Global ETD Search

211	HOW TO BE A BAD HOST FOR VIRUSES BY UNDERSTANDING THE COMPLEXITIES OF HOST LIPID-VIRAL PROTEIN INTERACTIONS Emily A David (17583603) 10 December 2023 (has links) <p dir="ltr">The recent global pandemic, COVID-19, has revealed to all the importance of understanding the complex relationship between viruses and hosts. Before COVID-19, I started my study of viral protein-host lipid interactions in the hemorrhagic fevers Ebola and Marburg viruses. These viruses contain a matrix protein that interacts with the plasma membrane to facilitate the formation of both authentic viruses and virus-like particles. My goal was to understand the limitations of their specific host lipid interactions. However, when the COVID-19 pandemic began, so to be our swift response in the development of a biosafety level 2 compatible model. This model can be used for studying severe acute respiratory distress syndrome 2 (SARS-CoV-2) assembly, egress, and entry. This model enabled exponentially greater access to more facilities to study the intricacies of SARS-CoV-2 assembly. With more access to studying the virus in a safe model, our goal is to push the understanding of viral assembly faster. I then began to take apart the individual pieces of the model and started to look at understanding the roles that they play independently. The membrane protein is the most abundant structural protein and I studied the specific lipid interactions of the soluble fraction of the protein. Physicians observed nucleocapsid protein mutations in the clinic with the increasing number of SARS-CoV-2 variants that are on the rise. The microscopy data collected can give us more insight into perhaps how the nucleocapsid protein induces the formation of filopodia structures at the plasma membrane. The envelope protein proved to be a challenge, but I determined a specific envelope and ceramide interaction in cells. The envelope protein was also causing the formation of microvesicles for an undefined function. I was able to determine the subcellular localization of the protein to the mitochondria. The localization to the mitochondria appears to induce depolarization of the mitochondria membrane action potential and induces the increase in mitochondria dysfunction signal, cytochrome c. Although the mitochondria were dysfunctional, there was no increase in apoptosis signal in the presence of the protein alone.</p> Enzymes Protein trafficking Virology Medical biochemistry - lipids Medical virology Cell physiology Basic pharmacology BSL-2 Virus-like particle VLP SARS-CoV-2 EBOV (Ebola virus) mitochondrial membrane localization lipid interaction studies ceramide scanning electron microscopy methodology SARS-CoV-2-N SARS-CoV-2-E SARS-CoV-2-M VP40
212	Creation, deconstruction, and evaluation of a biochemistry animation about the role of the actin cytoskeleton in cell motility Kevin Wee (11198013) 28 July 2021 (has links) <p>External representations (ERs) used in science education are multimodal ensembles consisting of design elements to convey educational meanings to the audience. As an example of a dynamic ER, an animation presenting its content features (i.e., scientific concepts) via varying the feature’s depiction over time. A production team invited the dissertation author to inspect their creation of a biochemistry animation about the role of the actin cytoskeleton in cell motility and the animation’s implication on learning. To address this, the author developed a four-step methodology entitled the Multimodal Variation Analysis of Dynamic External Representations (MVADER) that deconstructs the animation’s content and design to inspect how each content feature is conveyed via the animation’s design elements.</p><p><br></p><p> </p><p>This dissertation research investigated the actin animation’s educational value and the MVADER’s utility in animation evaluation. The research design was guided by descriptive case study methodology and an integrated framework consisting of the variation theory, multimodal analysis, and visual analytics. As stated above, the animation was analyzed using MVADER. The development of the actin animation and the content features the production team members intended to convey via the animation were studied by analyzing the communication records between the members, observing the team meetings, and interviewing the members individually. Furthermore, students’ learning experiences from watching the animation were examined via semi-structured interviews coupled with post- storyboarding. Moreover, the instructions of MVADER and its applications in studying the actin animation were reviewed to determine the MVADER’s usefulness as an animation evaluation tool.</p><p><br></p><p> </p><p>Findings of this research indicate that the three educators in the production team intended the actin animation to convey forty-three content features to the undergraduate biology students. At least 50% of the student who participated in this thesis learned thirty-five of these forty-three (> 80%) features. Evidence suggests that the animation’s effectiveness to convey its features was associated with the features’ depiction time, the number of identified design elements applied to depict the features, and the features’ variation of depiction over time.</p><p><br></p><p>Additionally, one-third of the student participants made similar mistakes regarding two content features after watching the actin animation: the F-actin elongation and the F-actin crosslink structure in lamellipodia. The analysis reveals the animation’s potential design flaws that might have contributed to these common misconceptions. Furthermore, two disruptors to the creation process and the educational value of the actin animation were identified: the vagueness of the learning goals and the designer’s placement of the animation’s beauty over its reach to the learning goals. The vagueness of the learning goals hampered the narration scripting process. On the other hand, the designer’s prioritization of the animation’s aesthetic led to the inclusion of a “beauty shot” in the animation that caused students’ confusion.</p><p><br></p><p> </p><p>MVADER was used to examine the content, design, and their relationships in the actin animation at multiple aspects and granularities. The result of MVADER was compared with the students’ learning outcomes from watching the animation to identify the characteristics of content’s depiction that were constructive and disruptive to learning. These findings led to several practical recommendations to teach using the actin animation and create educational ERs.</p><p><br></p><p> </p><p>To conclude, this dissertation discloses the connections between the creation process, the content and design, and the educational implication of a biochemistry animation. It also introduces MVADER as a novel ER analysis tool to the education research and visualization communities. MVADER can be applied in various formats of static and dynamic ERs and beyond the disciplines of biology and chemistry.</p> Biochemistry Design Education Media Studies Animal Cell and Molecular Biology Computer Graphics Higher Education Time-Series Analysis Education not elsewhere classified Computer Gaming and Animation Education Assessment and Evaluation Educational Technology and Computing chemistry education research Chemistry Education biochemistry education biology education research Education research methodology data visualization methods data visualizations Visual analytics Tableau Desktop Animation (Cinematography) multimedia research Research methods Mixed method research design media research Science education - assessment Movie analysis
213	TARGETED DELIVERY OF BONE ANABOLICS TO BONE FRACTURES FOR ACCELERATED HEALING Jeffery J H Nielsen (8787002) 21 June 2022 (has links) <div>Delayed fracture healing is a major health issue involved with aging. Therefore, strategies to improve the pace of repair and prevent non-union are needed in order to improve patient outcomes and lower healthcare costs. In order to accelerate bone fracture healing noninvasively, we sought to develop a drug delivery system that could safely and effectively be used to deliver therapeutics to the site of a bone fracture. We elected to pursue the promising strategy of using small-molecule drug conjugates that deliver therapeutics to bone in an attempt to increase the efficacy and safety of drugs for treating bone-related diseases.</div><div>This strategy also opened the door for new methods of administering drugs. Traditionally, administering bone anabolic agents to treat bone fractures has relied entirely on local surgical application. However, because it is so invasive, this method’s use and development has been limited. By conjugating bone anabolic agents to bone-homing molecules, bone fracture treatment can be performed through minimally invasive subcutaneous administration. The exposure of raw hydroxyapatite that occurs with a bone fracture allows these high-affinity molecules to chelate the calcium component of hydroxyapatite and localize primarily to the fracture site.</div><div>Many bone-homing molecules (such as bisphosphonates and tetracycline targeting) have been developed to treat osteoporosis. However, many of these molecules have toxicity associated with them. We have found that short oligopeptides of acidic amino acids can localize to bone fractures with high selectivity and with very low toxicity compared to bisphosphonates and tetracyclines.</div><div>We have also demonstrated that these molecules can be used to target peptides of all chemical classes: hydrophobic, neutral, cationic, anionic, short, and long. This ability is particularly useful because many bone anabolics are peptidic in nature. We have found that acidic oligopeptides have better persistence at the site of the fracture than bisphosphonate-targeted therapeutics. This method allows for a systemic administration of bone anabolics to treat bone fractures, which it achieves by accumulating the bone anabolic at the fracture site. It also opens the door for a new way of treating the prevalent afflictions of broken bones and the deaths associated with them.</div><div>We further developed this technology by using it to deliver anabolic peptides derived from growth factors, angiogenic agents, neuropeptides, and extracellular matrix fragments. We found several promising therapeutics that accelerated the healing of bone fractures by improving the mineralization of the callus and improving the overall strength. We optimized the performance of these molecules by improving their stability, targeting ligands, linkers, dose, and dosing frequency.</div><div>We also found that these therapeutics could be used to accelerate bone fracture repair even in the presence of severe comorbidities (such as diabetes and osteoporosis) that typically slow the repair process. We found that, unlike the currently approved therapeutic for fracture healing (BMP2), our therapeutics improved functionality and reduced pain in addition to strengthening the bone. These optimized targeted bone anabolics were not only effective at healing bone fractures but they also demonstrated that they could be used to speed up spinal fusion. Additionally, we demonstrated that acidic oligopeptides have potential to be used to treat other bone diseases with damaged bone.</div><div>With these targeted therapeutics, we no longer have to limit bone fracture healing to casts or invasive surgeries. Rather, we can apply these promising therapeutics that can be administered non-invasively to augment existing orthopedic practices. As these therapeutics move into clinical development, we anticipate that they will be able to reduce the immobilization time that is the source of so many of the deadly complications associated with bone fracture healing, particularly in the elderly.</div> Genetics not elsewhere classified Nanobiotechnology Animal behaviour Clinical microbiology Endocrinology Nanomedicine Regenerative medicine (incl. stem cells) Solid state chemistry Molecular medicine Proteins and peptides Solution chemistry Radiation and matter Biomaterials Biomechanical engineering bone fracture healing Anabolic Agents/pharmacology Targeted Drug DeliveryDesign Bone fracture targeted drugs Bone theapeutics targeted therapies Growth factors Neuropepetides Extracellular matrix Extracellular matrix fragments spinal fusion Angiogensis Acidic oligopeptides osteoporosic fractures diabetic bone fractures Integrin alpha 5 Acelerated Bone fracture healing Hydroxyapatite Hydroxyapatite targeting Targeting peptides Peptide drugs osteomyelitis (OM) Rheumatoid arthritis Bone fractures osteogenic therapies osteotropic osteotropic nanomedicines osteoinductive capacity Molecular Medicine Organic Chemistry Proteins and Peptides Radiochemistry Solid State Chemistry Solution Chemistry Biochemistry Animal Behaviour Biological Engineering Biotechnology Genetics Medicine Pharmacology Biomaterials Biomechanical Engineering Biomechanics Endocrinology Nanobiotechnology Nanomedicine
214	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760
215	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760

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