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- All Subjects: Biochemistry
Description
It has been well established that mitochondria play a critical role in the pathology of Friedreich's Ataxia. This disease is believed to be caused by a deficiency of frataxin, which research suggests is responsible for iron sulfur cluster assembly. This incomplete assembly of iron sulfur clusters is believed to be linked with dysfunctional complexes in the mitochondrial respiratory chain, increased oxidative stress, and potential cell death. Increased understanding of the pathophysiology of this disease has enabled the development of various therapeutic strategies aimed at restoring mitochondrial respiration. This thesis contains an analysis of the biological activity of several classes of antioxidants against oxidative stress induced by diethyl maleate in Friedreich's Ataxia lymphocytes and CEM leukemia cells. Analogues of vitamin E α-tocopherol have been shown to protect cells under oxidative stress. However, these same analogues show various levels of inhibition towards the electron transport chain complex I. Bicyclic pyridinols containing a ten carbon substituent provided favorable cytoprotection. N-hydroxy-4-pyridone compounds were observed to provide little protection. Similarly, analogues of CoQ10 in the form of pyridinol and pyrimidinol compounds also preserved cell viability at low concentrations.
ContributorsJaruvangsanti, Jennifer (Author) / Hecht, Sidney (Thesis advisor) / Woodbury, Neal (Committee member) / Skibo, Edward (Committee member) / Arizona State University (Publisher)
Created2012
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 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
Description
With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in which photons of light are captured, converted into chemically useful forms, and stored for biological use, an investigation into the reaction center protein, specifically into its cascade of cofactors, was undertaken. The purpose of this experimentation was to advance our knowledge and understanding of how differing protein environments and variant cofactors affect the spectroscopic aspects of and electron transfer kinetics within the reaction of Rh. sphaeroides. The native quinone, ubiquinone, was extracted from its pocket within the reaction center protein and replaced by non-native quinones having different reduction/oxidation potentials. It was determined that, of the two non-native quinones tested—1,2-naphthaquinone and 9,10- anthraquinone—the substitution of the anthraquinone (lower redox potential) resulted in an increased rate of recombination from the P+QA- charge-separated state, while the substitution of the napthaquinone (higher redox potential) resulted in a decreased rate of recombination.
ContributorsSussman, Hallie Rebecca (Author) / Woodbury, Neal (Thesis director) / Redding, Kevin (Committee member) / Lin, Su (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Enzyme Replacement Therapy (ERT) is a treatment often used for patients with disorders that affect the production of various enzymes within the body, such as Cystic Fibrosis and Fabry Disease. ERT involves the use of artificially-produced enzymes, which can be derived from humans, pigs, and bacteria. Generally, enzymes derived from porcine and bacterial sources are much less expensive and more accessible than those derived from a human source. This, and the ethical implications that porcine enzymes carry, make the decision of choosing treatment simple to some and complex to others. Ethically, human-derived enzymes are often considered more ethical, while not conflicting with religious beliefs and practices as porcine-derived enzymes do.
In order to further compare porcine and human-derived enzymes, a determination of the enzyme effectiveness was done via digestion simulation. The digestion for both the human and porcine-derived enzymes consisted of three steps: oral, gastric, and intestinal. After the digestion, the absorbance for each enzyme class as well as a dilution curve of the formula used was read and recorded. Using the standard dilution curve and the absorbance values for each unknown, the formula and thus enzyme concentration that was lost through the reaction was able to be calculated.
The effectiveness of both the human and porcine enzymes, determined by the percent of formula lost, was 18.2% and 19.7%, respectively, with an error of 0.6% from the spectrophotometer, and an error of about 10% from the scale used for measuring the enzymes. This error was likely due to the small mass required of the enzymes and can be prevented in the future by performing the experiment at a larger scale.
In order to further compare porcine and human-derived enzymes, a determination of the enzyme effectiveness was done via digestion simulation. The digestion for both the human and porcine-derived enzymes consisted of three steps: oral, gastric, and intestinal. After the digestion, the absorbance for each enzyme class as well as a dilution curve of the formula used was read and recorded. Using the standard dilution curve and the absorbance values for each unknown, the formula and thus enzyme concentration that was lost through the reaction was able to be calculated.
The effectiveness of both the human and porcine enzymes, determined by the percent of formula lost, was 18.2% and 19.7%, respectively, with an error of 0.6% from the spectrophotometer, and an error of about 10% from the scale used for measuring the enzymes. This error was likely due to the small mass required of the enzymes and can be prevented in the future by performing the experiment at a larger scale.
ContributorsBlevins, Brianna R (Author) / Martin, Thomas (Thesis director) / McILwraith, Heide (Committee member) / College of Integrative Sciences and Arts (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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
The devastating 2014 Ebola virus outbreak in Western Africa demonstrated the lack of therapeutic approaches available for the virus. Although monoclonal antibodies (mAb) and other molecules have been developed that bind the virus, no therapeutic has shown the efficacy needed for FDA approval. Here, a library of 50 peptide based ligands that bind the glycoprotein of the Zaire Ebola virus (GP) were developed. Using whole virus screening of vesicular stomatitis virus pseudotyped with GP, low affinity peptides were identified for ligand construction. In depth analysis showed that two of the peptide based molecules bound the Zaire GP with <100 nM KD. One of these two ligands was blocked by a known neutralizing mAb, 2G4, and showed cross-reactivity to the Sudan GP. This work presents ligands with promise for therapeutic applications across multiple variants of the Ebola virus.
ContributorsRabinowitz, Joshua Avraam (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Particulate Guanylyl Cyclase Receptor A (pGC-A) is an atrial natriuretic peptide receptor, which plays a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain of pGC-A interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop a method that can regulate pGC-A, structural information regarding its intact form is necessary. Currently, only the extracellular domain structure of rat pGC-A has been determined. However, structural data regarding the transmembrane domain, as well as functional intracellular domain regions, need to be elucidated.This dissertation presents detailed information regarding pGC-A expression and optimization in the baculovirus expression vector system, along with the first purification method for purifying functional intact human pGC-A. The first in vitro evidence of a purified intact human pGC-A tetramer was detected in detergent micellar solution. Intact pGC-A is currently proposed to function as a homodimer. Upon analyzing my findings and acknowledging that dimer formation is required for pGC-A functionality, I proposed the first tetramer complex model composed of two functional subunits (homodimer). Forming tetramer complexes on the cell membrane increases pGC-A binding efficiency and ligand sensitivity.
Currently, a two-step mechanism has been proposed for ATP-dependent pGC-A signal transduction. Based on cGMP functional assay results, it can be suggested that the binding ligand also moderately activates pGC-A, and that ATP is not crucial for the activation of guanylyl cyclase. Instead, three modulators can regulate different activation levels in intact pGC-A.
Crystallization of purified intact pGC-A was performed to determine its structure. During the crystallization condition screening process, I successfully selected seven promising initial crystallization conditions for intact human pGC-A crystallization. One selected condition led to the formation of excellent needle-shaped crystals. During the serial crystallography diffraction experiment, five diffraction patterns were detected. The highest diffraction resolution spot reached 3 Å.
This work will allow the determination of the intact human pGC-A structure while also providing structural information on the protein signal transduction mechanism. Further structural knowledge may potentially lead to improved drug design. More precise mutation experiments could help verify the current pGC-A signal transduction and activation mechanism.
ContributorsZhang, Shangji (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
Description
The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems.
This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli.
This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli.
ContributorsZhou, Yu (Ph.D.) (Author) / Yan, Hao (Thesis advisor) / Green, Alexander (Thesis advisor) / Woodbury, Neal (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2019
Description
Glycans are monosaccharide-based heteropolymers that are found covalently attached to many different proteins and lipids and are ubiquitously displayed on the exterior surfaces of cells. Serum glycan composition and structure are well known to be altered in many different types of cancer. In fact, glycans represent a promising but only marginally accessed source of cancer markers. The approach used in this dissertation, which is referred to as “glycan node analysis”, is a molecularly bottom-up approach to plasma/serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, β1-6 branching, and core fucosylation as single analytical signals.
The diagnostic utility of this approach as applied to lung cancer patients across all stages as well as prostate, serous ovarian, and pancreatic cancer patients compared to certifiably healthy individuals, nominally healthy individuals and/or risk-matched controls is reported. Markers for terminal fucosylation, α2-6 sialylation, β1-4 branching, β1-6 branching and outer-arm fucosylation were most able to differentiate cases from controls. These markers behaved in a stage-dependent manner in lung cancer as well as other types of cancer. Using a Cox proportional hazards regression model, the ability of these markers to predict progression and survival in lung cancer patients was assessed. In addition, the potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.
Plasma samples from former bladder cancer patients with currently no evidence of disease (NED), non-muscle invasive bladder cancer (NMIBC), and muscle invasive bladder cancer (MIBC) along with certifiably healthy controls were analyzed. Markers for α2-6 sialylation, β1-4 branching, β1-6 branching, and outer-arm fucosylation were able to separate current and former (NED) cases from controls; but NED, NMIBC, and MIBC were not distinguished from one another. Markers for α2-6 sialylation and β1-6 branching were able to predict recurrence from the NED state using a Cox proportional hazards regression model adjusted for age, gender, and time from cancer. These two glycan features were found to be correlated to the concentration of C-reactive protein, a known prognostic marker for bladder cancer, further strengthening the link between inflammation and abnormal plasma protein glycosylation.
The diagnostic utility of this approach as applied to lung cancer patients across all stages as well as prostate, serous ovarian, and pancreatic cancer patients compared to certifiably healthy individuals, nominally healthy individuals and/or risk-matched controls is reported. Markers for terminal fucosylation, α2-6 sialylation, β1-4 branching, β1-6 branching and outer-arm fucosylation were most able to differentiate cases from controls. These markers behaved in a stage-dependent manner in lung cancer as well as other types of cancer. Using a Cox proportional hazards regression model, the ability of these markers to predict progression and survival in lung cancer patients was assessed. In addition, the potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.
Plasma samples from former bladder cancer patients with currently no evidence of disease (NED), non-muscle invasive bladder cancer (NMIBC), and muscle invasive bladder cancer (MIBC) along with certifiably healthy controls were analyzed. Markers for α2-6 sialylation, β1-4 branching, β1-6 branching, and outer-arm fucosylation were able to separate current and former (NED) cases from controls; but NED, NMIBC, and MIBC were not distinguished from one another. Markers for α2-6 sialylation and β1-6 branching were able to predict recurrence from the NED state using a Cox proportional hazards regression model adjusted for age, gender, and time from cancer. These two glycan features were found to be correlated to the concentration of C-reactive protein, a known prognostic marker for bladder cancer, further strengthening the link between inflammation and abnormal plasma protein glycosylation.
ContributorsRoshdiferdosi, Shadi (Author) / Borges, Chad R (Thesis advisor) / Woodbury, Neal (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2018
Description
Though DNA nanostructures (DNs) have become interesting subjects of drug delivery, in vivo imaging and biosensor research, however, for real biological applications, they should be ‘long circulating’ in blood. One of the crucial requirements for DN stability is high salt concentration (like ~5–20 mM Mg2+) that is unavailable in a cell culture medium or in blood. Hence DNs denature promptly when injected into living systems. Another important factor is the presence of nucleases that cause fast degradation of unprotected DNs. The third factor is ‘opsonization’ which is the immune process by which phagocytes target foreign particles introduced into the bloodstream. The primary aim of this thesis is to design strategies that can improve the in vivo stability of DNs, thus improving their pharmacodynamics and biodistribution.
Several strategies were investigated to address the three previously mentioned limitations. The first attempt was to study the effect length and conformation of polyethylene glycol (PEG) on DN stability. DNs were also coated with PEG-lipid and human serum albumin (HSA) and their stealth efficiencies were compared. The findings reveal that both PEGylation and albumin coating enhance low salt stability, increase resistance towards nuclease action and reduce uptake of DNs by macrophages. Any protective coating around a DN increases its hydrodynamic radius, which is a crucial parameter influencing their clearance. Keeping this in mind, intrinsically stable DNs that can survive low salt concentration without any polymer coating were built. Several DNA compaction agents and DNA binders were screened to stabilize DNs in low magnesium conditions. Among them arginine, lysine, bis-lysine and hexamine cobalt showed the potential to enhance DN stability.
This thesis also presents a sensitive assay, the Proximity Ligation Assay (PLA), for the estimation of DN stability with time. It requires very simple modifications on the DNs and it can yield precise results from a very small amount of sample. The applicability of PLA was successfully tested on several DNs ranging from a simple wireframe tetrahedron to a 3D origami and the protocol to collect in vivo samples, isolate the DNs and measure their stability was developed.
Several strategies were investigated to address the three previously mentioned limitations. The first attempt was to study the effect length and conformation of polyethylene glycol (PEG) on DN stability. DNs were also coated with PEG-lipid and human serum albumin (HSA) and their stealth efficiencies were compared. The findings reveal that both PEGylation and albumin coating enhance low salt stability, increase resistance towards nuclease action and reduce uptake of DNs by macrophages. Any protective coating around a DN increases its hydrodynamic radius, which is a crucial parameter influencing their clearance. Keeping this in mind, intrinsically stable DNs that can survive low salt concentration without any polymer coating were built. Several DNA compaction agents and DNA binders were screened to stabilize DNs in low magnesium conditions. Among them arginine, lysine, bis-lysine and hexamine cobalt showed the potential to enhance DN stability.
This thesis also presents a sensitive assay, the Proximity Ligation Assay (PLA), for the estimation of DN stability with time. It requires very simple modifications on the DNs and it can yield precise results from a very small amount of sample. The applicability of PLA was successfully tested on several DNs ranging from a simple wireframe tetrahedron to a 3D origami and the protocol to collect in vivo samples, isolate the DNs and measure their stability was developed.
ContributorsBanerjee, Saswata (Author) / Yan, Hao (Thesis advisor) / Angell, Austen (Committee member) / Woodbury, Neal (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2018