Life Sciences Commons^™

The Phase Behavior Of Ubqln Proteins And Implications For Protein Quality Control, Yiran Yang

Dissertations - ALL

The ubiquitin-proteasome system (UPS) and autophagy are essential pathways for maintaining protein quality control (PQC) in cells. Misfolded proteins and large aggregates are cleared by UPS and autophagy signaled by ubiquitin (Ub) or polyubiquitin (polyUb) chains. Shuttle proteins facilitate cargo transporting by interacting with both ubiquitin and degradation machineries. Previously, our lab discovered that the shuttle protein Ubiquilin-2 (UBQLN2) is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. LLPS is a biophysical process by which proteins separate themselves from the surrounding aqueous solution by forming protein-rich droplets. The overarching goals of this work are …

Direct Single Molecule Imaging Of Enhanced Diffusion For Enzymes And Enzyme-Conjugated Dna Origami, Mengqi Xu

Dissertations - ALL

Enzymes have been shown to diffuse faster in the presence of their substrates. Recently, we revealed new insights into this process of enhanced diffusion using single-particle tracking (SPT) with total internal reflection fluorescence (TIRF) microscopy. We found that the mobility of individual enzymes was enhanced three-fold in the presence of the substrate, and the motion remained Brownian. We showed that the relative increase in diffusion is independent of the total enzyme concentrations; and the oligomerization state of enzymes did not change during the catalytic turnover. These experiments ruled out the possibility that the enhanced enzyme diffusion was caused by the …

Intrinsically Disordered Protein Regions In Human Evolution And As Therapeutic Targets, Karen Paco Mendivil

KGI Theses and Dissertations

Intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) fail to form stable structures but have important biological functions via interacting with various molecular partners (proteins, DNA, RNA, glycosaminoglycans). We hypothesized that IDPRs are potential targets for therapeutics development because they are reservoirs of evolutionary innovation, and they play crucial roles in adaptation to pathogens.

We first studied the evolution of IDPRs in the human proteome and compared it with the proteome of non-human primates. We have found that evolutionary young protein-coding genes have included low conserved regions in the N-terminal part of proteins, and such regions are linked to high …

Slo2.1 Channels: A New Molecular Mechanism To Regulate Uterine Excitability, Juan Jose Ferreira

Arts & Sciences Electronic Theses and Dissertations

At the end of pregnancy, the uterus transitions from a non-contractile state to a highly contractile state. Two processes primarily drive this transition. First, from the 28th week of pregnancy until labor, the resting membrane potential of uterine (myometrial) smooth muscle cells (MSMCs) gradually becomes more positive (depolarizes) (Parkington et al. 1999). Second, at the end of pregnancy, MSMCs express more oxytocin receptors and become more sensitive to oxytocin (Kimura et al. 1996). However, the detailed mechanisms by which these processes occur have not been determined. My central hypothesis was that the Na+-activated K+ channel SLO2.1 plays a key role …

Computational Methods For Analysis Of Data For Conformational And Phase Equilibria Of Disordered Proteins, Jared Michael M Lalmansingh

Arts & Sciences Electronic Theses and Dissertations

Intrinsically disordered proteins and regions (IDPs / IDRs) are a class of proteins with diverse conformational heterogeneity that do not fold into a tertiary structure due to the lack of a native structural state. Consequently, disordered proteins are remarkably flexible and exhibit multivalent properties that enable them to adopt myriad functional roles within the cell such as: signaling transduction, transcription, enzymatic catalysis, translation, and many more. Due to their multivalency, some IDPs undergo monomeric and heterotypic interactions which can drive phase separation. Such IDPs can form membraneless organelles with specific regulatory roles within the cell which include, but are not …

On The Challenges And Rewards Of Analyzing Molecular Dynamics At The Terabyte And Millisecond Scale, Justin Roy Porter

Arts & Sciences Electronic Theses and Dissertations

Molecular Dynamics (MD) and Markov state models (MSMs) are powerful tools for estimating and concisely representing the conformational ensemble accessible to biological macromolecules, particularly proteins. Conformational ensembles are of special importance biological function, both in health and disease, because biology derives from molecules’ entire conformational distribution rather than any single structure. Consequently, MD is poised to become a powerful tool for personalized medicine and for the study of molecular sequence-function relationships generally. However, because of their hyperdimensionality and size, just generating MD datasets and Markov state models (MSMs) that represent biologically relevant molecules is a substantive technical challenge. Then, even …

Regulatory Effects Of The E. Coli Recbcd Nuclease Domain On Dna Unwinding Kinetics, Nicole Fazio

Arts & Sciences Electronic Theses and Dissertations

I have examined the effects of deleting the nuclease domain of the E. coli helicase RecBCD on the rates of ATP-independent DNA melting, single stranded (ss) DNA translocation, and double stranded (ds) DNA unwinding by RecBCD. The canonical role of the nuclease domain is DNA degradation, but the removal of this domain showed unexpected effects on other RecBCD activities including DNA binding, melting, and unwinding. This thesis presents a mechanistic study of DNA unwinding by RecBCD and a RecBCD variant with the nuclease domain deleted (RecBΔnucCD). I examined the rates of ssDNA translocation and dsDNA unwinding by RecBCD and RecBΔnucCD …

A Review Of Calcineurin Biophysics With Implications For Cardiac Physiology, Ryan B. Williams

Theses and Dissertations

Calmodulin is a prevalent calcium sensing protein found in all cells. Three genes exist for calmodulin and all three of these genes encode for the exact same protein sequence. Recently mutations in the amino acid sequence of calmodulin have been identified in living human patients. Thus far, patients harboring these mutations in the calmodulin sequence have only displayed an altered cardiac related phenotype. Calcineurin is involved in many key physiological processes and its activity is regulated by calcium and calmodulin. In order to assess whether or not calcineurin contributes to calmodulinopathy (a pathological state arising from dysfunctional calmodulin), a comprehensive …

Granulins In Norm And Neurodegenerative Pathologies, Anukool Bhopatkar

Dissertations

Granulins (GRNs) are small, cysteine-rich modules produced from the proteolytic cleavage of the precursor protein called progranulin (PGRN). GRNs are present in the form of seven tandem repeats within the precursor and are known to be produced in the extracellular and in lysosomal environments. In physiology, PGRN and GRNs plays pleiotropic roles such as neuronal growth and differentiation, immunomodulation, wound healing. Recent studies have implicated pathological role for PGRN in Alzheimer disease (AD) and frontotemporal dementia (FTD) but specific mechanism(s) remains unclear. However, potential interactions between GRNs and Ab42 and TDP-43 seem like a plausible underlying mechanism. Studies presented here …

Utilizing Computational Approaches To Study Tubulin- Tubulin Electrostatic Interactions, Tolulope Ayodeji Ale

Open Access Theses & Dissertations

Microtubules play significant roles in transporting cargos within the cell, as well as the cell cycle. They are essentially the highway for the transportation of cell organelles. Understanding the interactions among microtubule components is crucial for the treatment of neurodegenerative diseases and cancer. In this study, we utilize a series of computational approaches to study the electrostatic interactions at the binding interfaces of interacting tubulin monomers. Our study revealed that the electrostatic attractive interactions in the αβ tubulin dimer is the strongest while the interaction between two αβ tubulin dimers in the longitude direction is the weakest. The result identified …

Liposomes: Production And Applications For Controlled Drug Delivery, Gamid Abatchev

Boise State University Theses and Dissertations

This dissertation recognizes the enormous potential presented by the ever-evolving development of liposomes as drug carriers and seeks to offer further investigation into their useful production and utilization. The first chapter presents the basic principles governing their formation by self-assembly in water solutions, briefly describes the most common production methods, and points out essential past advances that led to their use as drug carriers. Chapter two exemplifies production of liposomes by the traditional methods of extrusion and sonication, detailing passive and active loading, as well as physical characterization by Dynamic Light Scattering, microscopy imaging, and fluorescence spectroscopy. In the next …

Deciphering Chemomechanical Couplings In Proteins Using Microsecond-Level Molecular Dynamics Simulations, Vivek Govind Kumar

Graduate Theses and Dissertations

All-atom molecular dynamics (MD) simulations combine the high temporal resolution of experimental methods like smFRET and spatial resolution of methods like x-ray crystallography, to provide a detailed dynamic picture of biomolecular processes. Here, microsecond-level atomistic MD simulations have been used to characterize chemomechanical couplings in human fibroblast growth factor 1 (hFGF1) and the spike proteins of SARS CoV-1 and SARS-CoV-2. hFGF1 is a globular signaling protein that is involved in several physiological processes ranging from cell proliferation to wound healing. Experimental studies have previously described the low proteolytic and thermal stability of hFGF1, in addition to the stabilizing role of …

Engineering Fluorescently Labeled Human Fibroblast Growth Factor One Mutants And Characterizing Their Photophysics Properties Towards Designing Fret Assays, Mamello Mohale

Graduate Theses and Dissertations

Human fibroblast growth factor one (hFGF1) belongs to a family of 22 FGF members produced by fibroblast cells. Cell signaling during physiological processes of angiogenesis and wound healing occurs when hFGF1 binds to its receptor (FGFR). However, when heterogenous homeostasis is not maintained, fibroblast cells exhibit excessive proliferation which can lead to a myriad of cancers. smFRET is an ultrasensitive distant dependent (1-10 nm) technique capable of resolving such heterogeneity in structural dynamics and binding affinities (Kd). Therefore, we successfully designed and characterized fluorescently labeled hFGF1 tracers which span the visible light region of the electromagnetic spectrum for use in …

Mechanistic Insight Into Β-Lactamase Catalysis, Inhibitor Design And Resistance, Michael Trent Kemp

USF Tampa Graduate Theses and Dissertations

The emergence of antibiotic resistance and spread of Gram negative bacteria poses a very real health threat to the public. The main mode of resistance within Gram negative bacteria is the production of β-lactamase enzymes that catalyze the breakdown of β-lactam antibiotics through a hydrolysis mechanism. Once the β-lactam ring is hydrolyzed and opened, the drug loses its efficacy, which allows for the bacteria to grow and proliferate uninhibited. These β-lactamase enzymes are organized into four categories based on the Ambler classification, with classes A, C and D being denoted as serine-based β-lactamase enzymes. Class B is composed of metalloenzymes …

Investigation Of Immobilized Enzymes In Confined Environment Of Mesoporous Host Matrices, Xiaoliang Wang

USF Tampa Graduate Theses and Dissertations

Enzyme immobilization in metal-organic frameworks (MOFs) as a promising strategy, is attracting the interest of scientists from different disciplines with the expansion of MOF’s development. Different from other traditional host materials, their unique strengths of high surface areas, large yet adjustable pore sizes, functionalizable pore walls, and diverse architectures make MOFs an ideal platform to investigate hosted enzymes, which is critical to the industrial and commercial process. In addition to the protective function of MOFs, the extensive roles of MOFs in the enzyme immobilization are being well-explored by making full use of their remarkable properties like well-defined structure, high porosity, …

Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo

Doctoral Dissertations

Long-term memory and learning are still poorly understood from a molecular and cellular standpoint. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric kinase that is involved in this remarkable process. However, the molecular details of its specific roles in these processes remains elusive. CaMKII activation-triggered subunit exchange presents a novel possible mechanism involved in long-term memory and learning by exchanging active subunits with other CaMKIIs. CaMKII subunit exchange also shows that exchanged CaMKIIs spread their phosphorylation state to newly synthesized CaMKIIs. This provides a long-lasting signal that might possibly be involved in long-term memory by escaping a cell’s …

Validation Of Arterial Spin Labeling For Longitudinal Monitoring And Differential Diagnosis Of Frontotemporal Dementia, Tracy Ssali

Electronic Thesis and Dissertation Repository

Frontotemporal dementia (FTD) is a devastating neurodegenerative disease characterized by a rapid decline in behavioural, language, and motor abilities. Advances in the understanding of FTD genetics and pathophysiology, and the subsequent development of novel disease modifying treatments have highlighted the need for tools to assess their efficacy. While structural magnetic resonance imaging (MRI) and functional imaging with ¹⁸F-flurodeoxyglucose (FDG) positron emission tomography (PET) are used for clinical diagnosis, structural changes are subtle at the early stages and PET imaging is expensive and access limited. Given the coupling of cerebral blood flow (CBF) to energy metabolism, an attractive alternative is …

Phase Imaging For Reducing Macrovascular Signal Contributions In High-Resolution Fmri, Olivia W. Stanley

Electronic Thesis and Dissertation Repository

High resolution functional MRI allows for the investigation of neural activity within the cortical sheet. One consideration in high resolution fMRI is the choice of which sequence to use during imaging, as all methods come with sensitivity and specificity tradeoffs. The most used fMRI sequence is gradient-echo echo planar imaging (GE-EPI) which has the highest sensitivity but is not specific to microvasculature. GE-EPI results in a signal with pial vessel bias which increases complexity of performing studies targeted at structures within the cortex. This work seeks to explore the use of MRI phase signal as a macrovascular filter to correct …

Exogenous Surfactant As A Delivery Vehicle For Intrapulmonary Therapeutics, Brandon J. Baer

Electronic Thesis and Dissertation Repository

As an organ system, the lung has unique advantages and disadvantages for direct drug delivery. Its contact with the external environment allows for the airways to be easily accessible to intrapulmonary delivery. However, its complex structure, which divides into more narrow airways with each branch, can make direct delivery to the remote alveoli challenging. The objective of this thesis was to overcome this issue by using exogenous surfactant, a lipoprotein complex used to treat neonatal respiratory distress syndrome, as a carrier for pulmonary therapeutics. It was hypothesized that therapeutics administered with a surfactant vehicle would display enhanced delivery to the …

Dual Control Of One Component Signaling: Mechanistic And Structural Insights Into El222 Active States, Uthama Phani R. Edupuganti

Dissertations, Theses, and Capstone Projects

Photoreceptors play a crucial role in signal transduction as specialized proteins which sense light as environmental stimuli and transduce the signal to control of downstream functions. Here we focus our attention on one class of these proteins, the Light-Oxygen-Voltage (LOV) domain, which is sensitive to blue light via an internally-bound flavin chromophore. Since their initial discovery in plant phototropins, many details of their photochemistry, chromophore interactions, and use with a diverse set of functional effectors have been described. However, several key details, especially a comprehensive understanding of signaling mechanism and its regulation, still remain elusive due in part to the …

Molecular Dynamics Simulations Of Self-Assemblies In Nature And Nanotechnology, Phu Khanh Tang

Dissertations, Theses, and Capstone Projects

Nature usually divides complex systems into smaller building blocks specializing in a few tasks since one entity cannot achieve everything. Therefore, self-assembly is a robust tool exploited by Nature to build hierarchical systems that accomplish unique functions. The cell membrane distinguishes itself as an example of Nature’s self-assembly, defining and protecting the cell. By mimicking Nature’s designs using synthetically designed self-assemblies, researchers with advanced nanotechnological comprehension can manipulate these synthetic self-assemblies to improve many aspects of modern medicine and materials science. Understanding the competing underlying molecular interactions in self-assembly is always of interest to the academic scientific community and industry. …

Machine Learning And Solvation Theory For Drug Discovery, Lieyang Chen

Dissertations, Theses, and Capstone Projects

Drug discovery is a notoriously expensive and time-consuming process; hence, developing computational methods to facilitate the discovery process and lower the associated costs is a long-sought goal of computational chemists. Protein-ligand binding, which provides the physical and chemical basis for the mechanism of action of most drugs, occurs in an aqueous environment, and binding affinity is determined not only by atomic interactions between the protein and ligand but also by changes in their interactions with surrounding water molecules that occur upon binding. Thus, a quantitative understanding of the roles water molecules play in the protein-ligand binding process is an essential …

The Structural And Functional Role Of Photosensing In Rgs-Lov Proteins, Zaynab Jaber

Dissertations, Theses, and Capstone Projects

Light provides organisms with energy and spatiotemporal information. To survive and adapt, organisms have developed the ability to sense light to drive biochemical effects that underlie vision, entrainment of circadian rhythm, stress response, virulence, and many other important molecularly driven responses. Blue-light sensing Light-Oxygen-Voltage (LOV) domains are ubiquitous across multiple kingdoms of life and modulate various physiological events via diverse effector domains. Using a small molecule flavin chromophore, the LOV domain undergoes light-dependent structural changes leading to activation or repression of these catalytic and non-catalytic effectors. In silico analyses of high-throughput genomic sequencing data has led to the marked expansion …

A Quantitative Assessment Of Hemodynamic And Histological Parameters In The Mdx Mouse Model, Sarah Hakim

Electronic Thesis and Dissertation Repository

Duchenne muscular dystrophy (DMD) is one of the most commonly inherited musculoskeletal disorders affecting 1 in 3500 boys worldwide. The disorder is due to a lack of functional dystrophin protein, which, in turn, results in a loss of cell membrane integrity in skeletal, cardiac muscle and the brain leading to changes in perfusion. As such, within DMD tissue, necrosis is commonly observed, and it has been previously hypothesized that this may be due to a reduction in regional blood supply. Here, we aim to measure hemodynamic changes during disease progression in DMD (Mdx/Utrn+/+) mice, using dynamic contrast enhanced …

The Role Of Excited States In Determining Β-Lactamase Function And Bacterial Fitness, Catherine Rae Knoverek

Arts & Sciences Electronic Theses and Dissertations

Proteins are macromolecular machines that play a role in nearly every biological process. They are dynamic molecules which adopt many different conformations as they fold into their 3D structures, interact with their binding partners, and perform their functions. The most probable (lowest energy) protein conformation is referred to as the ground state, and this is often assumed to be the state determined by experimental methods such as x-ray crystallography. However, proteins also adopt higher energy excited states which can have significant probabilities. As these excited states are notoriously difficult to find and study, it is unclear if excited states contribute …

Dynamics Of Protein-Dna Interactions Characterized By Atomic Force Microscopy, Yaqing Wang

Theses & Dissertations

This thesis describes the nanoscale studies of protein-DNA interactions with different complexities using atomic force microscopy (AFM). One of the systems deals with DNA replication rescue. To maintain the genetic integrity, replication machinery needs to minimize the error rate, repair the damages, and restart the stalled replication caused by the attacks from the environment and inside the cell. The stalled replication rescue is orchestrated by a series of proteins, for example, the DNA helicases PriA and RecG and the ssDNA binding protein (SSB).

We demonstrated that SSB stimulates the restart process in two aspects. First, SSB facilitates the binding of …

Hydrodynamic Characteristics Of The Shortfin Mako Shark (Isurus Oxyrinchus) For Two Caudal Fin Morphologies, Matthew C. Rodriguez

Electronic Theses and Dissertations

No abstract provided.

Mechanisms Of Connexin-46 And -50 Intercellular Channel Function And Stability By Molecular Dynamics Simulations, Bassam George Haddad

Dissertations and Theses

Gap junctions make up a class of intercellular channels that characteristically connect the cytoplasm of directly apposed cells through large assemblies, or plaques, constituted by a multitude of intercellular channels. Gap junction mediated intercellular communication is critical for a variety of physiological functions, from coordinating electrical impulses in the heart and brain to maintaining homeostasis in most tissues. There are 21 isoforms of connexins, the constituent subunit of the gap junction, expressed in a tissue dependent manner. Gap junctions formed from different isoforms exhibit distinct biophysical properties, such as gating kinetics and sensitivity, as well as unique permeability and selectivity …

Modulation Of Protein Dynamics By Ligand Binding And Solvent Composition, Richard J. Lindsay

Doctoral Dissertations

Many proteins undergo conformational switching in order to perform their cellular functions. A multitude of factors may shift the energy landscape and alter protein dynamics with varying effects on the conformations they explore. We apply atomistic molecular dynamics simulations to a variety of biomolecular systems in order to investigate how factors such as pressure, the chemical environment, and ligand binding at distant binding pockets affect the structure and dynamics of these protein systems. Further, we examine how such changes should be characterized. We first investigate how pressure and solvent modulate ligand access to the active site of a bacterial lipase …

Study Of Binding Induced Mechanical Stabilization Of Proteins Using A Single Molecule Approach, Narayan Prasad Dahal

Theses and Dissertations

Proteins operating under force are involved in several biological processes and perform multiple roles. While the structures and roles of numerous proteins are ubiquitous, their involvement in binding-induced stabilization is currently poorly understood. Most protein systems operating under force interact with their binding partners in a force-dependent manner. Such systems are related to bacterial adhesion, cellular mechano-transduction, and muscle contraction. With a goal of understanding mechanical stability induced through ligand binding, I used single-molecule magnetic tweezers to study several protein systems. This approach involves protein engineering and hetero-covalent attachment chemistry, which, combined with magnetic tweezers, allows us to characterize the …