Physical Sciences and Mathematics Commons^™

Structure And Function Of Proteins Investigated By Crystallographic And Spectroscopic Time-Resolved Methods, Namrta Purwar

Theses and Dissertations

Biomolecules play an essential role in performing the necessary functions for life. The goal of this thesis is to contribute to an understanding of how biological systems work on the molecular level. We used two biological systems, beef liver catalase (BLC) and photoactive yellow protein (PYP). BLC is a metalloprotein that protects living cells from the harmful effects of reactive oxygen species by converting H2O2 into water and oxygen. By binding nitric oxide (NO) to the catalase, a complex was generated that mimics the Cat-H2O2 adduct, a crucial intermediate in the reaction promoted by the catalase. The Cat-NO complex is …

Transcending Microbial Source Tracking Techniques Across Geographic Borders: An Examination Of Human And Animal Microbiomes And The Integration Of Molecular Approaches In Pathogen Surveillance In Brazil And The United States, Amber Mae Koskey

Theses and Dissertations

Waterborne illnesses, attributed to the ingestion or contact with contaminated water, present a significant global health concern. Surface water sources can be impacted by wide array of pollution inputs, but fecal pollution generates the most significant and acute threat to human health. Therefore, the detection of fecal bacteria in surface water sources remains an important public health objective. Current surface water monitoring employs the use of fecal indicator bacteria (FIB) including E. coli and enterococci as proxies for pathogenic organisms carried in fecal pollution. These traditional indicators, detected by culture-based microbiological methods, do not discriminate fecal sources from another. New …

Evaluation Of Vdr-Coactivator Inhibitors Using Biochemical And Cell-Based Assays, Athena Marie Baranowski

Theses and Dissertations

ABSTRACT

EVALUATION OF VDR–COACTIVATOR INHIBITORS USING BIOCHEMICAL AND CELL–BASED ASSAYS

by

Athena Baranowski

The University of Wisconsin–Milwaukee, 2013

Under the Supervision of Dr. Alexander Arnold

The vitamin D receptor (VDR) is a ligand–dependent transcription factor, which belongs to the nuclear receptor superfamily. VDR–mediated gene regulation is governed by coregulators (coactivators and corepressors). VDR coregulator binding inhibitors (CBIs), which were discovered using high throughput screening (HTS), were evaluated using cell–based assays and biochemical assays to determine their ability to inhibit the interaction between VDR and steroid receptor coactivator–2 (SRC–2). Determining their ability to inhibit the VDR–SRC–2 interaction can lead to the …

Phylogeographic Inference Of Insular Mule Deer (Odocoileus Hemionus) Divergence In North America's Desert Southwest, Ona Alminas

Theses and Dissertations

Though mule deer (Odocoileus hemionus) persist in robust populations throughout most of their North American distribution, nearly 60% of their historic range in México has declined due to habitat loss and unregulated hunting. Two of the six subspecies inhabiting México's deserts and Baja California peninsula are of conservation concern, occurring on land bridge islands in the Pacific Ocean (O. h. cerrosensis on Cedros Island: threatened) and in the Sea of Cortés (O. h. sheldoni on Tiburón Island: endangered). Focusing on the desert southwest (n=449 deer), we obtained 1,611 bp of mtDNA sequence (control region: 583 bp; cytochrome b gene: 1,028 …

Protein Association In Living Cells Using Fret Spectrometry: Application To G-Protein Coupled Receptors, Suparna Patowary

Theses and Dissertations

Recent advancements in fluorescence microscopy coupled with newly developed fluorescent tags have transformed Fluorescence (Förster) Resonance Energy Transfer (FRET) into a powerful tool studying in vivo molecular interactions with improved spatial (angstrom) resolution. Though widely used to study protein-protein interactions, generalizing and testing the FRET theory for oligomeric complexes containing multiple donors and acceptors has only become possible in recent years. Therefore, many aspects of it are yet unexplored.

In this work, we tested the kinetic theory of FRET using linked fluorescent proteins located in the cytoplasm or at the plasma membrane. We used a novel method developed in our …

Mathematical Modeling Of Physiological Characteristics In Female Soccer Athletes, Thomas S. Goeppinger

Theses and Dissertations

Intermittent sports create challenges regarding performance measurement. Quantification of various physiological characteristics can lead to increased performance and injury reduction throughout a season of competition. Currently, a variation of an athletes' heart rate is the primary physiological characteristic used for quantifying load on the athlete. With increasing technology, we have the ability to gather additional characteristics regarding the physicality of athletes during competition. This study statistically compares various models using these new characteristics as predictors to the athletes' lactate concentration in their blood. From this comparison, we determine which physiological characteristic(s) best represent the performance and fatigue of these athletes. …

Phytoplankton Life History Events: Resting Stages And Physiological Cell Death, Christine R. Kozik

Theses and Dissertations

Understanding and predicting changes in phytoplankton populations requires knowledge of key life history processes such as recruitment from benthic resting stages and losses due to sedimentation and cell death. Currently, these processes are poorly understood in freshwater systems. Phytoplankton resting stage and cell death life history events were separately examined in two freshwater systems in Wisconsin, four northern lakes and an urban pond. In the norther lakes, sedimentation and benthic recruitment were examined using sediment and recruitment traps that were sampled weekly over two summers. Sedimentation and benthic recruitment contributed little to changes in standing crop chl a, but rather …

Zinc Chemical Biology: The Pursuit Of The Intracellular Targets Of Zinquin, Andrew Nowakowski

Theses and Dissertations

Fluorescent sensors have been a main microscopic tools used to understand Zn2+ physiology on a cellular level. The use of the fluorescent Zn2+ sensor Zinquin (ZQ) and its analogues have revealed that transient Zn2+ is a chief component in a variety of biochemical pathways. Yet, little work has been performed to validate the exact targets of Zinquin in a cellular environment. The goals of this investigation are to determine the types of Zinquin reactions that take place in the cell as well as the identities of its cellular targets.

It has been hypothesized that Zinquin reacts with free Zn2+ within …

Nearshore Benthic Oxygen Dynamics In Lake Michigan, Emily H. Tyner

Theses and Dissertations

The intense colonization of the Laurentian Great Lakes by dreissenid mussels has profoundly changed ecosystem processes, particularly benthic oxygen dynamics. Dissolved oxygen concentrations in mussel beds, sloughed Cladophora mats, and sediment indicate that hypoxia forms and disappears in some substrata (ephemeral Cladophora mats), while occurring consistently in others (depositional areas of sloughed Cladophora). Dissolved organic carbon concentrations are high (mean: 143 ± ± 28 ppm) in depositional Cladophora mats but lower (˂ 10 ppm) in most other environments. Field sampling and laboratory experiments suggest that under conditions of low water velocity and thick Cladophora cover, hypoxia may develop atop …

Towards The Use Of Time-Resolved X-Ray Crystallography In Mechanistic Studies Of Cytochrome C Nitrite Reductase From Shewanella Oneidensis, Matthew David Youngblut

Theses and Dissertations

A high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "Small Tetra-heme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-C strain, generated approximately 20 mg crude ccNiR/L culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR exhibited nitrite and hydroxylamine reductase activities comparable to those of E. …

High-Throughput Approaches For The Assessment Of Factors Influencing Bioavailability Of Small Molecules In Pre-Clinical Drug Development, Megan Marie Mccallum

Theses and Dissertations

A bioactive molecule must pass many hurdles to be designated as a "good" pharmaceutical lead or hit compound. It should have a significant activity, selectivity, bioavailability, and metabolic half-life. Many factors have been identified that influence the free drug concentration or bioavailability of orally administered drugs in the earliest development stages. In vitro pre-clinical assays have been developed to measure these parameters. The small molecule properties that are investigated here include aqueous solubility, permeability, reactivity (electrophilicity), small molecule-protein binding, and displacement of protein-bound molecules (drug-drug interactions). The development of rapid and miniaturized assays to quantify these factors is presented herein. …

Methods In Metallomics, Proteomics, And Toxicology: Development And Applications Of Laser Ablation Inductively Coupled Plasma Mass Spectrometry And Native Sds-Page, William John Wobig

Theses and Dissertations

Polyacrylamide gel electrophoresis (PAGE) is a bio-analytical method used to separate proteins in solution into an array of individual bands of proteins in a gel matrix. Current PAGE methods, however, have severe limitations in simultaneously maintaining a protein's native structure and association with transition metals while providing adequate resolution. Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) provides a means to perform trace to ultra-trace level inorganic analysis of solid samples such as dried PAGE gels containing metallo-protein arrays. Current LA-ICP-MS methods involving the analysis of PAGE gels, however, have been limited in their effective use by inadequate limits of …

The Shifting Importance Of Competition And Facilitation Along Diversity, Environmental Severity, And Plant Ontogenetic Gradients, Alexandra Wright

Theses and Dissertations

Ecological theory and empirical studies have focused heavily on the importance of competition in plant communities. Competition can help explain species coexistence, the maintenance of species diversity, and biological invasions. Competition for resources appears to be ubiquitous among coexisting organisms. This overwhelming focus on competition over the past one hundred years may have overshadowed the importance of positive interactions (facilitation). Growing near your neighbors involves competition for resources, but it also involves alteration of a shared microclimate. Neighboring plants have the capacity to increase shade, decrease air temperatures, increase humidity, and increase shallow soil moisture in their local environment. In …