Matching Items (24)
Filtering by

Clear all filters

133471-Thumbnail Image.png
Description
Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been

Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been slow due to limited structural information on Rca. Previous efforts in the lab were directed towards solving the structure of spinach short-form Rca using X-ray crystallography, given that it had notably high thermostability in the presence of ATP-γS, an ATP analog. However, due to disorder within the crystal lattice, an atomic resolution structure could not be obtained, prompting us to move to negative stain electron microscopy (EM), with our long-term goal being the use of cryo-electron microscopy (cryo-EM) for atomic resolution structure determination. Thus far, we have screened different Rca constructs in the presence of ATP-γS, both the full-length β-isoform and truncations containing only the AAA+ domain. Images collected on preparations of the full-length protein were amorphous, whereas images of the AAA+ domain showed well-defined ring-like assemblies under some conditions. Procedural adjustments, such as the use of previously frozen protein samples, rapid dilution, and minimizing thawing time were shown to improve complex assembly. The presence of Mn2+ was also found to improve hexamer formation over Mg2+. Calculated class averages of the AAA+ Rca construct in the presence of ATP-γS indicated a lack of homogeneity in the assemblies, showing both symmetric and asymmetric hexameric rings. To improve structural homogeneity, we tested buffer conditions containing either ADP alone or different ratios of ATP-γS to ADP, though results did not show a significant improvement in homogeneity. Multiple AAA+ domain preparations were evaluated. Because uniform protein assembly is a major requirement for structure solution by cryo-EM, more work needs to be done on screening biochemical conditions to optimize homogeneity.
ContributorsHernandez, Victoria Joan (Author) / Wachter, Rebekka (Thesis director) / Chiu, Po-Lin (Committee member) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
134935-Thumbnail Image.png
Description
The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature

The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.
ContributorsPaxton, Rebecca Joanne (Author) / Stephanopoulos, Nicholas (Thesis director) / He, Ximin (Committee member) / Gould, Ian (Committee member) / Materials Science and Engineering Program (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
148326-Thumbnail Image.png
Description

Stress for college students is nothing new and as more kids go to college the number of cases are on the rise. This issue is apparent at colleges across the nation including Arizona State University. StreetWise aims to help students prevent or appropriately deal with stress through interactive lessons teaching

Stress for college students is nothing new and as more kids go to college the number of cases are on the rise. This issue is apparent at colleges across the nation including Arizona State University. StreetWise aims to help students prevent or appropriately deal with stress through interactive lessons teaching students life skills, social skills, and emotional intelligence.<br/>In order to prove the value of our service, StreetWise conducted a survey that asked students about their habits, thoughts on stress, and their future. Students from Arizona State University were surveyed with questions on respondent background, employment, number one stressor, preferred learning method, and topics that students were interested in learning. We found that students’ number one stressor was school but was interested in learning skills that would prepare them for their future after graduation. We used the results to make final decisions so that StreetWise could offer lessons that students would get the most value out of. This led to us conducting a second survey which included mock ups of the website, examples of interactive lesson plans, and an overview of the app. Students from the first survey were surveyed in addition to new respondents. This survey was intended for us to ensure that our service would maintain its value to students with the aesthetic and interface that we envisioned.

ContributorsWard, William Henry (Co-author) / Ahir, Hiral (Co-author) / Compton, Katherine (Co-author) / Byrne, Jared (Thesis director) / Hall, Rick (Committee member) / Materials Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
148328-Thumbnail Image.png
Description

Stress for college students is nothing new and as more kids go to college the number of cases are on the rise. This issue is apparent at colleges across the nation including Arizona State University. StreetWise aims to help students prevent or appropriately deal with stress through interactive lessons teaching

Stress for college students is nothing new and as more kids go to college the number of cases are on the rise. This issue is apparent at colleges across the nation including Arizona State University. StreetWise aims to help students prevent or appropriately deal with stress through interactive lessons teaching students life skills, social skills, and emotional intelligence.<br/>In order to prove the value of our service, StreetWise conducted a survey that asked students about their habits, thoughts on stress, and their future. Students from Arizona State University were surveyed with questions on respondent background, employment, number one stressor, preferred learning method, and topics that students were interested in learning. We found that students’ number one stressor was school but was interested in learning skills that would prepare them for their future after graduation. We used the results to make final decisions so that StreetWise could offer lessons that students would get the most value out of. This led to us conducting a second survey which included mock ups of the website, examples of interactive lesson plans, and an overview of the app. Students from the first survey were surveyed in addition to new respondents. This survey was intended for us to ensure that our service would maintain its value to students with the aesthetic and interface that we envisioned.

ContributorsAhir, Hiral V (Co-author) / Compton, Katherine (Co-author) / Ward, William (Co-author) / Byrne, Jared (Thesis director) / Hall, Rick (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
148322-Thumbnail Image.png
Description

The field of biomedical research relies on the knowledge of binding interactions between various proteins of interest to create novel molecular targets for therapeutic purposes. While many of these interactions remain a mystery, knowledge of these properties and interactions could have significant medical applications in terms of understanding cell signaling

The field of biomedical research relies on the knowledge of binding interactions between various proteins of interest to create novel molecular targets for therapeutic purposes. While many of these interactions remain a mystery, knowledge of these properties and interactions could have significant medical applications in terms of understanding cell signaling and immunological defenses. Furthermore, there is evidence that machine learning and peptide microarrays can be used to make reliable predictions of where proteins could interact with each other without the definitive knowledge of the interactions. In this case, a neural network was used to predict the unknown binding interactions of TNFR2 onto LT-ɑ and TRAF2, and PD-L1 onto CD80, based off of the binding data from a sampling of protein-peptide interactions on a microarray. The accuracy and reliability of these predictions would rely on future research to confirm the interactions of these proteins, but the knowledge from these methods and predictions could have a future impact with regards to rational and structure-based drug design.

ContributorsPoweleit, Andrew Michael (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Chiu, Po-Lin (Committee member) / School of Molecular Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
168314-Thumbnail Image.png
Description
Cryogenic Electron Microscopy (Cryo-EM) is a method that can be used for studying the structure of biological systems. Biological samples are frozen to cryogenic temperatures and embedded in a vitreous ice when they are imaged by electrons. Due to its ability to preserve biological specimens in near-native conditions, cryo-EM has

Cryogenic Electron Microscopy (Cryo-EM) is a method that can be used for studying the structure of biological systems. Biological samples are frozen to cryogenic temperatures and embedded in a vitreous ice when they are imaged by electrons. Due to its ability to preserve biological specimens in near-native conditions, cryo-EM has a significant contribution to the field of structural biology.Single-particle cryo-EM technique was utilized to investigate the dynamical characteristics of various protein complexes such as the Nogo receptor complex, polymerase ζ (Polζ) in yeast and human integrin ⍺vβ8-pro-TGFβ1-GARP complex. Furthermore, I proposed a new method that can potentially improve the sample preparation for cryo-EM. The Nogo receptor complex was expressed using baculovirus expression system in sf9 insect cells and isolated for structural studies. Nogo receptor complex was found to have various stoichiometries and interactions between individual proteins. A structural investigation of the yeast apo polymerase ζ holoenzyme was also carried out. The apo Polζ displays a concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. Furthermore, a lysine residue that obstructs the DNA-binding channel in apo Polζ was found and suggested a gating mechanism. In addition, cryo-EM studies of the human integrin ⍺vβ8-pro-TGFβ1-GARP complex was conducted to assess its dynamic interactions. The 2D classifications showed the ⍺vβ8-pro-TGFβ1-GARP complex is highly flexible and required several sample preparation techniques such as crosslinking and graphene oxide coating to improve protein homogeneity on the EM grid. To overcome challenges within the cryo-EM technique such as particle adsorption on air-water interface, I have documented a collaborative work on the development and application of lipid monolayer sandwich on cryo-EM grid. Cryogenic electron tomography (cryo-ET) along with cryo-EM were used to study the characteristics of lipid monolayer sandwich as a potential protective layer for EM grid. The cryo-ET results demonstrated that the thickness of lipid monolayer is adequate for single-particle cryo-EM processing. Furthermore, there was no appearance of preferred orientations in cryo-EM and cryo-ET images. To establish that this method is actually beneficial, more data must be collected, and high-resolution structures of protein samples must be obtained using this methodology.
ContributorsTruong, Chloe Du (Author) / Chiu, Po-Lin (Thesis advisor) / Liu, Wei (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
168308-Thumbnail Image.png
Description
Structural-based drug discovery is becoming the essential tool for drug development withlower cost and higher efficiency compared to the conventional method. Knowledge of the three-dimensional structure of protein targets has the potential to accelerate the process for screening drug candidates. X-ray crystallography has proven to be the most used and indispensable technology in

Structural-based drug discovery is becoming the essential tool for drug development withlower cost and higher efficiency compared to the conventional method. Knowledge of the three-dimensional structure of protein targets has the potential to accelerate the process for screening drug candidates. X-ray crystallography has proven to be the most used and indispensable technology in structural-based drug discovery. The provided comprehensive structural information about the interaction between the disease-related protein target and ligand can guide the chemical modification on the ligand to improve potency and selectivity. X-ray crystallography has been upgraded from traditional synchrotron to the third generation, which enabled the surge of the structural determination of macromolecular. The introduction of X-ray free electron laser further alleviated the uncertain and time-consuming crystal size optimization process and extenuated the radiation damage by “diffraction before destruction”. EV-D68 2A protease was proved to be an important pharmaceutical target for acute flaccid myelitis. This thesis reports the first atomic structure of the EV-D68 2A protease and the structuresof its two mutants, revealing it adopting N-terminal four-stranded sheets and C-terminal six-stranded ß-barrels structure, with a tightly bound zinc atom. These structures will guide the chemical modification on its inhibitor, Telaprevir. Integrin ⍺Mβ2 is an integrin with the α I-domain, related to many immunological functions including cell extravasation, phagocytosis, and immune synapse formation, so studying the molecular ligand-binding mechanism and activation mechanism of ⍺Mβ2 is of importance. This thesis uncovers the preliminary crystallization condition of ⍺Mβ2-I domain in complex with its ligand Pleiotrophin and the initial structural model. The structural model shows consistency with the previous hypothesis that the primary binding sites are metal iondependent adhesion sites on ⍺Mβ2-I domain and the thrombospondin type-1 repeat (TSR) domains of Pleiotrophin. Drug molecules with high potency and selectivity can be designed based on the reported structures of the EV-D68 2A protease and ⍺Mβ2-I domain in the future.
ContributorsLiu, Chang (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
168752-Thumbnail Image.png
Description
The work in this dissertation progressed the research of structural discovery for two targets critical in the fight of infectious disease. Francisella lipoprotein 3 (Flpp3) is a virulent determinant of tularemia and was the first protein of study. The proteins soluble domain was studied using a hybrid modeling theory that

The work in this dissertation progressed the research of structural discovery for two targets critical in the fight of infectious disease. Francisella lipoprotein 3 (Flpp3) is a virulent determinant of tularemia and was the first protein of study. The proteins soluble domain was studied using a hybrid modeling theory that used small angle X-ray scattering (SAXS) in combination with computation analysis to generate a SAXS-refined structure. The SAXS-refined structure closely resembled the NMR structure (PDB: 2MU4) which contains a hydrophobic cavity inside the protein that could be used for drug discovery purposes. The full-length domain of Flpp3 purified from the outer membrane of E. coli was also studied with a combination of biophysical characterization methods. Mass spectrometry and western blot analysis confirmed Flpp3 being translocated to the outer membrane, while SDS-PAGE confirmed the purity of Flpp3 in the monomeric form after size exclusion chromatography. Using Circular Dichroism (CD) the monomeric form of Flpp3 was shown to be almost fully refolded into having a primarily β-stranded secondary structure. This information advances the progress of both tularemia research and outer membrane protein research as no natively folded outer membrane protein structures have been solved for F. tularensis.The second protein worked on in this dissertation is the nonstructural protein 15 from SARS-CoV-2, also called NendoU. Nsp15 is an endoribonuclease associated with aiding the virus responsible for the current COVID-19 pandemic in evasion of the immune system. An inactive mutant of Nsp15 was studied with both negative stain electron microscopy and cryogenic electron microscopy (Cryo-EM) in the presence of RNA or without RNA present. The initial findings of negative stain electron microscopy of Nsp15 with and without RNA showed a difference in appearance. Negative stain analysis of Nsp15 is in the presence of a 5nt RNA sequence in low salt conditions shows a conformational change when compared to Nsp15 without RNA present. As well the presence of RNA appeared to shift the electron density in Cryo-EM studies of Nsp15. This work advances the research in how Nsp15 may bind and cleave RNA and aid in the evasion of the host cell immune system.
ContributorsGoode, Matthew (Author) / Fromme, Petra (Thesis advisor) / Guo, Jia (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2022
168754-Thumbnail Image.png
Description
Infectious diseases are the third leading cause of death in the United States and the second leading cause of death in the world. This work aims to advance structural studies of vital proteins involved in the infection process of both a bacterial and a viral infectious disease in hopes of

Infectious diseases are the third leading cause of death in the United States and the second leading cause of death in the world. This work aims to advance structural studies of vital proteins involved in the infection process of both a bacterial and a viral infectious disease in hopes of reducing infection, and consequently, fatality rates. The first protein of interest is OspA, a major outer surface protein in Borrelia burgdorferi – the causative bacterium of Lyme disease. Previous functional studies of OspA allude to both a role in colonization of B. burgdorferi in the tick vector and in evasion of the human immune system. This work describes the first ever structural studies of OspA as it is seen by the immune system: in the outer membrane. OspA was expressed in and purified from the outer membrane of Escherichia coli prior to characterization via circular dichroism (CD), native polyacrylamide gel electrophoresis, and electron microscopy. Characterization studies of OspA provide the first evidence of multimeric formation of OspA when translocated to the outer membrane, which presents a new perspective from which to build upon for the design of vaccinations against Lyme disease. The second protein of interest is nonstructural protein 15 (Nsp15), a protein responsible for facilitating immune system evasion of SARS-CoV-2 – the virus responsible for the COVID-19 pandemic. Nsp15 functions to enzymatically cleave negative sense viral RNA to avoid recognition by the human immune system. The work described in this dissertation is dedicated to the electron microscopy work utilized to reveal structural information on an inactive variant of Nsp15 bound to RNA sequences. Negative stain electron microscopy was used to verify Nsp15 structural integrity, as well as reveal a low-resolution image of structural deviation when RNA is bound to Nsp15. Cryo-electron microscopy was performed to solve structural density of Nsp15 without RNA to a resolution of 3.11 Å and Nsp15 bound to 5-nucleotides of RNA to a resolution of 3.99 Å. With further refinement, this structure will show the first structural data of Nsp15 bound to a visible RNA sequence, revealing information on the binding and enzymatic activity of Nsp15.
ContributorsKaschner, Emily (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra T (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2022
Description

Alzheimer’s disease (AD) is a common neurodegenerative disorder affecting approximately 10% of people aged 65 and up and 30-50% over 85. In pathological AD representations, a way to recognize early onset AD is the increased levels of pro-NGF in BFCNs that come from the downregulation of NGF with age. Pro-NGF

Alzheimer’s disease (AD) is a common neurodegenerative disorder affecting approximately 10% of people aged 65 and up and 30-50% over 85. In pathological AD representations, a way to recognize early onset AD is the increased levels of pro-NGF in BFCNs that come from the downregulation of NGF with age. Pro-NGF has a higher affinity for p75NTR, which binds and participates in the pro-NGF-p75NTR-sortilin complex sequentially cleaved by α- and γ-secretase. Pro-NGF triggers apoptosis through the cleavage of the intracellular membrane by γ-secretase. Since γ-secretase physically cleaves off the intramembrane portion that promotes TNF- and Fas-dependent apoptotic signaling pathways, it has a crucial role in AD and must be better understood. This research aims to understand better and visualize γ-secretase and its actions, specifically with its interactions with the substrate p75NTR in the RIP process. To analyze γ-secretase function, the proteins must be produced and analyzed through the protein expression protocol. During protein production, DNA, cell concentrations, and optical density measurements were difficult to produce due to the incompetency of e. coli cells (DH5α), contamination of the Sf9 insect cell culture, and decreased viability of aged insect cells. We identified the problems and improved the conditions for future project development.

ContributorsRapacz, Elizabeth (Author) / Chiu, Po-Lin (Thesis director) / Van Horn, Wade (Committee member) / Munk, Barbara (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution & Social Change (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05