Life Sciences Commons^™

A Rational Design Approach To Developing Second Generation Fabry Disease Treatments, Matthew Metcalf

Doctoral Dissertations

Fabry disease is an X-linked lysosomal storage disorder that affects approximately 1 in 40,000 males in its classical form and as many as 1:4,600 in its late-onset form [1]. The disease is caused by mutations in the gene encoding α- galactosidase (α-GAL), which results in deficient levels of α-GAL activity in the lysosomes of patients [2, 3]. This lack of enzymatic activity causes macromolecular substrates to accumulate in tissues, and can result in a wide range of symptoms such as impaired renal and cardiac function [4]. The severity of disease is linked to the amount of residual …

Investigating The Accumulation, Sub-Organ Distribution, And Biochemical Effects Of Nanomaterials Using Mass Spectrometry, Kristen Nicole Sikora

Doctoral Dissertations

Gold nanoparticles (AuNPs) are attractive materials for use in various biomedical applications, such as therapeutic delivery, due to their unique chemical properties and modular tunability. Mass spectrometry methods, including laser desorption/ionization mass spectrometry (LDI-MS) and inductively coupled plasma mass spectrometry (ICP-MS) have been successfully used to evaluate the distribution of AuNPs in complex biological systems. As new AuNP-based materials are developed for applications in therapeutic delivery, it is essential to simultaneously develop analytical techniques that can comprehensively assess their behavior in vivo. In this dissertation, novel mass spectrometric methods have been developed and utilized to evaluate the uptake, distribution, …

Engineering Mesothelin-Binding Proteins As Targeted Cancer Diagnostics And Therapeutics, Allison Rita Sirois

Doctoral Dissertations

Cancer is a significant global health concern; and traditional therapies, including chemotherapeutics, are often simultaneously toxic yet ineffective. There is a critical need to develop targeted cancer therapeutics which specifically inhibit molecules or molecular pathways essential for tumor growth and maintenance. Furthermore, a targeted therapy is only effective when a patient's tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a necessary counterpart of targeted therapies. Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including triple-negative breast, pancreatic, ovarian, liver, and lung, with limited expression in normal tissues. Aberrant MSLN expression promotes tumor …

Controlled Membrane Remodeling By Nanospheres And Nanorods: Experiments Targeting The Design Principles For Membrane-Based Materials, Sarah Zuraw-Weston

Doctoral Dissertations

In this thesis we explore two experimental systems probing the interactions of nanoparticles with lipid bilayer membranes. Inspired by the ability of cell membranes to alter their shape in response to bound particles, we report two experimental studies: one of nanospheres the other of long, slender nano-rods binding to lipid bilayer vesicles and altering the membrane shape. Our work illuminates the role of particle geometry, particle concentration, adhesion strength and membrane tension in how membrane morphology is determined. We combine giant unilamellar vesicles with oppositely charged nanoparticles, carefully tuning adhesion strength, membrane tension and particle concentration. In the case of …

Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma

Doctoral Dissertations

The mucosal barrier in the intestine is vital to maintain selective absorption of nutrients while protecting internal tissues and maintaining symbiotic relationship with luminal microbiota. This bio-barrier consists of a cellular epithelial barrier and an acellular mucus barrier. Secreted mucus regulates barrier function via in situ biochemical and biophysical interaction with luminal content that continually evolves during digestion and absorption. Increasing evidence suggests that a mucus barrier is indispensable to maintain homeostasis in the gastrointestinal tract. However, the importance of mucus barrier is largely underrated for in vitro mucosal tissue modeling. The major gap is the lack of experimental material …

Computer Simulations Of Biological Systems: From Protein Dynamics To Drug Discovery, Rupesh Agarwal

Doctoral Dissertations

Computational biophysics methods such as molecular dynamics (MD) simulations are often used in combination with experimental techniques like neutron scattering, NMR, and FTIR to explore protein conformational landscapes. With the improvements in experimental techniques, there is also a need to continually optimize the MD forcefield parameters to make precise predictions that match experimental results. To complement many of these experiments, an accurate model of deuteration is frequently required, but has been elusive. In our work, we developed a novel method to capture isotope effects in classical MD simulations by re-parameterization of the bonded terms of the CHARMM forcefield using quantum …

Probing Structure, Function And Dynamics In Bacterial Primary And Secondary Transporter-Associated Binding Proteins, Shantanu Shukla

Doctoral Dissertations

Substrate binding proteins (SBPs) are ubiquitous in all life forms and have evolved to perform diverse physiological functions, such as in membrane transport, gene regulation, neurotransmission, and quorum sensing. It is quite astounding to observe such functional diversity among the SBPs even when they are restricted by their fold space. Therefore, the SBPs are an excellent set of proteins that can reveal how proteins evolution novel function in a structurally conserved/constrained fold. This study attempts to understand the phenomenon of affinity and specificity evolution in SBPs by combining a set of biochemical, biophysical, and structural studies on the SBPs involved …

The Effect Of Nuclear Perturbations On The 3d Organization Of The Genome, Rosela Golloshi

Doctoral Dissertations

Cells in our body experience constant mechanical forces that influence biological functions such as growth and development. The nucleus has been implicated as a key mechanosensor and can directly influence chromatin organization and epigenetic alterations leading to gene expression changes. However, the mechanism by which such mechanical forces lead to genomic alterations and expression of mechanosensitive genes is not fully understood. The work presented in this dissertation investigates the effect of mechanical and epigenetic perturbations on the 3D genome organization. To investigate this 3D genome folding, we use Chromosome Conformation Capture followed by high throughput sequencing (Hi-C) (Chapter-1) which identifies …

Root Phosphomonoesterase As A Vital Component Of Increasing Phosphorus Availability In Tropical Forests, Kristine Grace Manno Cabugao

Doctoral Dissertations

Tropical forests, relative to other terrestrial ecosystems, exchange the largest amount of carbon with the atmosphere and also constitute a significant carbon sink. However, nutrient limitation, particularly of phosphorus (P), could limit growth of tropical forests and their function with the global carbon cycle. Thus, understanding root mechanisms to acquire P is necessary to representing the P cycle and corresponding interactions with plant growth. A large portion of total soil P in tropical forests occurs in organic forms, only accessible through root and microbial production of phosphatase enzymes. These phosphatase enzymes mineralize organic P into orthophosphate, the form of P …

Investigating Transmembrane-Lipid Interactions Of Epha2 And Ph Responsive Peptides, Katherine M. Stefanski

Doctoral Dissertations

Single-pass membrane receptor signaling plays vital roles in human development and maintaining homeostasis. These membrane receptors can also have causative functions in several diseases including cancer. Much is known about the structure and signaling outcomes of these receptors but the mechanistic details of how they pass an extracellular signal across the membrane and into cytoplasm via the transmembrane (TM) domain is unclear. It is further unknown how or if interactions with membrane lipids facilitate and/or regulate these events. Here we use the TYPE7 peptide to target the TM region of a receptor tyrosine kinase, EphA2. EphA2 engages in both tumorigenic …

Iojap: Morphological And Physiological Phenotype Characterization In Arabidopsis, Thomas Jay Payne

Doctoral Dissertations

IOJAP protein is found in all organisms that contain a ribosome of bacterial origin. The majority of studies suggest that IOJAP plays a role in translation, although this has yet to be thoroughly investigated in plants. Using Arabidopsis thaliana, an extensive phenotype characterization of iojap mutants was performed. Many processes of plant growth were slightly impaired at optimal temperature (22˚C) but became severely hindered at low temperature (12˚C and 4˚C). These cold temperature defects manifested in an overall reduction of plant growth as well as variegation, chlorosis, leaf hyponasty, as well as reduced maximum quantum yield (F_v/F …

Using Second Harmonic Generation To Study Gram-Positive Bacterial Membranes, Lindsey N. Miller

Doctoral Dissertations

Understanding how small-molecules, such as drugs, interact with bacterial membranes can quickly unravel into much more perplexing questions. No two bacterial species are alike, especially when comparing their membrane compositions which can even be altered by incorporating fatty acids from their surrounding environment into their lipid-membrane composition. To further complicate the comparison, discrete alterations in small-molecule structures can result in vastly different membrane-interaction outcomes, giving rise to the need for more "label-free" studies when analyzing drug mechanisms. The work presented in this dissertation highlights the benefits to using nonlinear spectroscopy and microscopy techniques for probing small-molecule interactions in living bacteria. …

Study Of The Role Of Biologically-Relevant, Labile Nickel Pools In The Maturation Of Nickel-Dependent Enzymes, Priyanka Basak

Doctoral Dissertations

Cellular nickel pools, comprised of static and labile pools of nickel complexes, play important roles in maintaining nickel homeostasis in various organisms (microbes, fungi, and plants), which utilize it as a cofactor of one or more nickel enzymes that catalyze specific reactions and are essential for their proper growth and survival in various ecological niches. Like other metals, tight regulation of cellular nickel levels is critical to prevent toxic effects of nickel deprivation, nickel overload, and ‘free’ nickel. While more static nickel pools include nickel tightly bound to nickel-dependent enzymes, nickel in the labile pool is exchangeable and weakly bound …

Needles In A Haystack Of Protein Diversity: Interrogation Of Complex Biological Samples Through Specialized Strategies In Bottom-Up Proteomics Uncover Peptides Of Interest For Diverse Applications, Manuel I. Villalobos Solis

Doctoral Dissertations

Peptide identification is at the core of bottom-up proteomics measurements. However, even with state-of the-art mass spectrometric instrumentation, peptide level information is still lost or missing in these types of experiments. Reasons behind missing peptide identifications in bottom-up proteomics include variable peptide ionization efficiencies, ion suppression effects, as well as the occurrence of chimeric spectra that can lower the efficacy of database search strategies. Peptides derived from naturally abundant proteins in a biological system also have better chances of being identified in comparison to the ones produced from less abundant proteins, at least in regular discovery-based proteomics experiments. This dissertation …

Origin Of Gene Specificity In The Nitrogen-Fixing Symbiosis, Christina Marie Stonoha-Arther

Doctoral Dissertations

Many legumes form a symbiosis with nitrogen-fixing bacteria found in the soil. This relationship is beneficial to both the plant and the bacteria; the plant receives nitrogen that is otherwise limited, and the bacteria receive fixed carbon. Upon sensing the bacteria, the plant forms a new organ (the nodule) where the bacteria are housed within the cells. Many genes are required for the proper formation and function of nodules; this dissertation is broadly focused on how genes required for nitrogen-fixing symbiosis are co-opted from other cellular processes and how they are specialized for symbiosis. Protein trafficking from the plant to …

Structure Function Analysis Of Mitochondrial Dna Polymerase Ic Reveals Multiple Roles In Kinetoplast Maintenance, Jonathan Miller

Doctoral Dissertations

Kinetoplastid organisms including medically relevant species like Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are distinguished by their single flagellum and unique mitochondrial DNA called kDNA, among other features. While there is heterogeneity in copy number and sequence classes among different species, kDNA is most often found as a network of thousands of DNA molecules catenated together. This unique biological property of disease-causing organisms has been a subject of study as a potential drug target since it is essential for parasite survival. Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and related diseases in other mammals and …

Structural Analysis Of Protein Therapeutics Using Covalent Labeling – Mass Spectrometry, Patanachai Limpikirati

Doctoral Dissertations

Using mass spectrometry (MS) to obtain information about a higher order structure of protein requires that a protein’s structural properties are encoded into the mass of that protein. Covalent labeling (CL) with reagents that can irreversibly modify solvent accessible amino acid side chains is an effective way to encode structural information into the mass of a protein, as this information can be read-out in a straightforward manner using standard MS-based proteomics techniques. The differential reactivity of proteins under two or more conditions can be used to distinguish protein topologies, conformations, and/or binding sites. CL-MS methods have been effectively used for …

Involvement Of Calcium-Dependent Protein Kinases And Phosphatases In Sperm Capacitation-Associated Events, Bidur Paudel

Doctoral Dissertations

ABSTRACT To acquire fertilizing ability, mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation^1,2. At the molecular level, capacitation is associated with a fast bicarbonate (HCO₃^-)-dependent activation of a unique type of soluble adenyl cyclase (sAC) and a consequent increase in cyclic AMP (cAMP) levels and PKA activation³. Activation of a cAMP/PKA pathway results in the phosphorylation of PKA substrates, which in turn initiates activation of several signaling cascades ultimately leading to an increase in phosphorylation on tyrosine residues (P-Tyr) of sperm axonemal proteins^4,5. Increase in …

Characterization Of Biodistribution Of Transferrin And Receptor Binding Mechanism By Mass Spectrometry, Hanwei Zhao

Doctoral Dissertations

Protein-based therapeutics have emerged as a key driver of rapid growth in drug development pipelines. However, developing such protein drugs is not straightforward in most cases, the existence of physiological barriers greatly restricts the efficient delivery of many therapeutic molecules, and therefore limits their clinical applications. A promising way to address this challenge takes advantage of certain transport protein which can effectively across and enhance the permeability of these barriers, such as transferrin (Tf) which can be internalized by malignant cells and cross physiological barriers via transferrin receptor (TfR)-mediated endocytosis and transcytosis. However, developing such products is impossible without successfully …

Tpr-Containing Proteins Control Protein Organization And Homeostasis For The Endoplasmic Reticulum, Jill Bradley-Graham

Doctoral Dissertations

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into several functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein-protein interactions, supporting interactions with a wide range of ligands or substrates. Nine TPR-containing proteins have been shown to localize to the ER and control protein organization and …

Covalent Labeling-Mass Spectrometry For Characterizing Protein-Ligand Complexes, Tianying Liu

Doctoral Dissertations

This dissertation focuses on applying covalent labeling (CL) and mass spectrometry (MS) for characterizing protein-ligand complexes. Understanding protein-ligand interactions has both fundamental and applied significance. Covalent labeling is a protein surface modification technique that selectively modifies solvent-exposed amino acid side chains of proteins. A covalent bond is formed between the functional groups of labeling reagent and protein’s side chain. One of the key factors that affects CL reactivity is a side chain’s solvent accessibility. Ligand binding protects residues on the protein surface from being labeled, and residues involved in ligand binding can be indicated via decreases in labeling extents. The …

Tools To Evaluate Nanodiamond-Mediated Delivery Of Tiopronin For Cataract Prevention, Justin Beltz

Doctoral Dissertations

”There is a growing demand for non-surgical means of cataract treatment. This dissertation presents three bodies of work that reflect the early-stage development of eye drop formulations aimed at delaying cataract progression. These formulations consist of the antioxidant 2-mercaptopropionylglycine (MPG) loaded onto nanodiamond particles.

Cataractogenesis is linked to oxidative damage to lens proteins. To investigate the potential of MPG for protection against oxidative damage, A549 cells were incubated in 0.6 mM tert-butylhydroperoxide (tBHP). Cells exposed to tBHP without MPG exhibited elevated levels of reactive oxygen species, which led to the depletion of the vital antioxidant glutathione and, ultimately, apoptosis. Co-administration …