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Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study in order to prevent HCV from harming people's health. The envelope protein 2 (E2) of HCV is thought to be a promising vaccine candidate because it can directly bind to a human cell receptor and plays a role in viral entry. However, the E2 protein production in cells is inefficient due to its complicated matured structure. Folding of E2 in the endoplasmic reticulum (ER) is often error-prone, resulting in production of aggregates and misfolded proteins. These incorrect forms of E2 are not functional because they are not able to bind to human cells and stimulate antibody response to inhibit this binding. This study is aimed to overcome the difficulties of HCV E2 production in plant system. Protein folding in the ER requires great assistance from molecular chaperones. Thus, in this study, two molecular chaperones in the ER, calreticulin and calnexin, were transiently overexpressed in plant leaves in order to facilitate E2 folding and production. Both of them showed benefits in increasing the yield of E2 and improving the quality of E2. In addition, poorly folded E2 accumulated in the ER may cause stress in the ER and trigger transcriptional activation of ER molecular chaperones. Therefore, a transcription factor involved in this pathway, named bZIP60, was also overexpressed in plant leaves, aiming at up-regulating a major family of molecular chaperones called BiP to assist protein folding. However, our results showed that BiP mRNA levels were not up-regulated by bZIP60, but they increased in response to E2 expression. The Western blot analysis also showed that overexpression of bZIP60 had a small effect on promoting E2 folding. Overall, this study suggested that increasing the level of specific ER molecular chaperones was an effective way to promote HCV E2 protein production and maturation.
ContributorsHong, Fan (Author) / Mason, Hugh (Thesis advisor) / Gaxiola, Roberto (Committee member) / Chang, Yung (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2011
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
This project examined the relationship of science teachers' knowledge about the laws relating to the teaching of creationism/evolution in public schools using multiple demographic factors. Overall, teachers correctly identified only 7 out of 10 "yes" or "no" answers about the laws, this score is only slightly better than the expected 5 out of 10 that would be obtained from guessing. Statistically significant results in differences in the overall score on the survey were found for three major variables. Teachers who say creationism should be taught in the classroom have a lower score than those who say it should not be taught in the classroom, with a large effect size. Teachers who teach biology or a life science had significantly higher scores than those who do not, with a small/medium effect size. Older teachers had significantly higher scores than younger teachers, with a small effect size. Identifying the demographic variables that effect teacher knowledge about the laws is the first step to determining how to educate teachers on the legality teaching of creationism/evolution in public school classrooms to avoid violations of the First Amendment.
ContributorsSorge, Aidan Bennet (Author) / Parker, John (Thesis director) / Lynch, John (Committee member) / School for the Future of Innovation in Society (Contributor) / Department of English (Contributor) / College of Integrative Sciences and Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Abstract
Purpose—Use a framework of genetic knowledge to investigate the association between the genotypes of various genes with phenotypes, specifically the traits of elite athletes, in order to establish a personal opinion on their relevance to athletic performance.
Methods—Assemble and analyze selected published scientific studies on genotype and athletic performance and lastly to formulate a personal opinion on the value of genetic testing of athletes. ACTN3, ACE, MSTN, and apoE were the genes selected for analyses.
Results—Two genes, ACTN3 and ACE, showed a significant relationship of genotype to phenotypic traits related to athletic performance. ApoE did not demonstrate a phenotypic association with athletic performance, however it showed a correlation with injury susceptibility leading to traumatic brain injury (TBI). MSTN did not show a phenotypic association with athletic performance.
Conclusion—When considering the multifactorial nature of athletics, each sport must be investigated individually due to the different individual requirements. ACTN3 and ACE are the most widely studied genes, therefore, considerable data on their relevance to athletic performance was easily obtained and supported a relationship between genotype and athletic performance.
Purpose—Use a framework of genetic knowledge to investigate the association between the genotypes of various genes with phenotypes, specifically the traits of elite athletes, in order to establish a personal opinion on their relevance to athletic performance.
Methods—Assemble and analyze selected published scientific studies on genotype and athletic performance and lastly to formulate a personal opinion on the value of genetic testing of athletes. ACTN3, ACE, MSTN, and apoE were the genes selected for analyses.
Results—Two genes, ACTN3 and ACE, showed a significant relationship of genotype to phenotypic traits related to athletic performance. ApoE did not demonstrate a phenotypic association with athletic performance, however it showed a correlation with injury susceptibility leading to traumatic brain injury (TBI). MSTN did not show a phenotypic association with athletic performance.
Conclusion—When considering the multifactorial nature of athletics, each sport must be investigated individually due to the different individual requirements. ACTN3 and ACE are the most widely studied genes, therefore, considerable data on their relevance to athletic performance was easily obtained and supported a relationship between genotype and athletic performance.
ContributorsMinto, Jordan Taylor- Lloyd (Author) / Steele, Kelly (Thesis director) / Penton, C. Ryan (Committee member) / College of Integrative Sciences and Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
It is presently believed that brown adipose tissue (BAT) is an important tissue in the control of obesity because it has the propensity to increase energy expenditure. The purpose of this study was to attempt to quantify the thermogenesis of BAT when four rats were exposed to a progression of low-fat to high-fat diet. Exogenous norepinephrine (NE) injections (dose of 0.25 mg/kg i.p.) were administered in order to elicit a temperature response, where increases in temperature indicate increased activity. Temperatures were measured via temperature sensing transponders that had been inserted at the following three sites: interscapular BAT (iBAT), the abdomen (core), and lower back (reference). Data showed increased BAT activity during acute (2-3 weeks) high fat diet (HFD) in comparison to low fat diet (LFD), but a moderate to marked decrease in BAT activity during chronic HFD (6-8 weeks) when compared to acute HFD. This suggests that while a HFD may initially stimulate BAT in the short-term, a long-term HFD diet may have negative effects on BAT activation.
ContributorsSivak, Hanna (Author) / Sweazea, Karen (Thesis director) / Herman, Richard (Committee member) / Caplan, Michael (Committee member) / School of Life Sciences (Contributor) / College of Integrative Sciences and Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
"Going back as far as the time of Hippocrates, ancient Egyptians, tribal African nations, and many other early civilizations, humans used herbal remedies to treat their ailments. One such remedy was willow bark, used in tea form, to treat rheumatism and fevers. This remedy was around for many thousands of years, along with other treatments containing salicylates, although this was not understood at the time. As time has gone on, the willow bark tea has been transformed into aspirin as we know it today. In addition to its medicinal uses, aspirin has become versatile in its uses, including use in homemade facial treatments and in the garden. As beneficial as aspirin has been, there are negative consequences to its use, particularly in young children, and it may have strange effects on gender when used by pregnant women. From such humble beginnings, aspirin has been shown to be more than a simple painkiller." Topics discussed in this paper include: the origins of aspirin and its use as a medical treatment, the beginnings of aspirin as it is known today, how aspirin interacts with the body, the specific chemical reactions that occur when aspirin is taken, aspirin as part of a heart health regimen, the possible uses of aspirin in treating cancer, general information about dosages and typical aspirin use, some side effects of aspirin use, and novel uses of aspirin that are not necessarily medical in nature. The beneficial nature of aspirin and the possibilities it presents are discussed alongside information about its potential limitations and negative effects.
ContributorsMontes, Ariana (Author) / Huffman, Holly (Thesis director) / Garg, Vikas (Committee member) / College of Integrative Sciences and Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Cardiovascular disease (CVD) remains the leading cause of mortality, resulting in 1 out of 4 deaths in the United States at the alarming rate of 1 death every 36 seconds, despite great efforts in ongoing research. In vitro research to study CVDs has had limited success, due to lack of biomimicry and structural complexity of 2D models. As such, there is a critical need to develop a 3D, biomimetic human cardiac tissue within precisely engineered in vitro platforms. This PhD dissertation involved development of an innovative anisotropic 3D human stem cell-derived cardiac tissue on-a-chip model (i.e., heart on-a-chip), with an enhanced maturation tissue state, as demonstrated through extensive biological assessments. To demonstrate the potential of the platform to study cardiac-specific diseases, the developed heart on-a-chip was used to model myocardial infarction (MI) due to exposure to hypoxia. The successful induction of MI on-a-chip (heart attack-on-a-chip) was evidenced through fibrotic tissue response, contractile dysregulation, and transcriptomic regulation of key pathways.This dissertation also described incorporation of CRISPR/Cas9 gene-editing to create a human induced pluripotent stem cell line (hiPSC) with a mutation in KCNH2, the gene implicated in Long QT Syndrome Type 2 (LQTS2). This novel stem cell line, combined with the developed heart on-a-chip technology, led to creation of a 3D human cardiac on-chip tissue model of LQTS2 disease.. Extensive mechanistic biological and electrophysiological characterizations were performed to elucidate the mechanism of R531W mutation in KCNH2, significantly adding to existing knowledge about LQTS2. In summary, this thesis described creation of a LQTS2 cardiac on-a-chip model, incorporated with gene-edited hiPSC-cardiomyocytes and hiPSC-cardiac fibroblasts, to study mechanisms of LQTS2.
Overall, this dissertation provides broad impact for fundamental studies toward cardiac biological studies as well as drug screening applications. Specifically, the developed heart on-a-chip from this dissertation provides a unique alternative platform to animal testing and 2D studies that recapitulates the human myocardium, with capabilities to model critical CVDs to study disease mechanisms, and/or ultimately lead to development of future therapeutic strategies.
ContributorsVeldhuizen, Jaimeson (Author) / Nikkhah, Mehdi (Thesis advisor) / Brafman, David (Committee member) / Ebrahimkhani, Mo (Committee member) / Migrino, Raymond Q (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2021
Description
Antibodies are naturally occurring proteins that protect a host during infection through direct neutralization and/or recruitment of the innate immune system. Unfortunately, in some infections, antibodies present unique hurdles that must be overcome for a safer and more efficacious antibody-based therapeutic (e.g., antibody dependent viral enhancement (ADE) and inflammatory pathology). This dissertation describes the utilization of plant expression systems to produce N-glycan specific antibody-based therapeutics for Dengue Virus (DENV) and Chikungunya Virus (CHIKV). The Fc region of an antibody interacts with Fcγ Receptors (FcγRs) on immune cells and components of the innate immune system. Each class of immune cells has a distinct action of neutralization (e.g., antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP)). Therefore, structural alteration of the Fc region results in novel immune pathways of protection. One approach is to modulate the N-glycosylation in the Fc region of the antibody. Of scientific significance, is the plant’s capacity to express human antibodies with homogenous plant and humanized N-glycosylation (WT and GnGn, respectively). This allows to study how specific glycovariants interact with other components of the immune system to clear an infection, producing a tailor-made antibody for distinct diseases. In the first section, plant-produced glycovariants were explored for reduced interactions with specific FcγRs for the overall reduction in ADE for DENV infections. The results demonstrate a reduction in ADE of our plant-produced monoclonal antibodies in in vitro experiments, which led to a greater survival in vivo of immunodeficient mice challenged with lethal doses of DENV and a sub-lethal dose of DENV in ADE conditions. In the second section, plant-produced glycovariants were explored for increased interaction with specific FcγRs to improve ADCC in the treatment of the highly inflammatory CHIKV. The results demonstrate an increase ADCC activity in in vitro experiments and a reduction in CHIKV-associated inflammation in in vivo mouse models. Overall, the significance of this dissertation is that it can provide a treatment for DENV and CHIKV; but equally importantly, give insight to the role of N-glycosylation in antibody effector functions, which has a broader implication for therapeutic development for other viral infections.
ContributorsHurtado, Jonathan (Author) / Chen, Qiang (Thesis advisor) / Arntzen, Charles (Committee member) / Borges, Chad (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2019
Description
The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
ContributorsDaer, René (Author) / Haynes, Karmella (Thesis advisor) / Brafman, David (Committee member) / Nielsen, David (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2017
Description
Synthetic gene networks have evolved from simple proof-of-concept circuits to
complex therapy-oriented networks over the past fifteen years. This advancement has
greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a
mechanism that cells use to achieve a discrete number of mutually exclusive states in
response to environmental inputs. However, complex contextual connections of gene
regulatory networks in natural settings often impede the experimental establishment of
the function and dynamics of each specific gene network.
In this work, diverse synthetic gene networks are rationally designed and
constructed using well-characterized biological components to approach the cell fate
determination and state transition dynamics in multistable systems. Results show that
unimodality and bimodality and trimodality can be achieved through manipulation of the
signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to
communicate with each other.
Moreover, a synthetic quadrastable circuit is also built and experimentally
demonstrated to have four stable steady states. Experiments, guided by mathematical
modeling predictions, reveal that sequential inductions generate distinct cell fates by
changing the landscape in sequence and hence navigating cells to different final states.
Circuit function depends on the specific protein expression levels in the circuit.
We then establish a protein expression predictor taking into account adjacent
transcriptional regions’ features through construction of ~120 synthetic gene circuits
(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.
These combined results illustrate applications of synthetic gene networks to
understand the cell fate determination and state transition dynamics in multistable
systems. A protein-expression predictor is also developed to evaluate and tune circuit
dynamics.
complex therapy-oriented networks over the past fifteen years. This advancement has
greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a
mechanism that cells use to achieve a discrete number of mutually exclusive states in
response to environmental inputs. However, complex contextual connections of gene
regulatory networks in natural settings often impede the experimental establishment of
the function and dynamics of each specific gene network.
In this work, diverse synthetic gene networks are rationally designed and
constructed using well-characterized biological components to approach the cell fate
determination and state transition dynamics in multistable systems. Results show that
unimodality and bimodality and trimodality can be achieved through manipulation of the
signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to
communicate with each other.
Moreover, a synthetic quadrastable circuit is also built and experimentally
demonstrated to have four stable steady states. Experiments, guided by mathematical
modeling predictions, reveal that sequential inductions generate distinct cell fates by
changing the landscape in sequence and hence navigating cells to different final states.
Circuit function depends on the specific protein expression levels in the circuit.
We then establish a protein expression predictor taking into account adjacent
transcriptional regions’ features through construction of ~120 synthetic gene circuits
(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.
These combined results illustrate applications of synthetic gene networks to
understand the cell fate determination and state transition dynamics in multistable
systems. A protein-expression predictor is also developed to evaluate and tune circuit
dynamics.
ContributorsWu, Fuqing (Author) / Wang, Xiao (Thesis advisor) / Haynes, Karmella (Committee member) / Marshall, Pamela (Committee member) / Nielsen, David (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2017