Biochemistry, Biophysics, and Structural Biology Commons^™

Alternative Pathway For Provision Of Acyl Coa Precursors For Fatty Acid Biosynthesis: Purification And Kinetic Characterization Of Phosphotransbutyrylase And Butyrate Kinase From Listeria Monocytogenes, Sirisha Sirobhushanam

Theses and Dissertations

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a disease characterized by gastroenteritis, meningitis, spontaneous miscarriages and high mortality rate among infected individuals. L. monocytogenes is a major concern in the food industry, due to its ability to grow at refrigeration temperatures and the zero tolerance policy of the FDA, resulting in expensive food recalls. Growth at low temperatures is aided in part by the high membrane content of branched-chain fatty acids (BCFAs) which imparts greater fluidity to the membrane. Mutants of L. monocytogenes impaired in BCFA biosynthesis display diminished growth at normal and low temperatures, exhibit lower tolerance …

Binding Of Oxygen To Human Hemoglobin Within The Erythrocyte Using Icam Spectrophotometry, Kyle K. Hill

Department of Chemistry: Dissertations, Theses, and Student Research

Many of the spectrophotometric techniques used to determine the properties of intracellular human hemoglobin cannot be utilized due to the turbidity of erythrocyte suspensions. An Integrating Cavity Absorption Meter, or ICAM, allows for absorption measurements of strongly scattering samples in the visible-light region of the spectrum. The spectrum of oxygenated hemoglobin within erythrocytes is significantly different from the absorption spectrum of oxygenated hemoglobin in solution. Studies of the oxygen binding to hemoglobin in erythrocytes allowed the four sequential binding constants (Adair constants) to be determined and compared with those of hemoglobin in solution. The Adair constants for hemoglobin in solution …

Effects Of Creatine, Leucine, And Ethanol On Glial Cell Line-Derived Neurotrophic Factor In C2c12 Skeletal Muscle Cells, Daniel Waters

Honors Theses

Glial Cell Line-Derived Neurotrophic Factor (GDNF) is a protein expressed throughout the mammalian nervous system that serves several protective functions. Originally discovered in the brain, where it supports the survival of dopaminergic neurons, GDNF has since been shown to be expressed in skeletal muscle and is vital for the maintenance of motor neurons. The relationship between muscle hypertrophy, and GDNF is not well understood. The purpose of this study was to examine the relationship between hypertrophy and GDNF by inducing hypertrophy in C2C12 skeletal muscle cells using leucine and creatine, inhibiting hypertrophy using ethanol and measuring GDNF content. As measured …

A Guided Inquiry To The Bromination Of Alkenes: An Effort To Foster Student Understanding Of Thin Layer Chromatography In The Organic Chemistry Laboratory, Casey Wright

Honors Theses

Many of the chemistry laboratories taught at Western Michigan University are “cookbook” labs, meaning that students are given an instruction manual with a prescribed set of instructions and a predicted outcome. Therein we present an introductory organic chemistry laboratory experiment to reinforce the student understanding of Thin Layer Chromatography which utilizes a guided inquiry format as part of an effort to move toward discovery based learning in our organic chemistry curriculum. Educational research was performed on classroom artifacts collected from the laboratory. We were able to gain an understanding of the struggles students face in applying general chemistry content knowledge …

The Pcna-Associated Protein Pari Negatively Regulates Homologous Recombination Via The Inhibition Of Dna Repair Synthesis, Peter Burkovics, Lili Dome, Szilvia Juhasz, Veronika Altmannova, Marek Sebesta, Martin Pacesa, Kasper Fugger, Claus Storgaard Sorensen, Marietta Y W T Lee, Lajos Haracska, Lumir Krejci

NYMC Faculty Publications

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after …

Novel Compound Heterozygous Mutations Expand The Recognized Phenotypes Of Fars2-Linked Disease, Melissa A. Walker, Kyle Mohler, Kyle W. Hopkins, Derek H. Oakley, David A. Sweetser, Michael Ibba, Matthew P. Frosch, Ronald L. Thibert

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Mutations in mitochondrial aminoacyl-tRNA synthetases are an increasingly recognized cause of human diseases, often arising in individuals with compound heterozygous mutations and presenting with system-specific phenotypes, frequently neurologic. FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase (mtPheRS), perturbations of which have been reported in 6 cases of an infantile, lethal disease with refractory epilepsy and progressive myoclonus. Here the authors report the case of juvenile onset refractory epilepsy and progressive myoclonus with compound heterozygous FARS2 mutations. The authors describe the clinical course over 6 years of care at their institution and diagnostic studies including electroencephalogram (EEG), brain magnetic resonance …

Doxycycline Resistance In Plasmodium Falciparum Linked To Single Nucleotide Polymorphisms In The Plasmodium Falciparum Apicoplast Small Subunit Ribosomal Rna (Pfssrrna) Gene, Amanda Wasko

Scholarly and Creative Works Conference (2015 - 2021)

Plasmodium falciparum, a protozoan parasite known as malaria, widely impacts human health; thus antimalarial drug investigations are critical. Doxycycline is a commonly used antimalarial prophylactic, but its mechanism of action is unclear. In prokaryotes, doxycycline works as an antibacterial by disrupting protein translation via the small subunit ribosome. Interestingly, P. falciparum has a small subunit ribosome of prokaryotic origins in the apicoplast, a plastid-like organelle. Therefore, we hypothesized that doxycycline works in P. falciparum by inhibiting protein synthesis via the small subunit ribosomal RNA and that mutations in the gene encoding the P. falciparum apicoplast small subunit ribosomal RNA …

Consensus On Consensus: A Synthesis Of Consensus Estimates On Human-Caused Global Warming, John Cook, Naomi Oreskes, Peter T. Doran, William R L Anderegg, Bart Verheggen, Ed W. Maibach, J Stuart Carlton, Stephan Lewandowsky, Andrew G. Skuce, Sarah A. Green, Dana Nuccitelli, Peter Jacobs, Mark Richardson, Bärbel Winkler, Rob Painting, Ken Rice

Department of Chemistry Publications

The consensus that humans are causing recent global warming is shared by 90%–100% of publishing climate scientists according to six independent studies by co-authors of this paper. Those results are consistent with the 97% consensus reported by Cook et al(Environ. Res. Lett. 8 024024) based on 11 944 abstracts of research papers, of which 4014 took a position on the cause of recent global warming. A survey of authors of those papers (N = 2412 papers) also supported a 97% consensus. Tol (2016 Environ. Res. Lett.11 048001) comes to a different conclusion using …

Cell Permeability Studies Of Aapeptides And Novel Molecular Probes For Ad, Ge Bai

USF Tampa Graduate Theses and Dissertations

Alzheimer's diseases(AD) has been discovered and under research for more than 70 years, However there is no cure for these progressive and devastating diseases. Based on the following hypothsis: Aß metabolite problem/over production result in the accumulation, and lead to aggregation is the cause of Alzheimer’s disease. AApeptide and Melatonin derivatives can bind to Aß and block the aggregation of β amyloid monomers, decrease the toxicity of Aß to neurons and slow the progressive of Alzheimer’s diseases. In addition, AApeptide which mimic transmembrane peptide Tat will have similar transmembrane function. We have set up our goals as follows: 1) Using …

Stabilin-Mediated Cellular Internalization Of Phosphorothioate-Modified Antisense Oligonucleotides (Asos), Colton M. Miller, Aaron J. Donner, Emma K. Blank, Andrew W. Egger, Brianna M. Kellar, Punit P. Seth, Edward N. Harris

UCARE: Research Products

Introduction: Antisense oligonucleotides (ASOs) are short chemically modified oligonucleotides (5-7.4 kDa) that can produce a pharmacological effect by binding to RNA and affecting intermediary metabolism. Over 35 phosphorothioate (PS) ASOs are at various stages of clinical development for use as therapeutic agents and pharmacological tools. Antisense therapy is a progressing area of research, as these small strands of nucleotide oligomers can be produced to silence genes that aggravate chronic disorders or infections. An important distinction for ASOs compared to DNA is the substitution of the phosphodiester (PO) backbone with the PS modification. This sulfur substitution allows for these polar polyanionic …

Photocatalytic Reduction Of Fumarate To Succinate On Zns Mineral Surfaces, Ruixin Zhou, Marcelo I. Guzman

Chemistry Faculty Publications

The reductive tricarboxylic acid (rTCA) cycle is an important central biosynthetic pathway that fixes CO₂ into carboxylic acids. Among the five reductive steps in the rTCA cycle, the two-electron reduction of fumarate to succinate proceeds nonenzymatically on the surface of photoexcited sphalerite (ZnS) colloids suspended in water. This model reaction is chosen to systematically study the surface photoprocess occurring on ZnS in the presence of [Na₂S] (1–10 mM) hole scavenger at 15 °C. Experiments at variable pH (5–10) indicate that monodissociated fumaric acid is the primary electron acceptor forming the monoprotic form of succinic acid. The following …

Investigation Of Respiratory Syncytial Virus Structural Determinants And Exploitation Of The Host Ubiquitin System, Jillian Nicole Whelan

USF Tampa Graduate Theses and Dissertations

Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal …

Effects Of Macromolecular Crowding On Ferredoxin And Ferredoxin-Nadp+ Reductase Kinetics, Danielle Bautista, David W. Seybert Ph.D.

Undergraduate Research and Scholarship Symposium

Macromolecular crowding refers to the intrinsic occurrence of high concentrations of macromolecules found within cells, which may alter biomolecular properties of other macromolecules. Simulating these environments in vitro should enable a more accurate understanding of these modifications. We have employed in vitro studies to specifically examine the effects of macromolecular crowding on biological redox reactions. The iron-sulfur protein ferredoxin (Fdx) and the FAD-containing flavoprotein ferredoxin-NADP+ reductase (FNR), from Spinacea oleracea, were utilized as a model electron transfer system. Utilizing NADPH as a reductant, the Fdx-FNR complex reduces mammalian cytochrome c as a surrogate electron acceptor. Reduction of the heme moiety …

Genetically Induced Production Of Secondary Metabolites In Bacillus Megaterium, Majie C. Foster, Jay L. Brewster, P. Matthew Joyner

Seaver College Research And Scholarly Achievement Symposium

Historically, the most important source of new antibiotic drug leads has been small organic compounds made by bacteria. Many antibiotics have been developed into pharmaceutical agents from these molecules (often called secondary metabolites) produced by soil bacteria. Bacillus species are soil bacteria known for producing various antimicrobials including gramicidin, bacitracin, surfactin, and others. Bacillus megaterium is a widely used model gram positive bacterium. Although there has been extensive research on this organism, little is known about its secondary metabolites. We hypothesized that the production of secondary metabolites in this organism could be induced by replacing promoters controlling the expression of …

Axonal Transport And Life Cycle Of Mitochondria In Parkinson's Disease Model, Hyun Sung

Open Access Dissertations

In neurons, normal distribution and selective removal of mitochondria are essential for preserving compartmentalized cellular function. Parkin, an E3 ubiquitin ligase associated with familial Parkinson’s disease, has been implicated in mitochondrial dynamics and removal. However, it is not clear how Parkin plays a role in mitochondrial turnover in vivo, and whether the mature neurons possess a compartmentalized Parkin-dependent mitochondrial life cycle. Using the live Drosophila nervous system, here, I investigate the involvement of Parkin in mitochondrial dynamics; organelle distribution, morphology and removal. Parkin deficient animals displayed less number of axonal mitochondria without disturbing organelle motility behaviors, morphology and metabolic state. …

A Unified Analytic Framework For Prioritization Of Non-Coding Variants Of Uncertain Significance In Heritable Breast And Ovarian Cancer, Eliseos J. Mucaki, Natasha G. Caminsky, Ami M. Perri, Ruipeng Lu, Alain Laederach, Matthew Halvorsen, Joan H. M. Knoll, Peter K. Rogan

Biochemistry Publications

Background

Sequencing of both healthy and disease singletons yields many novel and low frequency variants of uncertain significance (VUS). Complete gene and genome sequencing by next generation sequencing (NGS) significantly increases the number of VUS detected. While prior studies have emphasized protein coding variants, non-coding sequence variants have also been proven to significantly contribute to high penetrance disorders, such as hereditary breast and ovarian cancer (HBOC). We present a strategy for analyzing different functional classes of non-coding variants based on information theory (IT) and prioritizing patients with large intragenic deletions.

Methods

We captured and enriched for coding and non-coding variants …

Mutant Study Of Sinorhizobium Meliloti Proline Utilization A (Puta), Jacob E. Wilkinson, John J. Tanner, Donald F. Becker

UCARE: Research Products

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or …

The Nmr Solution Structure And Function Of Rpa3313: A Hypothetical Protein From R. Palustris, Austin J. Lowe, Jonathan Catazaro, Cheryl Arrowsmith, Robert Powers

UCARE: Research Products

Protein function elucidation often relies heavily on amino acid sequence analysis and other bioinformatics approaches. The reliance is further extended to structure homology modeling for ligand docking and protein-protein interaction mapping. However, sequence analysis of RPA3313 exposes a large, unannotated class of hypothetical proteins mostly from the Rhizobiales order. In the absence of sequence and structure information, further functional elucidation of this class of proteins has been significantly hindered. A high quality NMR structure of RPA3313 reveals that the protein forms a novel split βαβ fold with a conserved ligand binding pocket between the first β-strand and the N-terminus of …

Genetically Incorporated Vinyl Sulfide For Various Bioorthogonal Reactions, Carly Faller, Xin Shang, Jiantao Guo

UCARE: Research Products

No abstract provided.

Analysis Of Histone Deacetylase Involvement In Pseudomonas Syringae-Triggered Chromatin Changes, Jennifer Wies, Victoria Shum, Karin V. Van Dijk

UCARE: Research Products

I had the great privilege of working with Dr. Karin van Dijk, through UCARE, researching the specific pathways through which histone deacetylation occurs in plants during the 2015-2016 academic year. My first year conducting research helped me develop my technical skills and learn about experimental protocols and how to conduct them. I would like to continue my research during the 2016-2017 academic year and now that I have a year of research experience, be able to further develop my skills and learn more about my research project.

Background

Pseudomonas syringae is a bacterial pathogen that is well known for causing …

Hair Analysis In Motor Vehicles, Jaclyn E. Humrich, Larry Barksdale

UCARE: Research Products

The purpose of this research was to examine the frequency of human and animal hair in motor vehicles in the Lincoln, NE area. We were looking specifically at animal hair to see if there is a significant difference in the distribution of animal hair in pet-owner and non pet-owner vehicles. Finding a significantly high distribution of animal hair in pet-owner vehicles would lead us to believe that an association could be made between a suspect owning an animal and their presence at a crime scene.

Our null hypothesis is that there is no significance in the distribution of cat/dog hairs …

Biophysical Studies Of The Allosteric Regulatory Mechanism Of Syk Tandem Sh2 Domains Interacting With Immunoreceptor Tyrosine-Based Activation Motifs, Chao Feng

Open Access Dissertations

The non-receptor spleen tyrosine kinase (Syk) is an important player in signal transduction from immunoreceptors to various downstream targets. It is widely expressed in both haematopoietic and epithelial cells. Syk disorder is closely related with many inflammatory and autoimmune diseases, as well as cancers.

Syk associates with immunoreceptors through its tandem SH2 domains (tSH2), which contain two SH2 domains connected by interdomain A. The association of Syk with immunoreceptors is regulated by Y130 phosphorylation in interdomain A. The unphosphorylated tSH2 can bind with the doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the cytoplasmic domains of immunoreceptors with very high …

Learning The Abc's Of Ribose Transport Using Biophysical Methods, Satchal K. Erramilli

Open Access Dissertations

ATP-binding cassette transporters comprise a large superfamily of proteins that are involved in a variety of biological phenomenon, from bacterial metabolism to cellular homeostasis, antigen-presentation, and drug resistance. These proteins are implicated in a variety of clinically relevant phenomenon, including the human diseases cystic fibrosis, macular degeneration, and cancer. Understanding their structure-function can guide therapeutics and contribute to our overall understanding of these biological phenomena.

This study focuses on understanding the motor protein of the bacterial ribose ABC transporter in the context of transport. This complex is required for the uptake of the nucleotide precursor, ribose. Using biophysical methods, we …

Characterization Of Cu-Rich Aggregates In Neurogenic Niches Of The Rodent Brain By X-Ray Fluorescence Microscopy, Brendan T. Sullivan

Open Access Dissertations

Copper is an essential element in the brain playing several critical roles ranging from neurotransmitter synthesis to ATP production. As Cu is typically present in micromolar concentrations and has a spatially capricious distribution in the brain, determining concentrations has historically been challenging. X-ray fluorescence microscopy (XRF) offers excellent spatial resolution (down to 30~nm) and detection limits (sub parts per million), making it an excellent tool for analyzing metal distributions in the brain. Using XRF, it is demonstrated that Cu-rich aggregates with concentrations in the hundreds of millimolar are present in the subventricular zone of rats and mice. As the subventricular …

Mechanistic Characterization Of Acetic Acid Resistance Enzymes Of Acetobacer Aceti, Jesse R. Murphy

Open Access Dissertations

Acetobacter aceti (A. aceti) is a Gram-negative, acidophilic bacterium that is used for the industrial production of acetic acid from ethanol. Oxidation of ethanol by membrane-bound oxidoreductases provides energy for A. aceti and the production of high concentrations of acetic acid is an effective defense mechanism. Acetic acid diffuses through cell membranes at low pH and effectively kills many bacteria, including E. coli, at low millimolar concentrations. The ability of A. aceti to thrive in molar concentrations of acetic acid is partially due to the twin subjects of this thesis, the acetic acid resistance factors AarA (citrate synthase, …

Fipronil Promotes Adipogenesis Via Ampkα-Mediated Pathway In 3t3-L1 Adipocytes, Quancai Sun, Jeremy Yang, Kyong-Sup Yoon, John M. Clark, Yeonhwa Park

SIUE Faculty Research, Scholarship, and Creative Activity

Emerging evidence suggests that organochlorine, organophosphorus and neonicotinoid insecticide exposure may be linked to the development of obesity and type 2 diabetes. However, there is no knowledge of the potential influence of fipronil, which belongs to the phenylpyrazole chemical family, on obesity. Thus, the goal of this study was to determine the role of fipronil in adipogenesis using 3T3-L1 adipocytes. Fipronil treatment, at 10 mM, increased fat accumulation in 3T3-L1 adipocytes as well as promoted key regulators of adipocyte differentiation (CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor gamma-γ), and key regulators of lipogenesis (acetyl-CoA carboxylase and fatty acid synthase). The …

Defining The Roles Of Serine Palmitoyltransferase-Interacting Proteins In The Regulation Of Sphingolipid Homeostasis, Athen N. Kimberlin

Department of Biochemistry: Dissertations, Theses, and Student Research

Sphingolipids are major structural components of the plasma membrane and endomembrane system. Research suggests that sphingolipids are involved with the formation of lipid microdomains, also known as lipid rafts, which may help to organize proteins within the membrane and may be important for membrane trafficking. Aside from their structural roles in membranes, sphingolipids and their metabolic products have been implicated in several cellular signaling responses like programmed cell death (PCD). Because of this, maintenance of sphingolipid homeostasis is critical for eukaryotic cell growth and development. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is the primary regulatory …

Synthesis And Mechanistic Investigations Of Transition Metal Complexes And Ligands For Chemotherapeutic Applications, Jimmie Lee Bullock Jr.

Chemistry & Biochemistry Theses & Dissertations

Transition metal complexes have played a critical role in antitumor therapy with many complexes incorporating platinum, ruthenium, and lanthanum having been investigated in preclinical and clinical trials. The best known transition metal therapeutic is cisplatin, which is utilized in nearly 50% of all cancer therapies, despite its significant toxic side effects. The toxic side effects of current FDA approved platinum-based chemotherapeutics are often overlooked due to the “special status” granted to these drugs due to their ability to fight, what is often considered an incurable disease with life expectancies often measured in months. Oncology drug development has therefore now focused …

Water Soluble Cationic Porphyrin Sensor For Detection Of Hg2+, Pb2+, Cd2+, And Cu2+, Christopher Orr, Miranda Uherek, Matibur Zamadar

Undergraduate Research Conference

Here we report the sensing properties of the aqueous solution of mesotetra(N-methyl-4-pyridyl)porphine tetrachloride (1) for simultaneous detection of toxic metal ions by using UV-vis spectroscopy. Cationic porphyrin 1 displayed different electronic absorptions in UV-vis region upon interacting with Hg2+, Pb2+, Cd2+, and Cu2+ ions in neutral water solution at room temperature. Quite interestingly, the porphyrin 1 showed that it can function as a single optical chemical sensor and/ or metal ion receptor capable of detecting two or more toxic metal ions, particularly, Hg2+, Pb2+, and Cd2+ ions coexisting in a water sample. Porphyrin 1 in an aqueous solution provides a …

Antimicrobial Properties Of Isomers Of Benzofuranylethanol, Nick Cheatwood, Jessica Bronstad, Michele Harris

Undergraduate Research Conference

A biotransformaDon reacDon is a chemical conversion of a substance into a desired product with the aid of whole, living cells containing the necessary enzymes. In addiDons to living cells, other substances, such as vegetable strips, will also catalyze biotransformaDon reacDons. Advantages of biotransformaDon reacDons include the following: the ability to recycle material, the use of less hazardous chemicals, the ability to compost vegetable strips, the use of inexpensive materials that are locally available, and the ability to produce a single isomer. The biotransformaDon reacDon for the conversion of benzofuranyl methyl ketone (BMK) to (-)-benzofuranylethanol (BMA) using carrot strips as …