Biochemistry Commons^™

Vitamin B12-Based Bioconjugate Probes For In Vitro And In Vivo Imaging, Anna Kahkoska

Renée Crown University Honors Thesis Projects - All

Cancer is the second leading cause of death in the United States. This year, an estimated 577,190 Americans will die as a result of this family of diseases. Finding cancer at its most treatable stage gives patients the greatest chance of recovery; novel imaging agents that target primary and metastasized tumors offer hope for improved prognoses in the future. Based on the hypothesis that vitamin B12 (B12) and its association with specific transport proteins could offer selective access to cancer cell lines, a series of B12-based imaging agents were synthesized, characterized, and assayed for both in vitro and in vivo …

Investigation Of Ghrelin Acylation By Ghrelin O-Acyltransferase, Edward P. Prybolsky

Renée Crown University Honors Thesis Projects - All

Ghrelin is a peptide hormone involved in hunger signaling and other physiological processes. To become active, ghrelin first must be acylated by ghrelin O-acyltransferase (GOAT). This enzyme carries out a post-translational modification that attaches an octanoyl group to the side chain of the serine 3 (S3) residue of ghrelin. With ghrelin linked to hunger signaling, memory, and other physiological processes, GOAT has been identified as a potential drug target. However, inhibitor design is difficult because not much is known regarding the active site structure and catalytic mechanism of GOAT. This study reports the design of a novel fluorescence-based assay that …

Development Of Protein Farnesyltransferase Variants With Altered Substrate Selectivity, Susan Zhang

Renée Crown University Honors Thesis Projects - All

Post-translational modifications play an essential role in regulating protein structure and function. Enzymes catalyzing these modifications must often recognize and modify multiple substrate proteins from among a plethora of non-substrates with similar structures and amino acid sequences. For example, protein farnesyltransferase (FTase) catalyzes the addition of an isoprenoid group to a cysteine near the C-terminus of a substrate protein and is proposed to modify a pool of substrates numbering more than one hundred. We seek to understand the interactions in the FTase active site that engender substrate selectivity. By mutating two residues within FTase, we have developed FTase variants with …

Design Of Metalloproteins For Catalysis And Bioimaging, Krystyna Zhezherya

Renée Crown University Honors Thesis Projects - All

Abstract

Metalloenzymes constitute a large subclass of proteins with diverse functions including metal-sensing, signal transduction pathways, and metal transport. In this report, I aim to study the intricate relationship between structure and function as it relates to metalloproteins in the fields of catalysis and bioimaging. For catalysis, I used existing platforms in addition to de novo design to study small scaffolds with varying secondary structure, and their function in catalyzing the hydrolysis of a p-nitrophenol ester. For bioimaging, I used an existing enzyme known as human carbonic anhydrase I, hCA, to develop a bioimaging agent that can be fused to …

Interaction Of Bacillus Anthracis Exosporium Protein Bcla With Complement Factor H And Spore Persistence In The Lung, Sarah A. Jenkins

Dissertations and Theses (Open Access)

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse …

A Cell Simulator Platform: The Cell Collective, Tomáš Helikar, B. Kowal, J. A. Rogers

Department of Biochemistry: Faculty Publications

Diseases are often a result of multiple malfunctions in complex, nonlinear biological/biochemical networks. As such, these processes are far more complicated to understand because they tend to give rise to functions that are emergent in nature, i.e., higher-level (mal)functions that are more than the sum of their parts. Systems biology provides a new approach to understanding biological systems and diseases from a holistic perspective.

What Can Biochemistry Students Learn About Protein Translation? Using Variation Theory To Explore The Space Of Learning Created By Some Common External Representations, Thomas J. Bussey

UNLV Theses, Dissertations, Professional Papers, and Capstones

Biochemistry education relies heavily on students' ability to visualize abstract cellular and molecular processes, mechanisms, and components. As such, biochemistry educators often turn to external representations to provide tangible, working models from which students' internal representations (mental models) can be constructed, evaluated, and revised. However, prior research has shown that, while potentially beneficial, external representations can also lead to alternative student conceptions.

Considering the breadth of biochemical phenomena, protein translation has been identified as an essential biochemical process and can subsequently be considered a fundamental concept for biochemistry students to learn. External representations of translation range from static diagrams to …

The Phosphoramidase Competency Of Prototypical Phosphatase Catalytic Motifs, Mark P. Haney

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Phosphorylation of proteins is ubiquitous. Phosphorylation can activate proteins, deactivate proteins, assist in signaling, or serve other roles depending upon the biochemical pathway. Attaching phosphate to proteins is accomplished by enzymes called kinases; removing phosphate from proteins is accomplished by enzymes called phosphatases. Cells must regulate their biochemical pathways, and the antipodal roles of kinases and phosphatases represent the yin-yang of phosphorylation.

Phosphorylation of proteins is known to occur on serine, threonine, and tyrosine. This creates a phosphoester bond. Phosphoester bonds have a phosphorus-oxygen (P-O) bond. The ability of phosphatases to cleave such phosphoester bonds is well studied. Phosphorylation of …

Spectroscopic Investigation Of The Interaction Of Fadh In Dna Photolyase With Uv-Damaged Dna, Kyle Luke Williams

Theses, Dissertations and Culminating Projects

Skin cancer is the most prevalent malignancy found in humans, with the diagnosis rate continuing to steadily increase. The primary cause of this disease is overexposure to harmful ultraviolet (UV) radiation from sunlight, which can induce damage to the nitrogenous bases in DNA via dimerization. The most prevalent UV-induced photoproducts in DNA are cyclobutane pyrimidine dimers (CPDs), most commonly between adjacent thymidines.

Organisms have implemented methods by which to repair these CPDs, the primary of which are nucleotide excision repair (NER) and photoreactivation by photolyases. Photolyases are blue-light activated flavoproteins that are more efficient at recognizing and repairing CPDs than …

Structure-Function Analysis Of Human Integrator Subunit-4, Anupama Sataluri

Dissertations and Theses (Open Access)

Structure-function analysis of human Integrator subunit 4

Anupama Sataluri

Advisor: Eric. J. Wagner, Ph.D.

Uridine-rich small nuclear RNAs (U snRNA) are RNA Polymerase-II (RNAPII) transcripts that are ubiquitously expressed and are known to be essential for gene expression. snRNAs play a key role in mRNA splicing and in histone mRNA expression. Inaccurate snRNA biosynthesis can lead to diseases related to defective splicing and histone mRNA expression. Although the 3′ end formation mechanism and processing machinery of other RNAPII transcripts such as mRNA has been well studied, the mechanism of snRNA 3′ end processing has remained a mystery until the recent …

Oxidation Of Thrombomodulin Methionine 388 In Cigarette Smokers, Samrat B. Thapa

Graduate Theses and Dissertations

This work tested the hypothesis that oxidation of methionine 388 in thrombomodulin is higher in cigarette smokers, and thus a likely contributor towards the hypercoagulable state in smokers. Thrombomodulin, a protein cofactor found on endothelial cell surfaces, regulates the activity of thrombin. Thrombin bound to thrombomodulin no longer converts fibrinogen to fibrin, but instead activates Protein C which, in turn, stops the coagulation cascade by inactivation of clotting factors. The oxidation of methionine 388 of thrombomodulin has been shown in vitro to dramatically decrease the anticoagulant cofactor activity of thrombomodulin. The blood of cigarette smokers is more prone to clot …

Expression, Production, And Purification Of Novel Therapeutic Proteins, Mckinzie Shea Fruchtl

Graduate Theses and Dissertations

Interest in the production of recombinant proteins consisting of collagen binding domain (CBD) fused to a bioactive material has increased due to the targeting/attachment capabilities of CBD. For example, CBD fusions can be applied to the reversing of bone density loss and the repair of the eardrum, specifically, by choosing an appropriate fusion partner (parathyroid hormone or epidermal growth factor). The production of CBD fusions was examined using batch and fed-batch culturing of Escherichia coli to express the fusion proteins, and affinity chromatography to isolate the final product.

Different medium formulations, feeding strategies, and induction methods were tested in order …

Characterization Of The Product Specificity And Kinetic Mechanism Of Protein Arginine Methyltransferase 1, Shanying Gui

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein enzymes perform a vast array of functions within living organisms, catalyzing various metabolic reactions including DNA replication, DNA repair, protein synthesis, etc. In order to maintain proper cellular functions, enzymes need to be accurately regulated under different circumstances. Specifically, enzymes can be modified after their creation to give them additional functions. These modifications can do a variety of things including activating (turning on) or inactivating (turning off) an enzyme, changing what proteins or molecules can interact with the enzyme, changing the enzyme’s location in the cell, and/or targeting the enzyme for destruction. This dissertation focuses on a single class …

Investigation Of The Oxidation/Reduction Of Prmt1, Substrate Interaction With Prmt1, And The Role Of Arginine Methylation In Rna Surveillance, Damon V. Nitzel

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Enzymes are the machines of our cells. Just like machines, it takes a lot of energy to create them, and they then serve only the function they were created for. If we want to change the function of a machine, we need to modify it. Similarly, enzymes can be modified after their creation to give them additional function. These modifications can do a variety of things including activating (on) or inactivating (off) an enzyme, changing the enzyme’s location in the cell, and targeting the enzyme for destruction. This thesis focuses on a single class of enzymes, protein arginine methyltransferases (PRMTs), …

Effects Of Ergot Alkaloids And Bovine Bodily Fluids On Cytochrome P450 Activity, Nicholas Scott Ezell

Graduate Theses and Dissertations

This thesis evaluates the Promega^TM P450-Glo assay (Promega^TM V9800) as a tool for quantifying ergot alkaloid concentration. Current techniques used for detection of ergot alkaloids are slow and expensive, do not detect all ergot alkaloids, or are not effective on bovine bodily fluids. The first study was conducted to determine effects of commercial ergot alkaloids (n = 6; 0 - 400 μM) on the Promega^TM P450-Glo assay. Cytochrome P450 (CYP450) activity in assay had a differential response to each ergot alkaloid and concentration. As concentrations of ergotamine, dihydroergotamine, ergocornine, and ergocryptine increased CYP450 activity was inhibited (P …

Increased Production And Extraction Efficiency Of Triacylglycerides From Microorganisms And An Enhanced Understanding Of The Pathways Involved In The Production Of Triacylglycerides And Fatty Alcohols, Robert M. Willis

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid (fat) content of cellular feedstocks, improve extraction efficiencies of lipids, and to understand the pathways involved in the production of fatty alcohols and triacylglycerides, compounds commonly used in many industrial processes, from microbial feedstocks. This work has been done in an attempt to increase the overall economic viability of microbial biofuels production.

The production of biofuels from microalgae used as a feedstock allows for the …

Nuclear Localization Of Cpi-17, A Protein Phosphatase-1 Inhibitor Protein, Affects Histone H3 Phosphorylation And Corresponds To Proliferation Of Cancer And Smooth Muscle Cells., Masumi Eto, Jason A Kirkbride, Rishika Chugh, Nana Kofi Karikari, Jee In Kim

Department of Molecular Physiology and Biophysics Faculty Papers

CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear …

Mesobiliverdin Ixα Enhances Rat Pancreatic Islet Yield And Function, Taihei Ito, Dong Chen, Cheng-Wei Tom Chang, Takashi Kenmochi, Tomonori Saito, Satoshi Suzuki, Jon Y. Takemoto

Chemistry and Biochemistry Faculty Publications

The aims of this study were to produce mesobiliverdin IXα, an analog of anti-inflammatory biliverdin IXα, and to test its ability to enhance rat pancreatic islet yield for allograft transplantation into diabetic recipients. Mesobiliverdin IXα was synthesized from phycocyanobilin derived from cyanobacteria, and its identity and purity were analyzed by chromatographic and spectroscopic methods. Mesobiliverdin IXα was a substrate for human NADPH biliverdin reductase. Excised Lewis rat pancreata infused with mesobiliverdin IXα and biliverdin IXα-HCl (1–100 μM) yielded islet equivalents as high as 86.7 and 36.5%, respectively, above those from non-treated controls, and the islets showed a high degree of …

Divergent Protein Motifs Direct Ef-P Mediated Translational Regulation In Salmonella And Escherichia Coli, Steven J. Hersch, Mengchi Wang, S. Betty Zou, Kyung-Mee Moon, Leonard J. Foster, Michael Ibba, William Wiley Navarre

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the …

Kinetics Of Atmospheric Reactions Of Biogenic Volatile Organic Compounds: Measurement Of The Rate Constant Ofthujone + Cl· At 296 K And Calculation Ofthe Equilibrium Constant For The Ho2ch2ch2o2· H2o Complex, Marie Coy Killian

Theses and Dissertations

Biogenic volatile organic compounds (VOCs) react with Cl and OH radicals and the resulting radicals combine with oxygen to form peroxy radicals RO2. Organic peroxy radicals can then react with NO to form NO2, a precursor of tropospheric ozone. The work presented here explored the initial reaction between Cl and thujone, a VOC emitted by Great Basin sagebrush. The rate constant for the reaction of thujone + Cl at 296 K was measured with the method of relative rates with FTIR for detection of reactants. LEDs were used to photolyze Cl2 to generate Cl in the reaction cell. Thujone was …

Determination Of Trace Metals, Volatile Organic Compounds, And Other Water Standards In Wmu Drinking Water, Tyler Walter

Honors Theses

The USEPA creates drinking water maximum contamination levels to support safety in all environments. Inductively coupled plasma-optical emission spectroscopy (ICP-OES), Watersafe® Drinking Water Test kits, and solid phase microextraction with gas chromatography were used to assess the safety of drinking water in three buildings (Davis Hall, the Chemistry Building, and the Dalton Center) on the campus of Western Michigan University. ICP-OES was used to determine the concentration of several trace metals (Cu, Zn, Fe, Mn, Ni, Pb, and As), Watersafe® Drinking Water Test kits were used to determine the concentration of common contaminants and conditions (E. coli, pesticides atrazine and …

Investigations Of Substrate Channeling In The Proline Oxidative Pathway, Nikhilesh Sanyal

Department of Biochemistry: Dissertations, Theses, and Student Research

In cell metabolism, substrate channeling is a phenomenon where the product of one reaction is transported to a second enzyme active site without equilibrating into bulk solvent. Chapter 1 reviews the rationale and evidence for substrate channeling with the specific example of proline metabolism. Oxidation of proline to glutamate is catalyzed in consecutive reactions by proline dehydrogenase (PRODH) and pyrroline-5-carboxylate dehydrogenase (P5CDH). The intermediate Δ¹-pyrroline-5-carboxylate reportedly tends to be labile and inhibitory towards several metabolic pathways.

One of the main objectives of this dissertation was to investigate substrate channeling between independent proline oxidative enzymes from Thermus thermophilus- …

Roman Religion, Medicine, And Disease, Stephanie Clark

Honors Theses

Medicine, as experienced in the Roman Empire, was rapidly evolving. Balancing the newly developed theories with the already-established religious belief sparked the beginning of medical secularization. There were separate worlds, that of science and medicine, and that of the every day public, that had to be bridged in a way that honored both traditions, yet kept their integrity. The development of science and medicine pioneered by Galen, a philosopher whose medical knowledge and theory guided practitioners for almost two millennia, hit levels of exploration and understanding in ancient Rome that warranted its basis as common medical theory. However, the world, …

Chemically Diverse Microtubule Stabilizing Agents Initiate Distinct Mitotic Defects And Dysregulated Expression Of Key Mitotic Kinases., Cristina C. Rohena, Jiangnan Peng, Tyler A. Johnson, Phillip Crews, Susan L. Mooberry

Natural Sciences and Mathematics | Faculty Scholarship

Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent …

Chemical Warfare Agents In Plants: Biodefensive Terpenes From Sagebrush, Belinda Stierman

College of Arts and Sciences Poster Presentations

To prevent being eaten, some plants create compounds that are toxic to herbivores, and herbivores respond by creating new ways to metabolize these toxins. For example, sagebrush produces bio-defensive terpenes to deter foraging by sage grouse. A recent investigation to characterize plant secondary metabolites (PSM's) in sagebrush resulted in a correlation between terpene concentration and nutritional content in local sagebrush to the habitat selection of sage grouse. This study identified many of the terpenes in sagebrush, but not all of them. A highly volatile and elusive terpene is suspected to be an important PSM that significantly affects sage grouse foraging. …

Activities Of Methionine-Γ-Lyase In The Acidophilic Archaeon “Ferroplasma Acidarmanus” Strain Fer1, M. A. Khan, Madeline M. Lopez-Munoz, Charles W. Kaspar, Kai F. Hung

Faculty Research & Creative Activity

Biogeochemical processes on exposed pyrite ores result in extremely high levels of sulfuric acid at these locations. Acidophiles that thrive in these conditions must overcome significant challenges, including an environment with proton concentrations at pH 3 or below. The role of sulfur metabolism in the archaeon “Ferroplasma acidarmanus” strain fer1’s ability to thrive in this environment was investigated due to its growth-dependent production of methanethiol, a volatile organic sulfur compound. Two putative sequences for methionine- γ-lyase (EC 4.4.1.11), an enzyme known to carry out α,γ-elimination on L-methionine to produce methanethiol, were identified in fer1. Bioinformatic analyses identified a conserved pyridoxal-5′-phosphate …

Activities Of Methionine-Γ-Lyase In The Acidophilic Archaeon “Ferroplasma Acidarmanus” Strain Fer1, M. Khan, Madeline Lopez-Munoz, Charles Kaspar, Kai Hung

Faculty Research & Creative Activity

Biogeochemical processes on exposed pyrite ores result in extremely high levels of sulfuric acid at these locations. Acidophiles that thrive in these conditions must overcome significant challenges, including an environment with proton concentrations at pH 3 or below. The role of sulfur metabolism in the archaeon “Ferroplasma acidarmanus” strain fer1’s ability to thrive in this environment was investigated due to its growth-dependent production of methanethiol, a volatile organic sulfur compound. Two putative sequences for methionine- γ-lyase (EC 4.4.1.11), an enzyme known to carry out α,γ-elimination on L-methionine to produce methanethiol, were identified in fer1. Bioinformatic analyses identified a conserved pyridoxal-5′-phosphate …

Multiscale Modeling Of Enzyme-Catalyzed Methanol Production By Particulate Methane Monooxygenase, Katherine K. Bearden

Doctoral Dissertations

In this work, the conversion of methane to methanol by the particulate Methane Monooxygenase (pMMO) enzyme is investigated using a multi-scale modeling approach. This enzyme participates in carbon cycling and aids in the removal of harmful atmospheric methane, converting it to methanol. The interaction between pMMO and a neighboring enzyme that is present in the same organism is studied, and the unknown pMMO active site is elucidated and tested for methane oxidation towards the production of methanol.

Fundamental knowledge of pMMO's mechanism is not fully understood. Understanding how this enzyme works in nature will provide information towards designing efficient synthetic …

Treating Cancer With Heat: Hyperthermia As Promising Strategy To Enhance Apoptosis, Kanwal Ahmed, Syed Faisal Zaidi

Department of Biological & Biomedical Sciences

The fundamental idea and the effects of heat on cancer cells are well known. However, the results obtained in therapy by hyperthermia (HT) alone have been only partially satisfactory. Treatment at temperatures between .40 and 44 degrees C is cytotoxic for cells in an environment with a low oxygen partial pressure and low pH, conditions that are found specifically within tumour tissue, due to insufficient blood perfusion. Under such conditions radiotherapy is less effective, and systemically applied cytotoxic agents will reach such areas in lower concentrations than in well-perfused areas. Therefore, clinically, it is preferred to use hyperthermia in combination …

Adenovirus Type 5 Virions Asymmetrically Lose Icosahedral Structure At Low Phs Of Endosomes, Jennifer Marie Jarboe

Honors Theses

After receptor mediated endocytosis into an endosome, human Adenovirus Type 5 (Ad5) undergoes structural changes that allow the virus to release a viral protein, pVI, that lyses the endosome and releases the virus core into the cytosol, where it travels to the nucleus to insert its genome. Structural studies of Ad5 at a range of pHs typical of endosomes (7.5-4.5) using a transmission electron microscope show that Ad5 undergoes asymmetrical loss of proteins from vertex regions at the pH of the late endosome (pH5.15). Following further acidification, Ad5 ejects core material preferentially through one vertex—indicating an asymmetry at one vertex …