Biochemistry Commons^™

The Affect Of Dha Concentration On Lipid Membrane Fluidity, Elise Rezabeck

Williams Honors College, Honors Research Projects

My proposed project will be creating large unilaminar vesicles with varying concentrations of DHA in the vesicles. The diffusion in and out of these vesicles will be tracked by using singular lipid molecules. These singular lipid molecules will be tagged with fluorescence and viewed using an internal reflection fluorescence microscope. The data collected over the course of this project will then be analyzed using Matlab. The result of the project will be to find the specific lipid diffusion constant of these unilaminar vesicles. The relationship between the concentration of DHA and lipid diffusion and membrane fluidity will be found as …

Effect Of An Oncogenic Her2 Mutant On Egfr Activity, Paige Kalik

Williams Honors College, Honors Research Projects

Studies have shown that HERs are involved in the progression of human cancers. Researchers have found that HER2 mutations in colorectal cancer are similar to mutations found in breast cancer. Recombinant antibodies are effective therapeutic treatments for cancers. Antibodies can also be used to determine how proteins function under normal and pathophysiological conditions while also identifying interacting partners. In this Honors Project, my aim is to investigate the effect of an oncogenic mutant of HER2. Considering the information above, the question researched in this project was: how does the S310F mutation affect the organization and function of HER2? The specific …

Effects Of Nicotine Vs Non-Nicotine E-Cigarettes On The Aggression Behavior Of Crayfish, Alex Kimble

Williams Honors College, Honors Research Projects

This study looks at the effects of e-cigarette vapor with and without nicotine on the aggression behavior of crayfish (Procambarus clarkii). The treatment groups were control, nicotine, and non-nicotine. The mass, sex, and condition of their appendages were recorded before random assignment. For the purpose of this study only male crayfish were used as there were not enough female crayfish. The crayfish were then randomly assigned to each group and contained ten crayfish that were subjected to the e-cigarette vapor infused water environment for a twenty-four hour period. The control group did not contain e-cigarette vapor and was exposed to …

Functionalization Of Chitosan Based Microparticles For In Vitro 3d Culture Of Human Liver Cells, William Imes

Williams Honors College, Honors Research Projects

Previous work involving 3D culture of human liver cells with fluorinated chitosan based microparticles has shown that their incorporation provides needed structural cues that the culture of cells alone does not provide, such as increased gas transport. However, using bare microparticles to grow large 3D cellular structures is not practical as they tend to collapse before any meaningful research can be done on them. It is proposed that this is because of the lack of extracellular matrix (ECM)components within pure cell culture in vitro, which in their absence cannot adequately facilitate biochemical communication and adhesion between cells. It is …

Sequential Block Copolymers Of Polyisobutylene And Polyolefins, Manwel Labib

Williams Honors College, Honors Research Projects

A two-step approach will be utilized to synthesize random polyolefin multiblock-copolymers. This strategy takes advantage of a,w-terminated telechelic polyolefins. Polypropylene (iPP)/polyethylene (PE) are picked because of their strength, and polyisobutylene (PIB) because of its hemocompatibility. This copolymer is theorized to make for robust bio-inert plastic, which can be used for surgical mesh implants.

Biodegradation Of Rubber Particles In Soil: Using Acclimated Bacteria Isolated From Kansas Soil To Degrade Cryogrinds In Slurry, Shane Graham

Williams Honors College, Honors Research Projects

This study investigated the viability of bioremediating rubber cryogrind using enriched indigenous bacteria. To begin the experiment, soils from three highway roadside locations in Kansas, KS 96 and West, KS 400 and 143^rd, and 199^th, were collected and transported to the lab to be studied. An initial soil characterization was run on the soil samples using distilled (DI) water mixture and 0.01 M CaCl₂ to assess conductivity. The soils were tested to gather a baseline of the relationship between pH and conductivity and the impact of its distance from the roadside. Bacteria were isolated from …

The Influence Of Hydrogen Peroxide On The Enrichment Of Fe(Iii) Reducing Bacteria From Acid Mine Drainage, Susami Seth

Williams Honors College, Honors Research Projects

It is hypothesized that the ocean of Europa, a Jupiter moon, hosts bacteria on its oceanic floor. Understanding how Fe(III) reducing bacteria (FeRB) from AMD utilize organic materials within its surrounding environment outlines how FeRB could thrive and tolerate extreme conditions. FeRB are known to tolerate metals and highly reactive oxidants species (ROS), but in this experiment, H₂O₂ was the experimental factor to further test FeRB tolerance. H₂O₂ is a common ROS and is damaging to living material such as proteins, DNA, and RNA. A range of H₂O₂ concentrations were fed …

Synthesis Of Rna Nucleotides Under Probable Prebiotic Conditions, Ryan Stimson

Williams Honors College, Honors Research Projects

RNA being composed of multiple covalently linked nucleotides is thought to have been a precursor to life circa 4.3-3.8 billion years ago. Non-enzymatically formed adenosine monophosphate (AMP), more specifically, is a vitally important subtopic of the self-assembly of the first RNA sequence. The goal of this study was to synthesize AMP non-enzymatically under benign conditions that are likely to have existed on early Earth. In this experiment, 3’,5’-cAMP was successfully formed using wet-dry cycles at 80°C paired with the minerals zeolite beta, hydroxyapatite, and aerosil 300 in the presence of adenosine, urea, and pyrophosphate. A nuclear magnetic resonance spectrometer was …

Investigating Iron Metabolism In Subarachnoid Hemorrhage Patients, Alexander A. Maynard, Gardenia Pacheco

Williams Honors College, Honors Research Projects

Subarachnoid hemorrhage (SAH) is a stroke characterized by bleeding into the subarachnoid space of the brain, typically resulting in high mortality rate.⁸ Delayed cerebral ischemia (DCI), characterized by vasospasms induced arterial constriction, occurs in roughly one third of the surviving patients.²⁰ The development of DCI and neurodegeneration could be linked to metabolic pathology that occurs after SAH, specifically iron induced changes in redox status. The oxidized environment induced by iron has the potential to functionally affect the ferroxidase ceruloplasmin (Cp), which is linked to neurodegeneration.¹⁵ Global LC-MS based metabolomics data revealed alterations in metabolism in the CSF …

Cloning The Vision Related G Protein Transducin For Live Cell Fluorescence Studies, Deanna M. Bowman

Williams Honors College, Honors Research Projects

G coupled protein receptors (GCPR) are one of the largest families of receptors and mediate a variety of biological responses. Rhodopsin is the largest family and aids in sight, the α-subunit of the GCPR complex in extremely important to the activation and downstream signaling effects of GCPR. The α-subunit contains a small trans-domain portion and in this project the sequence of that portion will be inserted into a vector containing a fluorescent tag. These vectors will then be used to collect fluorescent cross correlation spectroscopy or FCCS data. The unit was cloned using assembly methods that include PCR and purification …

Mutations In Rhodopsin Transmembrane Helix 5 And Dimerization Interface, Margaret Pinkevitch

Williams Honors College, Honors Research Projects

G protein coupled receptors (GPCRs) are membrane proteins with many functional roles. This family of receptors is expansive, with several subfamilies. Here, a member of subfamily A, rhodopsin (Rho) is studied. Mutations in rhodopsin have been linked to the disease retinitis pigmentosa (RP). Some of these mutations have been found to disrupt the dimerization/oligomerization of Rho at the TM4/TM5 proposed dimerization interface, causing pathogenesis of RP. In this study, two mutations of transmembrane helix 5 were investigated for changes in dimerization and stability, cell trafficking, and signaling. It was found that the V209M and F220C mutations did not result in …

Detection Of Semi-Polar Vitamins And Nutrients In Biological Samples, Jennifer Janovick

Williams Honors College, Honors Research Projects

Metabolomics is used to examine metabolite fluctuations in biological systems and enables the diagnosis of metabolic disorders and the identification of new therapeutic targets.¹ Liquid chromatography-mass spectrometry (LC-MS) was utilized to develop a new method using a T3 column to separate small molecule nutrients and vitamins in a tissue sample. Initial tests of column performance used standard solutions; the amount of metabolites identified and the elution profile of these metabolites was examined. Both the standard solution and sample of brain tissue were tested with the final method developed. Six metabolites were identified in the standard and 31 were identified …

Sporadic Appearance Of Paralytic Spongiform Neurodegeneration In A Colony Of Inbred Mice Is Associated With Cns Retrovirus Expression, Magdalene Durbak

Williams Honors College, Honors Research Projects

Abstract

Exogenous and endogenous retroviruses (RV) have been known to induce vacuolar central nervous system neuropathology. Two mice which were juvenile inbred Rocky Mountain White (IRW), showed signs of spontaneous degenerate motor neuron disease. Signs included wasting, unkempt fur, adduction reflex upon tail elevation, kyphosis, and stilted gait by postnatal day 18 (P18). Brains and spinal cords were examined with H&E staining and stained with a broadly reactive retrovirus antibody. When compared to brains of mice with normal vacuolar pathology as well as mice infected with prototypic non-neurovirulent RV, Fr57E and prototypic neurovirulent RV FrCasE, the subject showed less severity …

Altering Oligomerization Of Epha2 Via Mutations In The Intracellular Domain, Ryan W. Lingerak

Williams Honors College, Honors Research Projects

Eph receptor tyrosine kinases (RTKs) are activated by membrane-bound ligands called ephrins. Eph RTKs are divided into two subclasses, each activated by a specific classes of the ligand ephrin. The overexpression of Eph receptors is correlated to cancer cell metastasis in several different types of cancers. Studies with the EphA2 extracellular domain (ECD) and ephrinA1 ligand have shown that upon binding of ephrin to the receptor, EphA2 undergoes increased oligomerization and activation. This indicates that oligomerization is intimately connected to kinase activity. High resolution crystal structures of the EphA2 ECD have revealed some details of these ligand bound oligomers, as …

Nanoparticle Targeting To The Central Nervous System, Emily Baker

Williams Honors College, Honors Research Projects

Drug delivery to the central nervous system is complicated by the blood-brain barrier, a vascular structure that prevents free diffusion of molecules into brain tissue. In this study, we examined the in vitro properties of a nanoparticle-based drug delivery system. We used several nanoparticle formulations, both LTP based and PLGA based, to test the cellular uptake and toxicity in microglial cells. Using immunofluorescence imaging, we show that LTP nanoparticles are taken up by microglia. We confirmed that our nanoparticle formulations are non-toxic by two cell viability assays. These results suggest that nanoparticle formulations may be a biocompatible method of delivering …

Effects Of Novel Fragment-Warhead Adducts In Situ And In Vitro With Glutaredoxin Orthologs., Andrew J. Caras

Williams Honors College, Honors Research Projects

Fragment-based drug design (FBDD) is a method of pharmaceutical research involving characterization of small “fragment” molecule interactions with a target protein. These fragments are optimized with the addition of a nucleophilic “warhead” moiety. Several novel fragment-warhead adducts have been synthesized in house by group member Ram Khattri Ph.D. The experiment investigates reactivity of these adducts with glutaredoxin protein (Grx) orthologs from Homo sapiens, Pseudomonas aeruginosa and Brucella melitensis. Three novel fragment-warhead adducts have been identified to cause weakly bacteriostatic and bacteriocidal effects, most with minimum inhibitory concentration values near 1.28 mg/mL. Discussed herein are molecular effects of these fragment-warhead …

Lipid-Protein Interactions Via Fluorescence Quenching In Small Unilamellar Vesicles, Talia Kordahi

Williams Honors College, Honors Research Projects

Studying peptide interactions with model membranes have aided in understanding protein-lipid interactions more clearly. In this report, fluorescent small unilamellar vesicles (SUVs) of a known composition were made. Two peptides, RND1 and MARCKS, were also fluorescently labeled and added to a solution of SUVs. The interactions between these were observed via fluorescence quenching, read out with a spectrofluorimeter. In addition, two environments of buffer were tested: 1X PBS and 0.1X PBS. After obtaining data, it was concluded that MARCKS quenched the SUVs more than RND1. This could be due to the presence of more lysine amino acids in MARCKS, which …

Investigation Of Dna Interaction With Bis-Cationic Imidazolium Salts, Kyle Daily 6440928

Williams Honors College, Honors Research Projects

Imidazolium salt compounds have shown promise in vitro against non-small cell lung cancer (NSCLC) lines, but the exact mechanism of activity these compounds is still unknown. Due to their planar aromatic structure, we hypothesized that our bis-cation imidazolium salt series could act through DNA intercalation similar to known intercalators ethidium bromide, acridine orange, and echinomycin. Here, we tested bis-cation imidazolium salts with linkers of 2, 4, 10, and 12 carbons for DNA intercalation via viscosity and fluorescent intercalator displacement (FID) assays. Despite some solubility issues that may have decreased results, we found that our butyl and dodecyl linked compounds had …

The Role Of Tgfβ1 And Ep Receptors In The Differentiation Of Normal And Diseased Lung Fibroblasts, Madison A. Hexter

Williams Honors College, Honors Research Projects

Fibroblasts, and their differentiation into myofibroblasts, are important in the pathology of several lung diseases including asthma and idiopathic pulmonary fibrosis (IPF). The differentiation process is dependent on several factors, such as soluble factors like transforming growth factor beta 1 (TGFβ1) and mechanical signals such as matrix stiffness. Additionally, the soluble arachidonic acid metabolite Prostaglandin E₂ (PGE₂) prevents TGFβ1 induced differentiation of fibroblasts to myofibroblasts, but the molecular mechanism is not completely understood¹⁵. Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive ion channel, has previously been implicated in the differentiation of cardiac fibroblasts^12,13. …

The Optimization Of Mitoneet Synthesis, Purification And Mutation Stability In The [2fe-2s] Cluster, Alexandria Defabio

Williams Honors College, Honors Research Projects

MitoNEET, a mitochondrial membrane protein with an unsymmetrical Fe-S cluster coordination, was compared to similar proteins within the Rieske and Ferredoxin families. Point mutations were introduced via site-directed mutagenesis to induce stability, greater retention of the cluster, and to uncover structural limitations of the protein. Mutations were made within the cluster coordination and within the hydrophobic core of the protein. Some of the cluster mutations displayed a natural affinity for nickel during immobilized metal-ion affinity chromatography. It is possible that these mutations aid in the formation of a chelate when exposed to nickel. Spectroscopic monitoring of changes in cluster stability …

Application Of Lipid Styryl Dye For Staining Intracytoplasmic Membranes In Gram-Negative Bacteria, Theodore J. Hammer

Williams Honors College, Honors Research Projects

Intracytoplasmic membranes are structures that form within cells which help facilitate a variety of different metabolic processes. This feature of intracellular membranes makes them particularly valuable for studying compartmentalization and cell dynamics in bacteria. In the past, transmission electron microscopy has been the primary method for imaging bacteria with intracytoplasmic membranes. Because transmission electron microscopy takes images of a cell in fixed slices, it’s impossible to follow a cell’s growth and development over time. Fluorescence microscopy is a particularly effective method of measurement that can combat these issues when evaluating live bacterial cells. Here, standard biochemical laboratory procedures were used …

Membrane Protein Spatial Organization In Mdck Cell Cultures, Rachel E. Neugebauer

Williams Honors College, Honors Research Projects

Studies on the organization of membrane proteins such as GPCRs in live cells help to draw important conclusions regarding cellular communication, and have become a significant topic in drug therapy development. Observing proteins in MDCK cells, which are able to form three-dimensional cultures, provides an intermediate step to better understanding the organization and behavior of these proteins in a tissue-like culture. In this study, fluorescent stains were used on fixed cells to show the morphological differences between MDCK cells in monolayer cultures and three-dimensional cultures, which form hollow, spherical cysts of tightly packed cells. Monolayer cells were transfected to view …

Evaluation Of The Signature Molecular Descriptor With Blosum62 And An All-Atom Description For Use In Sequence Alignment Of Proteins, Lindsay M. Aichinger

Williams Honors College, Honors Research Projects

This Honors Project focused on a few aspects of this topic. The second is comparing the molecular signature kernels to three of the BLOSUM matrices (30, 62, and 90) to test the accuracy of the mathematical model. The kernel matrix was manipulated in order to improve the relationship by focusing on side groups and also by changing how the structure was represented in the matrix by increasing the initial height distance from the central atom (Height 1 and Height 2 included).

There were multiple design constraints for this project. The first was the comparison with the BLOSUM matrices (30, 62, …

Sharp Rna Recognition Motif Optimizations, Extensions, And Mutations For Use In 2d And 3d Nmr Experiments, Shaun M. Christie

Williams Honors College, Honors Research Projects

SMRT/HDAC Associated Repressor Protein interacts with the long noncoding RNA, produced by SRA, by binding at the RRMs. Three projects were done to prepare the truncated proteins for use in 2D and 3D NMR experiments. The first focuses on RRM 3 and its optimization during the purification process. The second focuses on RRM 2-4, which was found to be missing two alpha helices that may be important for protein stability. These helices can also interact with RRM 3 as well due to the tight association of RRMs 3 and 4. The two step PCR extension of RRM 2-4 was assumed …