Matching Items (67)
Description
Heliobacteria are an anaerobic phototroph that require carbon sources such as pyruvate, <br/>lactate, or acetate for growth (Sattley, et. al. 2008). They are known for having one of the <br/>simplest phototrophic systems, the central component of which is a Type I reaction center (RC) <br/>that pumps protons to generate the electrochemical gradient for making ATP. Heliobacteria <br/>preform cyclic electron flow (CEF) with the RC in the light but can also grow chemotropically in <br/>the dark. Many anaerobes like heliobacteria, such as other members of the class Clostridia, <br/>possess the capability to produce hydrogen via a hydrogenase enzyme in the cell, as protons can <br/>serve as an electron acceptor in anaerobic metabolism. However, the species of heliobacteria <br/>studied here, H. modesticaldum have been seen to produce hydrogen via their nitrogenase <br/>enzyme but not when this enzyme is inactive. This study aimed to investigate if the reason for <br/>their lack of hydrogen production was due to a lack of an active hydrogenase enzyme, possibly <br/>indicating that the genes required for activity were lost by an H. modesticaldum ancestor. This <br/>was done by introducing genes encoding a clostridial [FeFe] hydrogenase from C. thermocellum<br/>via conjugation and measuring hydrogen production in the transformant cells. Transformant cells <br/>produced hydrogen and cells without the genes did not, meaning that the heliobacteria ferredoxin <br/>was capable of donating electrons to the foreign hydrogenase to make hydrogen. Because the <br/>[FeFe] hydrogenase must receive electrons from the cytosolic ferredoxin, it was hypothesized <br/>that hydrogen production in heliobacteria could be used to probe the redox state of the ferredoxin <br/>pool in conditions of varying electron availability. Results of this study showed that hydrogen <br/>production was affected by electron availability variations due to varying pyruvate <br/>concentrations in the media, light vs dark environment, use acetate as a carbon source, and being <br/>provided external electron donors. Hydrogen production, therefore, was predicted to be an <br/>effective indicator of electron availability in the reduced ferredoxin pool.
ContributorsVilaboy, Tatum (Author) / Redding, Kevin (Thesis director) / Ghirlanda, Giovanna (Committee member) / School of Life Sciences (Contributor) / School of Criminology and Criminal Justice (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
This dissertation describes work on three projects concerning the design and implementation of instrumentation used to study potential organic electronic devices. The first section describes the conducting atomic force microscope (CAFM) in the study of the mechanical and electronic interactions between DNA bases and nucleosides. Previous STM data suggested that an STM tip could recognize single base pairs through an electronic interaction after a functionalized tip made contact with a self assembled monolayer then was retracted. The conducting AFM was employed in order to understand the mechanical interactions of such a system and how they were affecting electrical responses. The results from the conducting AFM showed that the scanning probe system was measuring multiple base-pair interactions, and thus did not have single base resolution. Further, results showed that the conductance between a single base-nucleoside pair is below the detection limit of a potential commercial sequencing device. The second section describes the modifications of a scanning probe microscope in order to study the conductance of single organic molecules under illumination. Modifications to the scanning probe microscope are described as are the control and data analysis software for an experiment testing the single molecule conductance of an organic molecule under illumination. This instrument was then tested using a novel charge-separation molecule, which is being considered for its potential photovoltaic properties. The experiments showed that the instrumentation is capable of detecting differences in conductance upon laser illumination of the molecule on a transparent conductive surface. The third section describes measurements using the illuminated CAFM, as well as the design and construction of an illuminated mercury drop electrode apparatus. Both instruments were tested by attempting to observe photovoltaic behavior in a novel self-organized film of the charge-separation molecules mentioned in the previous paragraph. Results and calculations show that the conducting AFM is not a useful tool in the examination of these organic photovoltaics, while the mercury drop apparatus measured photovoltaic effects in the film. Although photovoltaic effects were measurable with the mercury drop electrode, it was found that the film exhibited very low photon-to-electron conversion efficiency (IPCE).
ContributorsKibel, Ashley Ann (Author) / Lindsay, Stuart M (Thesis advisor) / Chamberlin, Ralph (Committee member) / Moore, Thomas (Committee member) / Ozkan, Sefika (Committee member) / Sankey, Otto (Committee member) / Arizona State University (Publisher)
Created2010
Description
In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method.
ContributorsFu, Qiang (Author) / Lindsay, Stuart M (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
Description
Coronavirus disease 2019 (COVID-19), an illness caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), has been responsible for significant social and economic
disruption, prompting an urgent search for therapeutic solutions. The spike protein of the virus
has been examined as an immunogenic target because of its role in viral binding and fusion
necessary for infection of host cells. Previous studies have identified a recombinant protein
(denoted as S1) that has been shown to potentially induce a neutralizing antibody response by
mimicking the structure of the SARS-CoV-2 spike protein. We have produced the S1 in plants
using agroinfiltration, a plant transformation technique whereby plasmid-containing
Agrobacterium tumefaciens is injected into Nicotiana benthamiana plants, resulting in transfer of
the desired gene from bacteria to plant cells. S1 was expressed to high levels within 5 days of
infiltration, and Western blot analysis showed recognition of the S1 by an anti-S1 antibody.
ELISA results exhibited increased binding activity to anti-S1 with increasing concentrations of
S1, indicating their specific interaction. This ongoing study will demonstrate the potential of a
plant-produced S1 as a vaccine, therapeutic, and diagnostic tool against COVID-19 that is not
only effective, but also cost-efficient and scalable in comparison to conventional mammalian cell
culture production methods.
coronavirus 2 (SARS-CoV-2), has been responsible for significant social and economic
disruption, prompting an urgent search for therapeutic solutions. The spike protein of the virus
has been examined as an immunogenic target because of its role in viral binding and fusion
necessary for infection of host cells. Previous studies have identified a recombinant protein
(denoted as S1) that has been shown to potentially induce a neutralizing antibody response by
mimicking the structure of the SARS-CoV-2 spike protein. We have produced the S1 in plants
using agroinfiltration, a plant transformation technique whereby plasmid-containing
Agrobacterium tumefaciens is injected into Nicotiana benthamiana plants, resulting in transfer of
the desired gene from bacteria to plant cells. S1 was expressed to high levels within 5 days of
infiltration, and Western blot analysis showed recognition of the S1 by an anti-S1 antibody.
ELISA results exhibited increased binding activity to anti-S1 with increasing concentrations of
S1, indicating their specific interaction. This ongoing study will demonstrate the potential of a
plant-produced S1 as a vaccine, therapeutic, and diagnostic tool against COVID-19 that is not
only effective, but also cost-efficient and scalable in comparison to conventional mammalian cell
culture production methods.
ContributorsNguyen, Katherine (Author) / Chen, Qiang (Thesis director) / Ghirlanda, Giovanna (Committee member) / Jugler, Collin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-12
Description
The oxygen sensitivity of hydrogenase is a large barrier in maximizing the efficiency of algal hydrogen production, despite recent efforts aimed at rewiring photosynthesis. This project focuses on the role of photosystem II (PSII) in extended hydrogen production by cells expressing the PSI-HydA1 chimera, with the goal of optimizing continuous production of photobiohydrogen in the green alga, Chlamydomonas reinhardtii. Experiments utilizing an artificial PSII electron
Therefore, it can be concluded that downstream processes are limiting the electron flow to the hydrogenase. It was also shown that the use of a PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), at sub-saturating concentrations under light exposure during growth temporarily improves the duration of the H2 evolution phase. The maximal hydrogen production rate was found to be approximately 32 nmol h-1 (µg Chl)-1. Although downregulation of PSII activity with DCMU improves the long-term hydrogen production, future experiments must be focused on improving oxygen tolerance of the hydrogenase as a means for higher hydrogen yields.
Therefore, it can be concluded that downstream processes are limiting the electron flow to the hydrogenase. It was also shown that the use of a PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), at sub-saturating concentrations under light exposure during growth temporarily improves the duration of the H2 evolution phase. The maximal hydrogen production rate was found to be approximately 32 nmol h-1 (µg Chl)-1. Although downregulation of PSII activity with DCMU improves the long-term hydrogen production, future experiments must be focused on improving oxygen tolerance of the hydrogenase as a means for higher hydrogen yields.
ContributorsO'Boyle, Taryn Reilly (Author) / Redding, Kevin (Thesis director) / Ghirlanda, Giovanna (Committee member) / Vermaas, Willem (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Typical eukaryotic organelles use membranes formed by lipid bilayers in order to compartmentalize their functions within the cell. However, cells also contain membraneless organelles formed by intrinsically disordered proteins (IDPs) via liquid-liquid phase separation. The organelles form localized compartments that separate their contents from the environment.1 Here, this mechanism is used to generate artificial membraneless organelles that comprise a chemical reaction. An IDP, DEAD-box helicase (Ddx4), was bioconjugated to an enzyme, horseradish peroxidase (HRP), through the use of a bifunctional chemical linker, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), in order to examine if the enzyme could be incorporated in droplets and whether its activity would be affected. The conjugation of HRP-SMCC (43.4 kDa) to Ddx4 (25.6 kDa) was successful: SDS-PAGE analysis confirmed the presence of a product that was within the range of a full conjugate.
ContributorsFavila, Saul Roberto (Author) / Ghirlanda, Giovanna (Thesis director) / Vaiana, Sara (Committee member) / Allen, James (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The successful reduction of CO2 and protons by a light-induced cobalt porphyrin/cytb562 hybrid metalloenzyme in water is reported. Incorporation of the porphyrin into a protein scaffold results in increases in CO and H2 production over naked porphyrin. Rational point mutations to the CoPPIX binding site of cytb562 modulate production, indicating possible further improvements in catalytic activity.
ContributorsGwerder, Noah D (Author) / Ghirlanda, Giovanna (Thesis director) / Williams, Peter (Committee member) / Mangone, Marco (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin that specifically recognizes mannose-rich moieties, serves as a useful model for studying these glycan-recognition mechanisms. This study seeks to improve CVN's glycan-binding affinity by conjugating a boronic acid functional group to the N-terminus via N-terminal specific reductive alkylation by way of a benzaldehyde handle. However, large discrepancies were observed when attempting to confirm a successful conjugation, and further work is necessary to identify the causes and solutions for these issues.
ContributorsDiep, Tristan H (Author) / Ghirlanda, Giovanna (Thesis director) / Redding, Kevin (Committee member) / Mills, Jeremy (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of this study was to evaluate the effectiveness of an algorithm developed to predict regions of high-binding on proteins as it applies to determining the regions of interaction between binding partners. This approach was applied to tumor necrosis factor alpha (TNFα), its receptor TNFR2, programmed cell death protein-1 (PD-1), and one of its ligand PD-L1. The algorithms applied accurately predicted the binding region between TNFα and TNFR2 in which the interacting residues are sequential on TNFα, however failed to predict discontinuous regions of binding as accurately. The interface of PD-1 and PD-L1 contained continuous residues interacting with each other, however this region was predicted to bind weaker than the regions on the external portions of the molecules. Limitations of this approach include use of a linear search window (resulting in inability to predict discontinuous binding residues), and the use of proteins with unnaturally exposed regions, in the case of PD-1 and PD-L1 (resulting in observed interactions which would not occur normally). However, this method was overall very effective in utilizing the available information to make accurate predictions. The use of the microarray to obtain binding information and a computer algorithm to analyze is a versatile tool capable of being adapted to refine accuracy.
ContributorsBrooks, Meilia Catherine (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Ghirlanda, Giovanna (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Circulating tumor DNA analysis has several potential applications in cancer diagnostics. However, results in literature vary considerably, often due to different blood collection methods and protocols. Several new products to address pre-analytical variables of cfDNA processing have recently become available, and little is understood about their effects on DNA quality and downstream applications. We evaluated the effects of blood collection protocols and DNA extraction kits on cfDNA yield, quality, and fragment size using droplet-digital PCR (ddPCR) and targeted deep sequencing. To quantify cfDNA yield and size distribution, we developed a multiplexed ddPCR assay with FAM-labeled short amplicons and TET-labeled long amplicons targeting housekeeping genes. After assay validation, we compared the performance of several commercially-available cfDNA extraction kits using control plasma samples and different blood collection protocols using paired plasma samples from healthy volunteers. To assess whether cell-stabilizing preservative in Streck tubes may induce low-abundance noise in cfDNA, we performed molecularly-tagged targeted deep sequencing and developed an informatics approach for enumeration and variant calling from uniquely tagged DNA fragments. We found a significant difference between extraction methods but no significant difference across blood collection protocols in cfDNA yield or size distribution. Sequencing results showed no significant evidence of preservative-induced cfDNA damage across tested blood collection protocols. In summary, the multiplexed ddPCR assay enabled quantitative assessment of cfDNA extraction methods and blood collection protocols, and allowed for normalization of input to create reproducible sequencing libraries. Our results suggest that plasma samples processed up to 72 hours following venipuncture in Streck Cell-free DNA tubes may be used for downstream sequencing of circulating tumor DNA in patients with cancer.
ContributorsMarkus, Havell (Author) / Murtaza, Muhammed (Thesis director) / Ghirlanda, Giovanna (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12