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Engineering Self-Assembling Proteins To Produce A Safe And Effective Vaccine For Porcine Reproductive And Respiratory Syndrome, Ondre H. Harper

Electronic Thesis and Dissertation Repository

Commercially available vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) provide some control over the virus but none are ideal since they either are not completely safe for use, lack efficacy in promoting long-lasting immunity or provide no protection from heterologous PRRSV strains. Innovative approaches to designing vaccines are being pursued to overcome these drawbacks. One example is the use of nanoparticles to present a dense array of antigenic epitopes to the immune system which can effectively stimulate antibody producing cells (B cells) and T cells, resulting in long-lasting immunity. Here, I genetically fused antigenic epitopes from PRRSV to …

The Characterization Of The Rat Bone Sialoprotein Knockout Phenotype, Benjamin Harvey

Electronic Thesis and Dissertation Repository

Bone sialoprotein (BSP) is an extracellular phosphoprotein that has been associated with mineralizing tissues, such as the periodontium, the fibrocartilaginous enthesis, and long bone formation. In this study, we characterized the phenotype of BSP-deficient rats. Histomorphometric analysis discovered a thinning of the acellular cementum in both 20 and 50 week-old Bsp^-/- rats with no related periodontal defects and an organized periodontal ligament. Analysis of the mature quadriceps tendon (QCT) enthesis determined that BSP and osteopontin are present in the calcified fibrocartilage of wild-type rats at 14 weeks. The developing QCT enthesis of Bsp^-/- rats appears similar in mineral …

Systematic Identification Of The Lysine Methylome Using Methyllysine Binding Domains, Wen Qin

Electronic Thesis and Dissertation Repository

Post-translational modifications (PTM) are vital regulators of protein function and homeostasis. The role of dynamic regulations of non-histone lysine methylated proteins (NHKMP) recently began to be recognized in DNA damage repair, apoptosis and transcriptional pathways. My goal was to identify components of the NHKMP network to understand its importance in a healthy versus diseased cellular state. I used membrane peptide arrays to systematically characterize nine naturally occurring lysine methyl binding domains (KMBD). Five KMBDs were chosen based on their overlapping specificities to achieve maximum coverage of lysine methylated peptides. These five KMBDs was used to enrich for methylated lysine peptides …

Excess No Stabilizes The Luminal Domain Of Stim2 In A Cys-Specific Manner Thereby Regulating Basal Calcium Homeostasis And Store-Operated Calcium Entry, Matthew Novello

Electronic Thesis and Dissertation Repository

Stromal-interaction molecule 2 (STIM2) is an endoplasmic reticulum (ER) membrane-inserted Ca²⁺-sensing protein which, together with the plasma membrane Ca²⁺ channel Orai1, regulates basal Ca²⁺ homeostasis and store-operated Ca²⁺ entry (SOCE). Recent evidence suggests that S-nitrosylation, which is the covalent attachment of a nitric oxide (NO) moiety to a cysteine thiol, can attenuate the function of the paralog STIM1 protein. Compared to STIM1, STIM2 also functions as a basal Ca²⁺ homeostatic feedback regulator. Therefore, the objective of my study was to evaluate the susceptibility of STIM2 to S-nitrosylation and the effects that this …

Synthesis Of Crosslinkable Poly(Ester Amide)S For Cell Encapsulation And Delivery, Yu Ting (Natalie) Liang

Electronic Thesis and Dissertation Repository

Tissue engineering using adipose-derived stromal cells (ASCs) shows promise for soft tissue regeneration. Biodegradable polymers are potential biomaterials as they support the growth and delivery of cells. Specifically, poly(ester amide)s (PEAs) are a class of biodegradable polymers with tunable structures that have been shown to exhibit low cytotoxicity and support the growth of various cell types. This thesis involved the development of new water soluble amino acid-based PEAs with crosslinkable moieties to enable formation of hydrogel scaffolds for ASC encapsulation. These hydrophilic phenylalanine-based and alanine-based PEAs were synthesized by solution polycondensation and photo-crosslinked into a series of hydrogels with and …

Early Warning Of Global Change Effects On Catchment Nutrient Exports, Haibin Dong

Electronic Thesis and Dissertation Repository

Global change scientists seek sentinels of change. On forested landscapes, first-order catchments serve as sentinels of global stressors and their effects on downstream surface waters. Here, I explored global stressors – including climate warming, hydrological intensification, and recovery from atmospheric acidic deposition – and their effects on nutrient exports in 22-year stream chemistry records from 41 forested first-order catchments in a network of North American long-term monitoring sites. First, I used multivariate autoregressive models to establish relationships between changes in global stressors and changes in catchment nutrient exports. Second, I analyzed the residuals of these relationships to determine if there …

Assessing The Structure-Function Relationships Of The Apolipoprotein(A) Kringle Iv Sub-Type 10 Domain, Matthew J. Borrelli

Electronic Thesis and Dissertation Repository

Elevated plasma lipoprotein(a) (Lp(a)) is the most prevalent heritable risk factor in the development of cardiovascular disease. The apolipoprotein(a) (apo(a)) component of Lp(a) is strongly implicated in the pathogenicity of Lp(a). It is hypothesized that the inflammatory potential of Lp(a)/apo(a) is mediated by the lysine binding ability of the apo(a) kringle IV10 (KIV10) domain, along with its covalently bound oxidized phospholipid (oxPL). Using targeted mutagenesis, two novel null alleles for the LPA gene that generate non-secretable apo(a) species have been identified, resulting from amino acid substitutions in the KIV10 domain. A potential mechanism by which KIV10 oxPL modification is enriched …

Structural And Functional Characterization Of Deinococcal Dna Damage Response A (Ddra), Filip Todorovic

Electronic Thesis and Dissertation Repository

Deinococci exhibit a remarkable resilience toward DNA damage through the actions of several unique proteins, including DdrA. Although DdrA is critical for damage resistance, little is known about its mechanism of action. Despite sharing sequence similarity with Rad52, DdrA has been reported to lack single-stranded DNA annealing activity. In order to better characterize DdrA, structural studies were undertaken with the primary objective of gaining insight into the mechanism by which DdrA functions. Significant progress was made toward elucidating the X-ray crystal structure; in particular, identifying suitable DdrA domain boundaries for successful expression, purification and crystallization. In addition, we demonstrate for …

Microvascular Stenosis In Critical Limb Ischemia: Role Of Partial Endothelial To Mesenchymal Transition, Jacqueline M. Chevalier

Electronic Thesis and Dissertation Repository

Critical limb ischemia (CLI) is a widespread and debilitating manifestation of atherosclerosis. Unfortunately, revascularization strategies are often precluded or unsuccessful, resulting in amputation. A major reason for treatment failure is likely co-existing abnormalities in ­­the microvasculature. However, the specific microvascular defects present in end-stage PAD in humans remain unknown.

The purpose of this study was to delineate abnormalities in the microvascular wall in the critically ischemic skeletal muscle of patients with CLI.

To elucidate the microvascular landscape in CLI, we studied human tibialis anterior and gastrocnemius muscles harvested from below-knee amputations of 10 individuals with CLI. Control muscles are from …

Regulation Of Rna Stability By Terminal Nucleotidyltransferases, Christina Z. Chung

Electronic Thesis and Dissertation Repository

The dysregulation of RNAs has global effects on all cellular pathways. The regulation of RNA metabolism is thus tightly controlled. Terminal RNA nucleotidyltransferases (TENTs) regulate RNA stability and activity through the addition of non-templated nucleotides to the 3′-end. TENT-catalyzed adenylation and uridylation have opposing effects; adenylation stabilizes while uridylation silences or degrades RNA. All TENT homologs were initially characterized as adenylyltransferases; the identification of caffeine-induced death suppressor protein 1 (Cid1) in Schizosaccharomyces pombe as an uridylyltransferase led to the reclassification of many TENTs as uridylyltransferases. Cid1 uridylates mRNAs that are subsequently degraded by the exonuclease Dis-like 3′-5′ exonuclease 2 (Dis3L2), …

Differentially Activating The Oncogenic Kinase Akt1, Nileeka Balasuriya

Electronic Thesis and Dissertation Repository

The proto-oncogene Akt/protein kinase B plays a pivotal role in cell growth and survival. Phosphorylation of Akt at Thr308 and Ser473 activates the kinase following growth factor stimulation. Delineating specific role of each activation site in Akt1 on kinase activation, inhibition and substrate selection remain elusive.

We designed a unique set of tools, relying on genetic code expansion with phosphoserine and in vivo enzymatic phosphorylation, to produce differentially phosphorylated Akt1 variants. We found that having both sites phosphorylated increased the apparent catalytic rate of the enzyme by 1500-fold relative to the unphosphorylated enzyme. This increment was mainly due to the …

Investigating The Rotary Mechanism Of Atp Synthase Using Molecular Dynamics Simulations, Angela Marcela Murcia Rios

Electronic Thesis and Dissertation Repository

F₁-ATPase is a motor protein that can use ATP hydrolysis to drive rotation of the central subunit. The γ C-terminal helix constitutes of the rotor tip that is seated in an apical bearing formed by the α₃β₃ head. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we develop a molecular dynamics (MD) strategy to model the off-axis forces acting on γ in F₁-ATPase. MD runs showed stalling of the …

Structure And Function Of Stomatin-Like Protein 2, Safee Mian

Electronic Thesis and Dissertation Repository

Stomatin-like protein 2 (SLP-2), a member of the SPFH superfamily, is a mitochondrial inner membrane protein required for optimal mitochondrial respiration. SLP-2 binds to the important mitochondrial phospholipid cardiolipin (CL) and has been proposed to mediate formation of CL-enriched microdomains that would foster respiratory chain supercomplex (RCS) formation and stability. However, little is known about how SLP-2 structure facilitates its cellular function. The goal of this thesis was to elucidate if and how SLP-2 oligomerizes and by what means does it bind CL.

Biophysical analysis of the expressed SLP-2 SPFH domain, either with or without flanking residues, indicates it to …

High Molecular-Weight Hyaluronan Prevents Basal Cell Carcinoma Via Promoting Apoptosis In Cancer-Initiating Adult Stem Cells, Violet Liu

Electronic Thesis and Dissertation Repository

Basal cell carcinoma (BCC), a keratinocyte cancer, is the most common human neoplasm worldwide. Although rarely metastatic, BCC is associated with high morbidity rates with globally rising incidence rates. Accompanying the increase in newly diagnosed cases, the societal cost for BCC treatment in Canada is also expected to inflate, exceeding over $900 million/year by 2031. Chronic UVB exposure has been identified as the primary carcinogen that causes activating mutations in the hedgehog signaling pathway. However, there are no effective preventative methods against BCC, since meta-analyses report sunscreen application does not reduce BCC in compliant patients. The native high molecular-weight hyaluronan …

Synthesis And Characterization Of American Ginseng Polysaccharides Nanoparticles, Vincent Lee

Electronic Thesis and Dissertation Repository

This project was concerned with the synthesis of nanoparticles (NPs) by microfluidics from bulk ginseng polysaccharides (PS) isolated from American ginseng to design a new delivery system to improve the bioavailability of PS. Physicochemical analyses showed products of nanosizing as unimodal spheres with a diameter of ~19 nm. Pharmacological characterization studies in vitro of these nanoparticles of PS (NPPS) have demonstrated heightened immunostimulatory activity, and enhanced penetration across skin cell monolayer, which could be considered as evidence of increased bioavailability. Studies using PS sub-fractions with different molecular weights for NPPS synthesis showed that molecular weights is one of the parameters …

Mass Spectrometry-Based Proteomics Analysis Of Bioactive Proteins In Emd That Modulate Adhesion Of Gingival Fibroblast To Improve Bio-Integration Of Dental Implants, David Zuanazzi Machado Jr

Electronic Thesis and Dissertation Repository

Titanium implants are used in dental practice to replace damaged or lost teeth. The implant needs to integrate with the surrounding gingiva to protect it against bacterial invasion that leads to implant loss. The biointegration is dependent on the implant surface that interacts with proteins from biological fluids to modulate tissues response. Tailoring the surface with specific proteins from the enamel matrix derivative (EMD) would be beneficial to improve the implant-gingiva interface since EMD can affect various cells including gingival fibroblasts. A surface-affinity approach using three different titanium surfaces and saliva was utilized as a model in combination with tandem …