Physics Commons^™

Biomolecular Function From Structural Snapshots, Roshanak Etemadpour

Theses and Dissertations

Biological molecules can assume a continuous range of conformations during function. Near equilibrium, the Boltzmann relation connects a particular conformation's free energy to the conformation's occupation probability, thus giving rise to one or more energy landscapes. Biomolecular function proceeds along minimum-energy pathways on such landscapes. Consequently, a comprehensive understanding of biomolecular function often involves the determination of the free-energy landscapes and the identification of functionally relevant minimum-energy conformational paths on these landscapes. Specific techniques are necessary to determine continuous conformational spectra and identify functionally relevant conformational trajectories from a collection of raw single-particle snapshots from, e.g. cryogenic electron microscopy (cryo-EM) …

Investigation Of G Protein-Coupled Receptor Quaternary Structure Through Fluorescence Micro-Spectroscopy And Theoretical Modeling: Interdependence Between Receptor-Receptor And Receptor-Ligand Interactions, Joel David Paprocki

Theses and Dissertations

Proteins are of high interest in biophysics research due to the important roles they play within cells, such as sensing of chemical (ions and small molecules) and physical (e.g., light) stimuli, providing structure, transporting ions/molecules, signaling, and intercellular communication. The studies described in this dissertation focus on a particular type of membrane proteins known as G protein-coupled receptors (GPCR), which play a key role in cellular response to external stimuli. We used the sterile 2 α-factor mating pheromone receptor (Ste2), a prototypical class D GPCR present within Saccharomyces cerevisiae (baker’s yeast). Ste2 is responsible for initiating the second messenger signal …

Nebulizer-Based Systems To Improve Pharmaceutical Aerosol Delivery To The Lungs, Benjamin M. Spence

Theses and Dissertations

Combining vibrating mesh nebulizers with additional new technologies leads to substantial improvements in pharmaceutical aerosol delivery to the lungs across therapeutic administration methods. In this dissertation, streamlined components, aerosol administration synchronization, and/or Excipient Enhanced Growth (EEG) technologies were utilized to develop and test several novel devices and aerosol delivery systems. The first focus of this work was to improve the poor delivery efficiency, e.g., 3.6% of nominal dose (Dugernier et al. 2017), of aerosolized medication administration to adult human subjects concurrent with high flow nasal cannula (HFNC) therapy, a form of continuous-flow non-invasive ventilation (NIV). The developed Low-Volume Mixer-Heater (LVMH) …

Macromolecular Structure Determination At X-Ray Free Electron Lasers From Single-Particle Imaging To Time-Resolved X-Ray Crystallography, Ishwor Poudyal

Theses and Dissertations

X-ray free-electron lasers (XFELs) open the possibility of obtaining diffraction information from a single biological macromolecule. This is because XFELs can generate extremely intense X-ray pulses which are so short that diffraction data can be collected before the sample is destroyed. By collecting a sufficient number of single-particle diffraction patterns from many tilts of a molecule relative to the X-ray beam, the three-dimensional electron density can be reconstructed ab-initio. The resolution and therefore the information content of the data will ultimately depend largely on the number of patterns collected at the experiment. We estimate the number of diffraction patterns required …

Snow-Albedo Feedback In Northern Alaska: How Vegetation Influences Snowmelt, Lucas C. Reckhaus

Theses and Dissertations

This paper investigates how the snow-albedo feedback mechanism of the arctic is changing in response to rising climate temperatures. Specifically, the interplay of vegetation and snowmelt, and how these two variables can be correlated. This has the potential to refine climate modelling of the spring transition season. Research was conducted at the ecoregion scale in northern Alaska from 2000 to 2020. Each ecoregion is defined by distinct topographic and ecological conditions, allowing for meaningful contrast between the patterns of spring albedo transition across surface conditions and vegetation types. The five most northerly ecoregions of Alaska are chosen as they encompass …

Time-Resolved Structural Enzymology At X-Ray Free Electron Lasers, Tyler Norwood

Theses and Dissertations

Within the last decade, X-ray Free Electron Lasers (XFELs) have emerged across the world. These XFELs produce X-ray pulses with a duration on the order of femtoseconds, each of which contains 1012 photons. Before the XFEL, the brightest X-ray sources were 3rd generation synchrotrons. While these facilities are still very important for many experiments, XFELs allow for time-resolved experiments with femtosecond time resolution and mixing experiments that are not possible at the synchrotron. Enzymatic processes have promising prospects for medicine which use proteins as drug targets and enhance our understanding of these important biomolecules.

A number of procedures are involved …

Characterization Of The Effects Of Heavy Charged Particle Exposure On The Radiation Resistant Bacterium Deinococcus Radiodurans, Todd A. Bryant

Theses and Dissertations

Although not an extremophile, Deinococcus radiodurans has proven to have exceptional resistance to ionizing radiation, specifically via gamma and X-ray photons. To date, no known experiments have bombarded the bacterium with charged particles larger than hydrogen. This study explored the effects on the organism’s ability to survive high linear energy transfer heavy-charged particle exposures of oxygen ions. It also investigated the effects of low linear energy transfer ultraviolet radiation on various mutants. Two uvrB mutants were created to ascertain the importance of the gene in single-strand break repair following gamma irradiation and two recF mutants were created to explore the …

Characterization Of Silicon Ion Exposure On Deinococcus Radiodurans, Richard F. Daughtry

Theses and Dissertations

Deinococcus radiodurans is a robust bacterium that is known for its extraordinary resistance to ionizing radiation. In general, many of the investigations of this bacterium's resistance have revolved around low linear energy transfer radiation, such as gamma and electron radiation. This study explored Deinococcus radiodurans's ability to survive high linear energy transfer radiation, specifically proton and neutron radiation. Deinococcus radiodurans was dehydrated to reduce the effects of low linear energy transfer radiation. The bacteria were exposed to both neutron and proton radiation of varying amounts and rehydrated. The resulting colonies were counted and compared to colonies of non-irradiated control samples …

Evaluation And Adaptation Of Live-Cell Interferometry For Applications In Basic, Translational, And Clinical Research, Kevin A. Leslie

Theses and Dissertations

Cell mass is an important indicator of cell health and status. A diverse set of techniques have been developed to precisely measure the masses of single cells, with varying degrees of technical complexity and throughput. Here, the development of a non-invasive, label-free optical technique, termed Live-Cell Interferometry (LCI), is described. Several applications are presented, including an evaluation of LCI’s utility for assessing drug response heterogeneity in patient-derived melanoma lines and the measurement of CD3+ T cell kinetics during hematopoietic stem cell transplantation. The characterization of mast cells during degranulation, the measurement of viral reactivation kinetics in Kaposi’s Sarcoma, and drug …

Biomedical Applications Of Mid-Infrared Spectroscopic Imaging And Multivariate Data Analysis: Contribution To The Understanding Of Diabetes Pathogenesis, Ebrahim Aboualizadeh

Theses and Dissertations

Diabetic retinopathy (DR) is a microvascular complication of diabetes and a leading cause of adult vision loss. Although a great deal of progress has been made in ophthalmological examinations and clinical approaches to detect the signs of retinopathy in patients with diabetes, there still remain outstanding questions regarding the molecular and biochemical changes involved. To discover the biochemical mechanisms underlying the development and progression of changes in the retina as a result of diabetes, a more comprehensive understanding of the bio-molecular processes, in individual retinal cells subjected to hyperglycemia, is required. Animal models provide a suitable resource for temporal detection …

Investigation Of Membrane Receptors’ Oligomers Using Fluorescence Resonance Energy Transfer And Multiphoton Microscopy In Living Cells, Ashish K. Mishra

Theses and Dissertations

Investigating quaternary structure (oligomerization) of macromolecules (such as proteins and nucleic acids) in living systems (in vivo) has been a great challenge in biophysics, due to molecular diffusion, fluctuations in several biochemical parameters such as pH, quenching of fluorescence by oxygen (when fluorescence methods are used), etc.

We studied oligomerization of membrane receptors in living cells by means of Fluorescence (Förster) Resonance Energy Transfer (FRET) using fluorescent markers and two photon excitation fluorescence micro-spectroscopy. Using suitable FRET models, we determined the stoichiometry and quaternary structure of various macromolecular complexes. The proteins of interest for this work are : (1) sigma-1 …

Symmetry And Reconstruction Of Particle Structure From Random Angle Diffraction Patterns, Sandi Wibowo

Theses and Dissertations

The problem of determining the structure of a biomolecule, when all the evidence from experiment consists of individual diffraction patterns from random particle orientations, is the central theoretical problem with an XFEL. One of the methods proposed is a calculation over all measured diffraction patterns of the average angular correlations between pairs of points on the diffraction patterns. It is possible to construct from these a matrix B characterized by angular momentum quantum number l, and whose elements are characterized by radii q and q’ of the resolution shells. If matrix B is considered as dot product of vectors, which …

Theoretical Investigation Of Interactions And Relaxation In Biological Macromolecules, Koki Yokoi

Theses and Dissertations

One of the major challenges posed to our quantitative understanding of structure, dynamics, and function of biological macromolecules has been the high level of complexity of biological structures. In the present work, we studied interactions between G protein-coupled receptors (GPCRs), and also introduced a theoretical model of relaxation in complex systems, in order to help understand interactions and relaxation in biological macromolecules.

GPCRs are the largest and most diverse family of membrane receptors that play key roles in mediating signal transduction between outside and inside of a cell. Oligomerization of GPCRs and its possible role in function and signaling currently …

Time-Resolved, Near Atomic Resolution Structural Studies At The Free Electron Laser, Jason James Tenboer

Theses and Dissertations

Time-resolved serial femtosecond crystallography (TR-SFX) employs X-ray free electron lasers (XFELs) to provide X-ray pulses of femtosecond (fs) duration with 1012 photons per pulse. These XFELs are more than a billion times more brilliant than 3rd generation synchrotron X-ray sources. For structure determination, protein crystals on the micrometer length scale (microcrystals) are injected into the X-ray beam and the resulting diffraction patterns are recorded on fast-readout pixel detectors. Although these intense pulses deposit enough energy to ultimately destroy the protein, the processes that lead to diffraction occur before the crystal is destroyed. This so-called diffraction-before-destruction principle overcomes radiation damage, which …

Oligomerization Of The Sterile-2 G-Protein Coupled Receptor In Yeast Cells In The Presence And Absence Of Alpha-Factor Pheromone Using Fluorescence Spectroscopy And Forster Resonance Energy Transfer Analysis, Joel David Paprocki

Theses and Dissertations

G-protein-coupled receptors (GPCRs) are the largest family of receptors that respond to a wide variety of extracellular stimuli, including molecular ligands such as odorants, neurotransmitters, and hormones, as well as physical agents sigh as light and pressure. The stimulation event results in initiating conformational changes in the structure of the receptor, which further results in the release of the heterotrimeric G-protein; the latter has a variety of functions within signaling pathways in cellular biology. The GPCR explored in this investigation is the Sterile 2 α-factor receptor (Ste2), whose natural function is that of a yeast mating pheromone receptor. Its natural …

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 …

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 …

Investigation Of The Quaternary Structure Of An Abc Transporter In Living Cells Using Spectrally Resolved Resonance Energy Transfer, Deo R. Singh

Theses and Dissertations

Förster resonance energy transfer (FRET) has become an important tool to study proteins inside living cells. It has been used to explore membrane protein folding and dynamics, determine stoichiometry and geometry of protein complexes, and measure the distance between two molecules. In this dissertation, we use a method based on FRET and optical micro-spectroscopy (OptiMiS) technology, developed in our lab, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of an ABC transporter in living cells. Specifically, the transporter we investigate originates …

In Vitro Toxicity Of Silver Nanoparticles In Human Lung Epithelial Cells, Christina R. Kearns

Theses and Dissertations

Nanotechnology is quickly becoming incorporated into everyday products and uses. Silver nanoparticles, specifically, are being used in commercial products, to include aerosols. The purpose of this research was to determine whether silver nanoparticles are toxic to human lung epithelial cells. Different types (coated vs. uncoated), concentrations (10, 50, 100, and 200 µg/mL) and sizes (coated 5 and 80nm, uncoated 10 and 80nm) of silver nanoparticles were used during this study. Toxicity measurements were completed through in vitro techniques. Another study was also completed on toxicity mechanisms by measuring the reactive oxygen species (ROS) generated. Results showed that silver nanoparticles induce …