. Uskoković, Tejal A. Dasai, Charles Hoover, Marija Vukomanović, Dragan P. Uskoković, Tejal A. Dasai

Pharmacy Faculty Articles and Research

Development of a material for simultaneous sustained and localized delivery of antibiotics and induction of spontaneous regeneration of hard tissues affected by osteomyelitis stands for an important clinical need. In this work, a comparative analysis of the bacterial and osteoblastic cell response to two different nanoparticulate carriers of clindamycin, an antibiotic commonly prescribed in the treatment of bone infection, one composed of calcium phosphate and the other comprising poly-(D,L-lactide-co-glycolide)-coated calcium phosphate, was carried out. Three different non-cytotoxic phases of calcium phosphate, exhibiting dissolution and drug release profiles in the range of one week to two months to one year, respectively, …

Synthesis And Biochemical Activities Of Antiproliferative Amino Acid And Phosphate Derivatives Of Microtubule-Disrupting Beta-Lactam Combretastatins, Niamh M. O'Boyle, Lisa M. Greene, Niall O. Keely, Shu Wang, Tadhg S. Cotter, Daniela M. Zisterer, Mary J. Meegan

Articles

The synthesis and biochemical activities of novel water-soluble β-lactam analogues of combretastatin A-4 are described. The first series of compounds investigated, β-lactam phosphate esters 7a, 8a and 9a, exhibited potent antiproliferative activity and caused microtubule disruption in human breast carcinoma-derived MCF-7 cells. They did not inhibit tubulin polymerisation in vitro, indicating that biotransformation was necessary for their antiproliferative and tubulin binding effects in MCF-7 cells. The second series of compounds, β-lactam amino acid amides (including 10k and 11l) displayed potent antiproliferative activity in MCF-7 cells, disrupted microtubules in MCF-7 cells and also inhibited the polymerisation of …

Novel Cis-Restricted Β-Lactam Combretastatin A-4 Analogues Display Anti-Vascular And Anti-Metastatic Properties In Vitro, Seema M. Nathwani, Lisa M. Greene, Linda Hughes, Miriam Carr, Niamh O'Boyle, Susan Mcdonnell, Mary J. Meegan, Daniela M. Zisterer

Articles

No abstract provided.

Nanoparticles Of Cobalt-Substituted Hydroxyapatite In Regeneration Of Mandibular Osteoporotic Bones, Nenad Ignjatović, Zorica Ajduković, Vojin Savić, Stevo Najman, Dragan Mihailović, Perica Vasilijević, Zoran Stojanović, Vuk Uskoković, Dragab Uskoković

Pharmacy Faculty Articles and Research

Indications exist that paramagnetic calcium phosphates may be able to promote regeneration of bone faster than their regular, diamagnetic counterparts. In this study, analyzed was the influence of paramagnetic cobalt-substituted hydroxyapatite nanoparticles on osteoporotic alveolar bone regeneration in rats. Simultaneously, biocompatibility of the material was tested in vitro, on osteoblastic MC3T3-E1 and epithelial Caco-2 cells in culture. The material was shown to be biocompatible and nontoxic when added to epithelial monolayers in vitro, while it caused a substantial decrease in the cell viability as well as deformation of the cytoskeleton and cell morphology when incubated with the osteoblastic …

Periplasmic Binding Proteins In Thermophiles: Characterization And Potential Application Of An Arginine-Binding Protein From Thermotoga Maritima: A Brief Thermo-Story, Alessio Ausili, Maria Staiano, Jonathan D. Dattelbaum, Antonio Varriale, Alessandro Capo, Sabato D'Auria

Chemistry Faculty Publications

Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima …

Structurally Diverse Hamigerans From The New Zealand Marine Sponge Hamigera Tarangaensis: Nmr-Directed Isolation, Structure Elucidation And Antifungal Activity, A. Jonathan Singh, Jonathan D. Dattelbaum, Jessica J. Field, Zlatka Smart, Ethan F. Woolly, Jacqueline M. Barber, Rosemary Heathcott, John H. Miller, Peter T. Northcote

Chemistry Faculty Publications

The NMR-directed investigation of the New Zealand marine sponge Hamigera tarangaensis has afforded ten new compounds of the hamigeran family, and a new 13-epi-verrucosane congener. Notably, hamigeran F (6) possesses an unusual carbon–carbon bond between C-12 and C-13, creating an unprecedented skeleton within this class. In particular, the structural features of 6, hamigeran H (10) and hamigeran J (12) imply a diterpenoid origin, which has allowed the putative biogenesis of three hamigeran carbon skeletons to be proposed based on geranyl geranyl pyrophosphate. All new hamigerans exhibited micromolar activity towards the HL-60 …

A Comprehensive, Multi-Scale Dynamical Model Of Erbb Receptor Signal Transduction In Human Mammary Epithelial Cells, Tomáš Helikar, Naomi Kochi, Bryan Kowal, Manjari Dimri, Mayumi Naramura, Srikumar M. Raja, Vimla Band, Hamid Band, Jim A. Rogers

Department of Biochemistry: Faculty Publications

The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1) have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins …

Biochemical And Cellular Mechanisms Of Retina And Retinal Pigment Epithelium Apoptosis, Srinivasa Rao Sripathi

Dissertations, Master's Theses and Master's Reports - Open

Oxidative stress, intense light exposure and oxygen imbalances such as hypoxic or hyperoxic conditions perturb mitochondria, nuclear function and further lead to cellular damage of retina and retinal pigment epithelial (RPE) cells. Our major aim is to understand the various biochemical and proteomic events that occur during the progression of retina and RPE cell death. The comprehensive objectives of this dissertation are to understand the functional aspects of protein expression, posttranslational modifications, protein or lipid binding changes, phenotypic, morphological alterations and their regulation during the retina and RPE apoptosis under oxidative stress. The entire study is divided into four chapters …

Oligodeoxynucleotide Synthesis Using Protecting Groups And A Linker Cleavable Under Non-Nucleophilic Conditions, Xi Lin

Dissertations, Master's Theses and Master's Reports - Open

Oligodeoxynucleotides (ODNs) containing latent electrophilic groups can be highly useful in antisense drug development and many other applications such as chemical biology and medicine, where covalent cross-linking of ODNs with mRNA, protein and ODN is required. However, such ODN analogues cannot be synthesized using traditional technologies due to the strongly nucleophilic conditions used in traditional deprotection/cleavage process.

To solve this long lasting and highly challenging problem in nucleic acid chemistry, I used the 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) function to protect the exo-amino groups on the nucleobases dA, dC and dG, and to design the linker between the nascent ODN and solid support. …

Determination Of Dimer Dissociation Constant Of Neural-Cadherin By Fluorescence Resonance Energy Transfer, Xiaoyun Z. Howard

Electronic Theses and Dissertations

No abstract provided.