Life Sciences Commons^™

Functional Analysis Of The Acetic Acid Resistance (Aar) Gene Cluster In Acetobacter Aceti Strain 1023, Elwood Mullins, T Joseph Kappock

Department of Biochemistry Faculty Publications

Vinegar production requires acetic acid bacteria that produce, tolerate, and conserve high levels of acetic acid. When ethanol is depleted, aerobic acetate overoxidation to carbon dioxide ensues. The resulting diauxic growth pattern has two logarithmic growth phases, the first associated with ethanol oxidation and the second associated with acetate overoxidation. The vinegar factory isolate Acetobacter aceti strain 1023 has a long intermediate stationary phase that persists at elevated acetic acid levels. Strain 1023 conserves acetic acid despite possessing a complete set of citric acid cycle (CAC) enzymes, including succinyl-CoA:acetate CoA-transferase (SCACT), the product of the acetic acid resistance (aar …

Study Of Coronavirus Protease Using Cfp-Yfp Fluorescent Assay, Caitlin E. Specht, Andrew Mesecar Ph.D., Katrina Molland Ph.D.

The Summer Undergraduate Research Fellowship (SURF) Symposium

Middle Eastern Respiratory Syndrome (MERS) is an emerging viral disease originating in the Arabian Peninsula with a current mortality rate of nearly fifty percent throughout Europe and Asia according to the World Health Organization. Characterization of this disease is being done to understand the basis of viral replication. One target for viral inhibition are replication proteases. Replication proteases are enzymes that cleave proteins specific to cell growth and reproduction that form the viral replicase complex making them an ideal target for viral replication inhibition. First, replication proteases were characterized using a fluorescence resonance energy transfer (FRET) construct by measuring the …

Cellular Uptake Mechanism Of Paclitaxel Nanocrystals, Iris K. Archer, Zhaohui Wang, Tonglei Li

The Summer Undergraduate Research Fellowship (SURF) Symposium

Therapeutic options for metastasized human cancer in current practice remain limited and, sadly, there is no cure for metastatic cancer. The typical approach, chemotherapy, has both low efficacy due to poor drug solubility, and cytotoxic side effects to healthy tissue when delivered indiscriminately. To address both of these issues, we are pursuing the use of nanocrystal formulations of current chemotherapeutic agents as delivery platforms. Herein, we have studied cellular uptake mechanisms in cancer cells of nanocrystals of a chemotherapeutic agent, paclitaxel. Our goal in this study is to determine whether the nanocrystals can be taken up via endocytosis, especially when …

Identification Of Set1 Target Genes, William Beyer, Scott D. Briggs

The Summer Undergraduate Research Fellowship (SURF) Symposium

The Set1 complex, a histone methyltransferase complex found in S. cerevisiae (budding yeast), is the only histone methyltransferase responsible for catalyzing methylation of histone H3 at Lysine 4. It possesses homologues in other species, humans included. While yeast only have the Set1 complex, the human homologues of the yeast Set1 complex include mixed-lineage leukemia family (MLL1-4), Set1 A, Set1 B, among others. MLL1-4 has been shown to play a role in transcription, cell type specification, and the development of leukemia. One application of characterizing the role of a protein is that the information gained can provide insight into the function …

Involvement Of Post-Transcriptional Histone Modifications In Chromosome Missegregation, Jessica N. Gabbard, Ann Kirchmaier

The Summer Undergraduate Research Fellowship (SURF) Symposium

Proper chromosome segregation during mitosis ensures the equal inheritance of parental DNA by two daughter cells. Errors in chromosome segregation result in aneuploidy, the inheritance of abnormal chromosome numbers. Aneuploidy is a characteristic of tumors cells; therefore, understanding the factors that cause chromosome missegregation will provide insight into carcinogenesis. Certain post-transcriptional histone modifications in centromeric and pericentromeric regions are associated with maintaining kinetochore integrity and ensuring proper chromosome segregation. However, whether loss or improper distribution of modifications directly or indirectly causes chromosome missegregation is yet to be determined. To compare the DNA content of mutated yeast strains relative to WT, …

Saccharomyces Cerevisiae Cdc7 Homology In Drosophila Melanogaster, Marcus R. Hosler, Robert E. Stephenson, Vikki M. Weake

The Summer Undergraduate Research Fellowship (SURF) Symposium

Saccharomyces cerevisiae Dbf4(Dumbbell former 4) and Cdc7(Cell Division Cycle 7) form a complex that phosphorylates Mcm2 (Minichromosome maintenance 2) to initiate DNA replication. Cdc7 is a target for cancer research because there is a Cdc7 ortholog in humans that is necessary for DNA replication and cell survival. Our goal is to characterise a putative Cdc7 homolog in Drosophila melanogaster (dCdc7). We have previously shown that expression of the known Drosophila Dbf4 ortholog, Chiffon, and dCdc7 can rescue yeast cells deficient in active Cdc7. Our hypothesis is that the dCdc7 is activated by Chiffon to phosphorylate MCM2. To test this hypothesis, …

Development Of A Metabolomic Method To Define The Phenylalanome In Arabidopsis Thaliana, Cole G. Wunderlich, Clint Chapple, Xu Li

The Summer Undergraduate Research Fellowship (SURF) Symposium

In the study of metabolomics, one of the greatest challenges can be accurately identifying compounds detected in biological extracts, especially when standards are not readily available. Current metabolomic methods are also limited in that they provide little to no information about a compound’s metabolic origin. In this study, we sought to address these issues by developing a novel metabolomic method that employs stable isotope feeding, LC-MS, Xcms, and an analytical software algorithm to study the ‘phenylalanome’ of Arabidopsis thaliana. Using this approach we were able to develop a method that, based on current results, is capable of detecting over …

The Effects Of Exogenous Extracellular Matrix And Substrate Stiffness On Mouse Tendon Cells In Vitro, Caleb J. Mcdaniel, Sarah Calve

The Summer Undergraduate Research Fellowship (SURF) Symposium

To improve the treatment of musculoskeletal injuries, a better understanding of the transitional environment in which progenitor cells form mature musculoskeletal constructs is necessary. This need arises because injury repair requires restructuring of tissue, similar to the initial tissue construction that occurs during embryonic development by progenitor cells. Differences in both the biochemical and mechanical environments between a transitional and a differentiated state are known to take place, but how these differences affect cell behavior had not yet been characterized in mammalian tendon cells. In order to investigate this, we have determined the effects of exogenous extracellular matrix and the …

Inkjet Printing Of Polarized Yeast Cells, Xiuyuan Yang, Kari Clase

The Summer Undergraduate Research Fellowship (SURF) Symposium

The motivation is to applying engineering knowledge to develop 3D bio-printing in inkjet printer (first stage--monolayer). To achieve the goal, there are three problems to solve. First, we have to figure out regulation of growth of target cells; inability to regulate the location and pattern of growing cells make us even unable to build 3D printer in the direct way. Second problem is how to protect of yeast cells from high temperature and viscous force when printing. The third issue is how to modify the inkjet printer especially the feeding system in order to implement printing on other materials rather …

Structural And Functional Characterization Of The Endosome-Associated Deubiquitinating Enzyme Amsh, Christopher Williamson Davies

Open Access Dissertations

The endosomal sorting complexes required for transport (ESCRT) machinery is a ubiquitin-dependent molecular mechanism made of up of four individual complexes: ESCRT-0, -I, -II, III, that is necessary for regulating the degradation of cell surface receptors directed towards the lysosome. Not only are the ESCRTs implicated in endosomal sorting and trafficking of proteins, its members also have roles in other important biological processes such as: cytokinesis, HIV budding, transcriptional regulation, and autophagy. As a function of its involvement in several processes throughout the cell, the ESCRT machinery is implicated in a wide variety of diseases including cancer, neurological disease, bacterial …

Characterization Of The Specificity And Functions Of The Protein Phosphatase Cdc14, Christie Eissler

Open Access Dissertations

Protein phosphorylation is perhaps the most ubiquitous posttranslational modification in eukaryotes and recent studies suggest that upwards of 75% of human proteins are phosphorylated. Many proteins are phosphorylated at multiple sites, often controlled by multiple kinases and phosphatases. Multisite phosphorylation can differentially affect the functional and regulatory cellular outcomes. For example, dephosphorylation of a protein at a particular site may inhibit nuclear localization of a protein while dephosphorylation of a different site may be necessary for enzymatic activation of a protein. Thus, multisite protein phosphorylation can complicate our understanding of the biological significance and the functional consequences of protein phosphorylation. …

Effects Of Dna Methylation Pattern On The Chromatin Structure, Isabel Cristina Jimenez Useche

Open Access Dissertations

DNA methylation plays an essential role in various biological processes such as stem cell differentiation, imprinting, X-chromosome inactivation, etc. Increased DNA methylation levels have been associated with chromatin compaction leading to gene silencing. For example, abnormal DNA methylation is associated with silencing of tumor suppressor genes and is observed in the onset of tumorigenesis. There is evidence suggesting that not all methylation events are relevant in chromatin compaction and the initiation of cancer. It seems that methylation at certain locations of the DNA might be key to start chromatin compaction and gene silencing, but the location of this methylation sites …

The Termite Digestome: Understanding The Digestive Physiology Involved In Lignocellulosic Biomass Degradation, Zachary John Karl

Open Access Dissertations

The purpose of this research was to advance the understanding of lower termite digestive physiology and discover potential biocatalysts that can aid in the degradation of lignocellulosic biomass. Various protein characterization and gene expression methods were used throughout this research in order to accomplish these objectives. The results of this dissertation indicate that: 1) termites and their symbionts act in a synergistic manner to degrade biomass in vitro, 2) the host fraction of the gut (i.e., foregut and midgut) is the likely site of glucose absorption, 3) the termite and its symbionts contribute specific enzymes to the digestive process, 4) …

Influence Of Cholesterol And Bilayer Asymmetry On Membrane Protein Distribution In Polymer-Tethered Raft-Mimicking Lipid Membranes, Noor Fueza Hussain

Open Access Dissertations

It is now widely recognized that lipid rafts, which are membrane domains enriched in cholesterol (CHOL) and sphingolipids (SL), play a significant functional role in the plasma membrane. Raft domains particularly affect membrane functionality by causing sequestering of membrane proteins. Underlying mechanisms of raft-associated membrane protein sequestration remain elusive, due to the complexity, transient nature, and small size of raft domains in cellular membranes. To address these challenges, this dissertation unveils the relationship between lipid raft composition and membrane protein sequestration and function using raft-mimicking model membrane mixtures comprised of coexisting liquid-ordered (lo) and liquid-disordered (ld) domains with reconstituted membrane …

Design, Synthesis, And Evaluation Of Molecular Inhibitors For Biologically Relevant Enzymes, Sarah Emma St. John

Open Access Dissertations

The work in this thesis details the design, synthesis, and biological evaluation of molecular inhibitors for the inhibition of biologically relevant enzymes. The first three chapters of this thesis concern the polyphenol resveratrol and its inhibition of the quinone reductase 2 (QR2) enzyme. The work on this subject resulted in the complete design, synthesis, biological and structural evaluation of a second generation library of resveratrol analogues. From this work we identified a novel resveratrol analogue that inhibits QR2 in a previously unknown binding orientation. The fourth chapter of this thesis details the de novo design of molecules for the inhibition …

Genetic Engineering Of Black Cherry (Prunus Serotina) For Reproductive Sterility And Insect Pest Resistance, Ying Wang

Open Access Dissertations

Black cherry (Prunus serotina Ehrh.) is one of the most valuable hardwoods for high- end cabinetry, furniture, architectural millwork, paneling, and veneer. However, the damage caused by cambial-mining insect pests triggers gummosis in black cherry, a non-specific defense response in which resinous gum is deposited at the site of injury. The gum defects dramatically decrease the yield of high-quality black cherry lumber, and the value can be reduced by as much as 90%. The goal of this project was to optimize the transformation and in vitro rooting system, and to develop transgenic black cherry for reproductive sterility and insect pest …

Gas-Phase Covalent And Non-Covalent Ion/Ion Chemistry Of Biological Macromolecules, John Robert Stutzman

Open Access Dissertations

Gas-phase ion/ion chemistry involves the interaction of oppositely charged ions inside of the mass spectrometer. During this gas-phase chemistry, particle transfer (i.e., proton and electron) or synthesis can occur at rapid reaction rates. Particle transfer represents a mature area of ion/ion chemistry, while selective covalent modification represents a fairly new area of gas-phase chemistry. Gas-phase covalent chemistry is based on traditional solution phase organic chemistry.

The work demonstrated in this dissertation greatly involves gas-phase covalent and non-covalent Schiff base chemistry on peptide and protein ions. The reagent dianion, 4-formyl 1,3-benzene disulfonic acid, has been used to covalently modify unprotonated primary …

Measuring And Modeling The Response Characteristics Of The Environmental Phosphate Transducer In Escherichia Coli, Chetan Sood

Open Access Dissertations

The PhoR/PhoB two-component system in Escherichia coli is a biological transducer that senses the limitation of environmental inorganic orthophosphate, the bacteria's preferred source of the essential nutrient phosphate, and transmits that information to the interior of the cell initiating a response that mitigates phosphate starvation. In the first part of this study, we present and apply a fluorescence microscopy technique to measure, in vivo, the dynamic response characteristics of the transducer with single-cell resolution. We report that the transience in the PhoR/PhoB TCS response is consistent with the transducer having a threshold sensitivity to the concentration of environmental phosphate, …

Biophysical Studies Of Cholesterol In Unsaturated Phospholipid Model Membranes, Justin Adam Williams

Open Access Dissertations

Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distributed to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. This research employs model membranes of well-defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega-3 (n-3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by …

Development Of Isotags For Nmr Based Metabolite Profiling And Applications, Fariba Tayyari

Open Access Dissertations

NMR spectroscopy is a powerful analytical tool for both qualitative and quantitative metabolite profiling analysis. However, accurate quantitative analysis of biological systems especially using one dimensional NMR has been challenging due to signal overlap. In contrast, the enhanced resolution and sensitivity offered by chemoselective isotope tags have enabled new and enhanced methods for detecting hundreds of quantifiable metabolites in biofluids using NMR spectroscopy or mass spectrometry. In this thesis we show improved sensitivity and resolution of NMR experiments imparted by 15N and 13C isotope tagging which enables the accurate analysis of plasma metabolites. To date, isotope tagging has been used …

Synthesis Of Novel Isoprenoid Diphosphate Analogs As Chemical Tools To Investigate Protein Geranylgeranylation, Kayla Jo Temple

Open Access Dissertations

Many proteins require prenylation in order to be biologically functional. Some such proteins include the small Ras and Rho GTPase superfamilies, nuclear lamins A and B, and the kinesin motor proteins CENP-E and F. Prenyltransferase (PTase) inhibition is currently being explored as a possible treatment not only for cancer but for a wide variety of other diseases.

Clinical studies revealed that the effectiveness of farnesyltransferase inhibitors (FTIs) to treat Ras-dependent tumors is determined by which isoform of Ras is overactive. Unfortunately the majority of Ras-dependent tumors have a mutation in either the N- or K-Ras isoforms; both of these isoforms …

Characterization Of Caxck31, A Bacterial Calcium/Proton Antiporter, Marc Robert Ridilla

Open Access Dissertations

To better understand a class of transporters known as Calcium/Cation Antiporters (CaCAs), the bacterial calcium/proton antiporter CAXCK31 was purified and characterized. New methods were developed for its heterologous overexpression and purification. These methods help to define stress responses to toxic membrane overproduction in E. coli and may be broadly applicable to studies of membrane proteins. The results from a variety of biochemical and biophysical experiments demonstrated that CAXCK31 exists as a dimer in the membrane and can be purified in the dimeric state. The methods used include chemical cross-linking, FRET, and SEC-MALS. In addition, various transport properties of CAXCK31, including …

Structural Studies On The Rubella Virus Capsid Protein And Its Organization In The Virion, Vidya Mangala Prasad

Open Access Dissertations

Rubella virus is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, rubella infection leads to severe damage in fetuses. Despite its medical importance, very little is known about the structure of the pleomorphic rubella virus as compared to its alphavirus relatives. The rubella capsid protein is a critical structural component of virions as well as a key factor in virus-host interactions. Three crystal structures of the structural domain of the rubella capsid protein have been described here. The polypeptide fold of the capsid protomer has not been observed previously. The capsid protein structure, along …

Development Of Tyrosine Kinase Peptide Biosensors And Methods For Detection, Andrew Michael Lipchik

Open Access Dissertations

New methods to monitor tyrosine kinase activity are critical for studying kinases in cell biology, drug discovery and the clinic. Peptide-based biosensors for detection of kinase activity utilitize a kinase specific artificial peptide substrate, which can report intercellular kinase activity through the incorporation of phosphate.

An artificial Syk substrate peptide was developed and incorporated with other functional modules to produce a Syk biosensor. These modules included a biotin-tag for affinity capture, a photo-cleavable amino acid to allow release of the substrate from the delivery module and the cell penetrating peptides TAT. A live cell kinase assay utilizing this biosensor was …

Generation And Statistical Modeling Of Active Protein Chimeras: A Sequence Based Approach, Nicholas Fico

Open Access Dissertations

Generation of active protein chimeras is a valuable tool to probe the functional space of proteins. Statistical modeling is the next logical step, allowing us to build a model of gene fragment replaceability between species. In this thesis I begin to develop the statistical tools that are needed to systematically describe combinatorial protein libraries. I present three sets of diverse chimeric protein libraries developed using sequence information. The statistical model of the human N-Ras and human K-Ras-4B genes reveal a set previously unidetifed surface residues on the N-Ras G-Domain that may be involved in cellular localization. Statistical modeling of a …

An Analysis Of The Impact Of Storage Temperature, Moisture Content & Duration Upon The Chemical Components & Bioprocessing Of Lignocellulosic Biomass, Arun Athmanathan

Open Access Dissertations

The successful utilization of lignocellulosic biomass as a feedstock for fuels and chemicals necessitates storage for 2-6 months. It is correspondingly important to understand the impact of storage parameters - moisture concentration, temperature and duration - on biomass quality.

As aerobic storage is the most viable large-scale solution, aerobic storage experiments were carried out with three projected bioenergy feedstocks - sweet sorghum (Sorghum bicolor) bagasse, corn (Zea mays) stover and switchgrass (Panicum virgatum). Stored samples of each were examined for dry matter loss and composition change to develop a material balance around carbohydrates and lignin.

A mean dry matter loss …

Biochemistry / Histone Protein Modification - Purdue University, Jake R. Carlson, Katherine Beavis

Data Curation Profiles Directory

The researcher is conducting research on the brain cells of drosophila larvae. Specifically, the goal is to sort nuclei from glial and neuronal cells from the brains of mutant and wild type drosophila larvae. She will do transcriptome and high throughput ChIP-seq analysis on the cell nuclei to examine the distribution of complexes that modify histones and how loss of those complexes affects transcription. The stages of her research involve a long period of methodology development, a shorter data collection period, and then data analysis. The lab notebook contains written documentation of all experiments and trials and links to data, …

Function And X-Ray Crystal Structure Of Escherichia Coli Yfde, Elwood Mullins, Kelly L. Sullivan, T. Joseph Kappock

Department of Biochemistry Faculty Publications

Many food plants accumulate oxalate, which humans absorb but do not metabolize, leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by converting it to oxalyl- CoA, which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl- CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria, including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT), YfdU (OXC), and YfdE, a class III CoA-transferase that is ~ 30% identical to YfdW. YfdW and …

Interaction Between Centromeric Histone H3 Variant And Shugoshin, Visarut Buranasudja

Open Access Theses

Precise and faithful segregation of chromosome segregation during mitosis depends on the ability of the cell to regulate chromosome bi-orientation on the mitotic spindle. Shugoshin (Sgo1), the protector of meiotic centromeric cohesin, is required for proper establishment of chromosome bi-orientation. Sgo1 plays a crucial role as part of a mitotic tension sensor between sister chromatids. Recently, Sgo1 has been reported to interact with histone H3 at the pericentromere region, as an important factor for tension sensing and chromosome segregation. However, the role of Sgo1 in tension sensing at centromere is still elusive. The centromere is the region of attachment of …

Interaction Between Maltose Binding Protein And Escherichia Coli Maltose Transporter, Yan Huang

Open Access Theses

The ATP-binding cassette (ABC) transporter superfamily is one of the largest families of transport proteins. The ABC transporters are responsible for selective permeability of solute across membranes energized by ATP hydrolysis, which occurs in all domains of life. Maltose transporter is an ABC importer that mediates maltose/maltodextrin uptake in bacteria and archaea. It is identified as an essential virulence factor in pathogenic species of Streptococcis pyogenes and Vibrio cholera (1, 2). Escherichia coli maltose transporter is a well-characterized system with crystal structures and exclusive biochemical studies available. Knowledge of the E. coli maltose transport mechanism will lead to a better …