Life Sciences Commons^™

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 …

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 …

Development Of A Starch-Based Mussel-Mimetic Adhesive Polymer, Jeffrey Kazimir De Kozlowski

Open Access Theses

Mussel-mimetic adhesive polymers have gained lots of attention for their strong adhesive strength, moisture resistance, and unique ability to crosslink. These properties are mainly attributed to the high content of catecholic 3,4-dihydroxyphenylalanine (DOPA) in mussel adhesive proteins. While there has been success in creating mussel-mimetic synthetic polymers, less effort has been given to create a renewable, green, biocompatible counterpart. This thesis explores the possibilities of starch-based mussel-mimetic adhesives. Carboxymethyl starch of various molecular weights and degree of substitution was synthesized and subsequent conjugation of dopamine to these polymers by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was investigated. The polymers suffered from very low substitution (DScatechol …

Biology And Ecology Of Glyphosate-Resistant Giant Ragweed, Kabelo Segobye

Open Access Theses

Giant ragweed (Ambrosia trifida L.) is a competitive annual plant found in disturbed landscapes and is the most troublesome weed in Indiana and the US Corn Belt. It is one of the most common and problematic weeds in corn and soybean production. The introduction of herbicide glyphosate, N-(phosphonomethyl) glycine in early 1970's provided farmers with a better and low-cost tool to control weeds. The use of glyphosate drastically increased after the development of glyphosate resistant agronomic crops in 1996 and was use as a post-emergence selective herbicide. This led to overreliance and repeated use of glyphosate for weed control especially …