Life Sciences Commons^™

Characterization Of Β-2-Microglobulin Pre-Amyloid Oligomers And Their Role In Amyloid Inhibition, Tyler M. Marcinko

Doctoral Dissertations

In dialysis patients, β-2 microglobulin (β2m) can aggregate and eventually form amyloid fibrils in a condition known as dialysis-related amyloidosis, which deleteriously affects joint, bone, and organ function, and eventually causes organ failure. To understand the early stages of the amyloid assembly process, we have employed a series of biophysical tools including chromatography, spectroscopy, and most especially, native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble pre-amyloid oligomeric species. We have also collaborated and integrated computational modeling to help better understand and rationalize the structural basis behind oligomerization. Recently, several small molecules have been identified …

Protein Detection And Structural Characterization By Mass Spectrometry Using Supramolecular Assemblies And Small Molecules, Bo Zhao

Doctoral Dissertations

Mass spectrometry (MS) has played an increasingly prominent role in proteomics and structure biology because it shows superior capabilities in identification, quantification and structural characterization of proteins. To realize its full potential in protein analysis, significant progress has been made in developing innovative techniques and reagents that can couple to MS detection. This dissertation demonstrates the use of polymeric supramolecular assemblies for enhanced protein detection in complex biological mixtures by MS. An amphiphilic random co-polymer scaffold is developed to form functional supramolecular assemblies for protein/ peptide enrichment. The influences of charge density and functional group pK_a on host-guest interactions …

Environmental Risk Factors For Inflammatory Bowel Disease: Triclosan And Other Consumer Antimicrobials, Katherine Z. Sanidad

Doctoral Dissertations

Inflammatory bowel disease (IBD) has become a serious health problem since the incidence and prevalence of IBD has dramatically increased throughout the world. There is evidence that environmental factors are primarily responsible for the increase of IBD, therefore, it is important to identify novel environmental risk factors to reduce the risk of IBD and its associated diseases. Antimicrobials used in consumer products might serve as environmental risk factors for IBD and its associated diseases. Triclosan (TCS), triclocarban (TCC), benzalkonium chloride (BAC), benzethonium chloride (BET), and chloroxylenol (PCMX) are widely used antimicrobial ingredients in consumer products and are ubiquitous contaminants in …

Rna-Seq And Mechanistic Enzymology Confirm Rna Self-Templated Extension By T7 Rna Polymerase And Suggest Novel Approaches Towards Improved In Vitro Rna Synthesis, Yasaman Gholamalipour

Doctoral Dissertations

Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyzed in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirmed that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. …

Mechanisms That Limit Oxidative Phosphorylation During High-Intensity Muscle Contractions In Vivo, Miles F. Bartlett

Doctoral Dissertations

Skeletal muscle oxidative capacity plays a critical role in human health and disease. Although current models of oxidative phosphorylation sufficiently describe skeletal muscle energetics during moderate-intensity contractions, much is still unknown about the mechanisms that control and limit oxidative phosphorylation during high-intensity contractions. In particular, the oxygen cost of force generation is augmented during exercise at workloads above the lactate threshold. Presently, it is unclear whether this augmentation in muscle oxygen consumption is driven by increased rates of oxidative ATP synthesis (ATP_OX) or by decreases in the efficiency of ATP_OX due to mitochondrial uncoupling. To address this …

Predicting Successful Chaperoning Of Fabry Disease Mutants Via Computation, Priyank Patel

Masters Theses

Fabry disease is an inherited X-linked recessive disorder caused by mutations in the galactosidase alpha (GLA) gene, leading to deficiencies in α-galactosidase A (α-GAL) enzyme production. α-GAL, a lysosomal glycosidase, catalyzes the removal of a terminal α-galactose; however, loss of α-GAL activity leads to accumulation of globotriaosylceramide (an endogenous substrate) and the eventual onset of the disease. Approved treatments for Fabry disease include enzyme replacement therapy and pharmacological chaperone therapy. In the latter treatment, 1-deoxygalactonojirimycin (DGJ), a pharmacological chaperone, is administered to Fabry disease patients, leading to increased enzymatic activity. The DGJ iminosugar acts as a competitive inhibitor …

The Effects Of Acidosis On Calcium Dependent Binding Of A Single Crossbridge, Matthew Unger

Masters Theses

Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because acidosis depresses the calcium (Ca²⁺) sensitivity and force production of muscle (18, 50). However, the molecular mechanisms behind this depression in Ca²⁺ sensitivity and force production are unknown. This gap in knowledge poses a significant challenge in generating a complete understanding of the fatigue process. To close this gap, the ability of myosin to bind to a single actin filament was measured under acidic conditions, in a laser trap assay, with and without regulatory proteins. Decreasing pH from 7.4 to 6.5 reduced the frequency …

Multiscale Simulations Of Intrinsically Disordered Proteins, Xiaorong Liu

Doctoral Dissertations

Intrinsically disordered proteins (IDPs) lack stable secondary and/or tertiary structures under physiological conditions. The have now been recognized to play important roles in numerous biological processes, particularly cellular signaling and regulation. Mutation of IDPs are frequently associated with human diseases, such as cancers and neuron degenerative diseases. Therefore, it is important to understand the structure, dynamics, and interactions of IDPs, so as to establish the mechanistic basis of how intrinsic disorder mediates versatile functions and how such mechanisms may fail in human diseases. However, the heterogeneous structural ensembles of IDPs are not amenable to high resolution characterization solely through experimental …

Ni Site Structure And Function In Biological Sensing And Enzyme Activity, Hsin-Ting Huang

Doctoral Dissertations

Ni(II) is one of the important cofactors involved in various enzyme functions. For organisms utilizing Ni(II), a regulation system is required to maintain Ni(II) homeostasis and prevent toxicity. The focus of this dissertation is on investigating the relationship between the Ni(II) site structure and the function of proteins, a Ni(II) sensor and a Ni(II) enzyme. RcnR, a Ni(II)/Co(II) sensor in E. coli, controls the expression of the Ni(II)/Co(II) exporter proteins, RcnAB. Due to the lack of structural information, the mechanism of metal induced allosteric regulation and metal selection is not fully elucidated. Results presented here show that binding of …

The Spatial Organization Of Mycobacterial Membrane, Julia Puffal

Doctoral Dissertations

Mycobacteria comprises a large group of organisms including the pathogenic species Mycobacterium tuberculosis, the causative agent of tuberculosis. A fast- growing saprophytic member of this genus, however, Mycobacterium smegmatis, is oftentimes used as a model organism for the pathogenic species. With a unique cell envelope architecture and unconventional polar growth, spatial coordination of cell envelope biosynthesis is vital for proper assembly of this complex structure. Here, we provide a comprehensive overview of known lateral heterogeneities in mycobacterial plasma membrane, with a particular focus on the intracellular membrane domain (IMD), a spatially distinct region of the plasma membrane with diverse functions. …

Studies On The Interaction And Organization Of Bacterial Proteins On Membranes, Mariana Brena

Masters Theses

Bacteria have developed various means of secreting proteins that can enter the host cell membrane. In this work I focus on two systems: cholesterol-dependent cytolysins and Type III Secretion.

Cholesterol is a molecule that is critical for physiological processes and cell membrane function. Not only can improper regulation lead to disease, but also the role cholesterol plays in cell function indicates it is an important molecule to understand. In response to this need, probes have been developed that detect cholesterol molecules in membranes. However, it has been recently shown that there is a need for probes that only respond to …

Bio-Inspired Polymers That Bind And Deliver Protein Cargo, Nicholas D. Posey

Doctoral Dissertations

Delivering functional proteins and antibodies into cells can allow researchers to probe the intracellular environment, discover new cellular pathways, and pioneer new therapeutics. However, the entry of exogenous, charged molecules, like proteins, into the cell is usually restricted by the membrane, thereby hindering intracellular delivery. Membrane permeable molecules such as cell penetrating peptides (CPPs) and protein transduction domains (PTDs) can be used to bypass the cell membrane and deliver protein into the cell, but these peptides involve iterative and laborious syntheses and are limited in terms of their chemical diversity. This dissertation work overall focuses on the design and synthesis …

Hybridized Polymeric Nano-Assemblies: Key Insights Into Addressing Mdr Infections, Ryan Landis

Doctoral Dissertations

Multidrug-resistant (MDR) bacteria contribute to more than 700,000 annual deaths world-wide. Millions more suffer from limb amputations or face high healthcare treatment costs where prolonged and costly therapeutic regimens are used to counter MDR infections. While there is an international push to develop novel and more powerful antimicrobials to address the impending threat, one particularly interesting approach that has re-emerged are essential oils, phytochemical extracts derived from plant sources. While their antimicrobial activity demonstrates a promising avenue, their stability in aqueous media, limits their practical use in or on mammals. Inspired by the versatility of polymer nanotechnology and the sustainability …

Probing Apoptotic Caspase Allostery And Exosite Interactions For Alternative Regulation, Derek J. Macpherson

Doctoral Dissertations

Programmed cell death, or apoptosis is a critical homeostatic pathway that monitors the balance of cell life and death. Apoptosis is regulated by a class of enzymes known as the cysteine aspartic proteases, or the caspases. The 12 human caspases that play important roles in the progression and regulation of apoptosis and inflammation. Caspases are tightly regulated by numerous factors including enzymatic activation, post-translational modifications, metal ligand binding, and protein modulation. Aberrant caspase activation and regulation has been implicated in the progression of numerous diseases such as proliferative diseases and neurodegeneration. The deeply entwined nature of caspases and apoptosis makes …

Active Site Design And Exploitation Of Allosteric Sites In Proteases, Maureen Hill

Doctoral Dissertations

Proteases are powerful enzymes with the intrinsic ability to proteolytically cleave other proteins, which often leads to a gain or loss of function. While proteases from the same family often share high similarity in the active site region, distal sites on the enzyme, such as allosteric sites, can differentiate closely related enzymes and allow for specific regulation. This dissertation investigates two different types of proteases, including the viral proteases from both Zika and dengue virus and the mammalian caspase proteases, and contributes significantly to the understanding of both allosteric sites and exosites in these enzymes. Flaviviruses, including Zika virus and …

Building The Outer Membrane Protein G (Ompg) Nanopore Library: From The Discrimination Of Biotin-Binding Proteins In Serum To Resolving Human Carbonic Anhydrase From Human Red Blood Cells, Bib Yang

Doctoral Dissertations

The use of pore-forming proteins (PFPs) in nanopore sensing has been fruitful largely due to their nanoscale size and the ease with which protein nanopores can be manipulated and consistently reproduced at a large scale. Nanopore sensing relies heavily on a steady ionic current afforded by rigid nanopores, as the change in current is indicative of analyte detection, revealing characteristics of the analyte such as its relative size, concentration, and charge, as well as the nanopore:analyte interaction. Rigid PFPs have been used in applications such as DNA sequencing, kinetic studies, analyte discrimination, and protein conformation dynamics at the single-molecule level. …

Nascent Dna Proteomics Analysis Uncovers Dna Replication Dynamics In The Human Pathogen Trypanosoma Brucei, Maria Rocha Granados

Doctoral Dissertations

DNA is the substrate of many cellular processes including DNA replication, transcription and chromatin remodeling. These processes are coordinated to maintain genome integrity and ensure accurate duplication of genetic and epigenetic information. Genome-wide studies have provided evidence of the relationship between transcription and DNA replication timing. A global analysis of DNA replication initiation in T. brucei showed that TbORC1 (subunit of the origin recognition complex, ORC) binding sites are located at the boundaries of transcription units. Although recent studies in T. brucei indicate functional links among DNA replication and transcription, the underlying mechanisms remain unknown. In this study, we adapted …

Protein Degradation Regulates Phospholipid Biosynthetic Gene Expression In Saccharomyces Cerevisiae, Bryan Salas-Santiago

Doctoral Dissertations

Transcriptional regulation of most phospholipid biosynthetic genes in Saccharomyces cerevisiae is coordinated by inositol and choline. Inositol affects phosphatidic acid (PA) intracellular levels. Opi1p interacts physically with PA and is the main repressor of the phospholipid biosynthetic genes. It is localized in the endoplasmic reticulum (ER) bound to the ER membrane protein Scs2p. When PA levels drop, Opi1p is translocated into the nucleus repressing most phospholipid biosynthetic genes. The OPI1 locus was identified in a screen looking for overproduction and excretion of inositol (Opi^-). Opi^- mutants are generally associated with a defect in …

Characterization Of The Motion Of Cellulose Synthase Protein Complexes In The Plant Cell Membrane, Nina Zehfroosh

Doctoral Dissertations

The polysaccharide cellulose is the main component of plant cell walls, so it is the most abundant polymer on Earth. While it is widely used in industry due to its remarkable properties, such as renewability and biodegradability, its biosynthesis is still not well understood. The large transmembrane protein Cellulose Synthase Complex (CSC) is responsible for synthesizing cellulose by polymerizing UDP glucose into the constituent glucan chains of cellulose. In this project, I used variable angle epi-fluorescence microscopy (VAEM) in combination with single-particle tracking to characterize the motion of GFP labeled CSCs in the hypocotyl of Arabidopsis thaliana (A. thaliana …

Chemoreceptor Dynamics And Signaling: Nmr Measurements Within Functional Complexes, Maryam Kashefi

Doctoral Dissertations

Bacteria employ remarkable membrane-bound nanoarrays to sense their environment and direct their swimming. Arrays consist of chemotaxis receptor trimers of dimers that are bridged at their membrane-distal tips by rings of two cytoplasmic proteins, a kinase CheA and a coupling protein CheW. It is widely accepted that ligand binding to the receptor causes a 2 Å piston motion of a helix that extends through the periplasmic and transmembrane domains. However, it is not clear how the signal propagates 200 Å further to control activity of the kinase bound at the tip of the receptor. Dynamic changes within the cytoplasmic domain …

Enhancing Nanopore Based Biosensening Technology Using Pore Forming Proteins, Christina M. Chisholm

Doctoral Dissertations

Pore forming proteins (PFPs) are membrane channels that are essential for various biological processes. For example, some PFPs act as gatekeepers of the cell, controlling the traffic of ions and macromolecules flowing into and out of cells; while others are involved in causing cell death (Reiner et al., 2012). Our fundamental understanding of PFPs determines our ability to employ these proteins for use in biomedical research and nanopore technology. Given their nanoscale dimensions, reproducibility and functionality these PFPs are widely used in the growing field of nanopore technology, particularly nanopore sensing (Reiner et al., 2012; Feng et al., 2015). These …