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Description
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
This study illustrates the abilities of the honeybee, Apis mellifera, to learn and differentiate between patterns solely off their spatial frequencies. Patterns were chosen based off of calculations derived from the measurements of the physical construction of the apposition compound eye, which led to predictions of what the bees could theoretically see. The hypothesis was then that bees would have a visual threshold where patterns with spatial frequencies that fall below this line should be easily distinguishable, and patterns above the threshold would have scores that mimic if the bees made choices randomly. There were 9 patterns tested, all with different spatial frequencies and in the colors of black, white, and gray. The bees were tested on their learning and pattern differentiation abilities with 10 pattern comparisons, with the lower frequency of the two being associated with an unscented sucrose solution reward. The results were surprising in that the previous studies pointing towards this visual threshold were inaccurate because of some of the patterns being learning in an intermediate ability. These intermediate scores suggest that the calculations predicting what the bees could see clearly were slightly wrong because it was more likely that the bees saw those images in more of a blur, which resulted in their intermediate score. Honeybees have served as a useful model organisms over the decades with studying learning involving visual information. This study lacked in its total numbers of trials and bees tested, which could have led to incomplete results, and this showing of an intermediate score and ability. Future studies should continue in order to advance this understanding of a perceptually and cognitively advance processing animal.
ContributorsBalsino, Brandon Bartholomew (Author) / Harrison, Jon (Thesis director) / Smith, Brian (Committee member) / Duell, Meghan (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Animals must learn to ignore stimuli that are irrelevant to survival, a process referred to as latent inhibition. The Amtyr1 gene has been shown through quantitative trait loci mapping to be linked to strong latent inhibition in honey bees. Here we implicate this G-protein coupled receptor for the biogenic amine tyramine as an important factor underlying this form of learning in honey bees. We show that dsRNA targeted to disrupt the tyramine receptors, specifically affects latent inhibition but not excitatory associative conditioning. Our results therefore identify a distinct reinforcement pathway for latent inhibition in insects.
ContributorsPetersen, Mary Margaret (Author) / Smith, Brian (Thesis director) / Wang, Ying (Committee member) / Sinakevitch, Irina (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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
Traumatic brain injury (TBI) most frequently occurs in pediatric patients and remains a leading cause of childhood death and disability. Mild TBI (mTBI) accounts for 70-90% of all TBI cases, yet its neuropathophysiology is still poorly understood. While a single mTBI injury can lead to persistent deficits, repeat injuries increase the severity and duration of both acute symptoms and long term deficits. In this study, to model pediatric repetitive mTBI (rmTBI) we subjected unrestrained juvenile animals (post-natal day 20) to repeat weight drop impact. Animals were anesthetized and subjected to sham or rmTBI once per day for 5 days. At 14 days post injury (PID), magnetic resonance imaging (MRI) revealed that rmTBI animals displayed marked cortical atrophy and ventriculomegaly. Specifically, the thickness of the cortex was reduced up to 46% beneath and the ventricles increased up to 970% beneath the impact zone. Immunostaining with the neuron specific marker NeuN revealed an overall loss of neurons within the motor cortex but no change in neuronal density. Examination of intrinsic and synaptic properties of layer II/III pyramidal neurons revealed no significant difference between sham and rmTBI animals at rest or under convulsant challenge with the potassium channel blocker, 4-Aminophyridine. Overall, our findings indicate that the neuropathological changes reported after pediatric rmTBI can be effectively modeled by repeat weight drop in juvenile animals. Developing a better understanding of how rmTBI alters the pediatric brain may help improve patient care and direct "return to game" decision making in adolescents.
ContributorsGoddeyne, Corey (Author) / Anderson, Trent (Thesis advisor) / Smith, Brian (Committee member) / Kleim, Jeffrey (Committee member) / Arizona State University (Publisher)
Created2014
Description
Immunotherapy has been revitalized with the advent of immune checkpoint blockade
treatments, and neo-antigens are the targets of immune system in cancer patients who
respond to the treatments. The cancer vaccine field is focused on using neo-antigens from
unique point mutations of genomic sequence in the cancer patient for making
personalized cancer vaccines. However, we choose a different path to find frameshift
neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on
frameshift antigens.
In this dissertation, I have summarized and characterized all the potential frameshift
antigens from microsatellite regions in human, dog and mouse. A list of frameshift
antigens was validated by PCR in tumor samples and the mutation rate was calculated for
one candidate – SEC62. I develop a method to screen the antibody response against
frameshift antigens in human and dog cancer patients by using frameshift peptide arrays.
Frameshift antigens selected by positive antibody response in cancer patients or by MHC
predictions show protection in different mouse tumor models. A dog version of the
cancer vaccine based on frameshift antigens was developed and tested in a small safety
trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell
immune responses. Further, I built the human exon junction frameshift database which
includes all possible frameshift antigens from mis-splicing events in exon junctions, and I
develop a method to find potential frameshift antigens from large cancer
immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer
diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that
ii
early treatment gives significantly better protection than late treatment and the correct
time point for treatment is crucial to give the best clinical benefit. A model for early
treatment is developed with these results.
Frameshift neo-antigens from microsatellite regions and mis-splicing events are
abundant at mRNA level and they are better antigens than neo-antigens from point
mutations in the genomic sequences of cancer patients in terms of high immunogenicity,
low probability to cause autoimmune diseases and low cost to develop a broadly effective
vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for
cancer vaccine development.
treatments, and neo-antigens are the targets of immune system in cancer patients who
respond to the treatments. The cancer vaccine field is focused on using neo-antigens from
unique point mutations of genomic sequence in the cancer patient for making
personalized cancer vaccines. However, we choose a different path to find frameshift
neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on
frameshift antigens.
In this dissertation, I have summarized and characterized all the potential frameshift
antigens from microsatellite regions in human, dog and mouse. A list of frameshift
antigens was validated by PCR in tumor samples and the mutation rate was calculated for
one candidate – SEC62. I develop a method to screen the antibody response against
frameshift antigens in human and dog cancer patients by using frameshift peptide arrays.
Frameshift antigens selected by positive antibody response in cancer patients or by MHC
predictions show protection in different mouse tumor models. A dog version of the
cancer vaccine based on frameshift antigens was developed and tested in a small safety
trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell
immune responses. Further, I built the human exon junction frameshift database which
includes all possible frameshift antigens from mis-splicing events in exon junctions, and I
develop a method to find potential frameshift antigens from large cancer
immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer
diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that
ii
early treatment gives significantly better protection than late treatment and the correct
time point for treatment is crucial to give the best clinical benefit. A model for early
treatment is developed with these results.
Frameshift neo-antigens from microsatellite regions and mis-splicing events are
abundant at mRNA level and they are better antigens than neo-antigens from point
mutations in the genomic sequences of cancer patients in terms of high immunogenicity,
low probability to cause autoimmune diseases and low cost to develop a broadly effective
vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for
cancer vaccine development.
ContributorsZhang, Jian (Author) / Johnston, Stephen Albert (Thesis advisor) / Chang, Yung (Committee member) / Stafford, Phillip (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2018
Description
Ant colonies provide numerous opportunities to study communication systems that maintain the cohesion of eusocial groups. In many ant species, workers have retained their ovaries and the ability to produce male offspring; however, they generally refrain from producing their own sons when a fertile queen is present in the colony. Although mechanisms that facilitate the communication of the presence of a fertile queen to all members of the colony have been highly studied, those studies have often overlooked the added challenge faced by polydomous species, which divide their nests across as many as one hundred satellite nests resulting in workers potentially having infrequent contact with the queen. In these polydomous contexts, regulatory phenotypes must extend beyond the immediate spatial influence of the queen.
This work investigates mechanisms that can extend the spatial reach of fertility signaling and reproductive regulation in three polydomous ant species. In Novomessor cockerelli, the presence of larvae but not eggs is shown to inhibit worker reproduction. Then, in Camponotus floridanus, 3-methylheptacosane found on the queen cuticle and queen-laid eggs is verified as a releaser pheromone sufficient to disrupt normally occurring aggressive behavior toward foreign workers. Finally, the volatile and cuticular hydrocarbon pheromones present on the cuticle of Oecophylla smaragdina queens are shown to release strong attraction response by workers; when coupled with previous work, this result suggests that these chemicals may underly both the formation of a worker retinue around the queen as well as egg-located mechanisms of reproductive regulation in distant satellite nests. Whereas most previous studies have focused on the short-range role of hydrocarbons on the cuticle of the queen, these studies demonstrate that eusocial insects may employ longer range regulatory mechanisms. Both queen volatiles and distributed brood can extend the range of queen fertility signaling, and the use of larvae for fertility signaling suggest that feeding itself may be a non-chemical mechanism for reproductive regulation. Although trail laying in mass-recruiting ants is often used as an example of complex communication, reproductive regulation in ants may be a similarly complex example of insect communication, especially in the case of large, polydomous ant colonies.
This work investigates mechanisms that can extend the spatial reach of fertility signaling and reproductive regulation in three polydomous ant species. In Novomessor cockerelli, the presence of larvae but not eggs is shown to inhibit worker reproduction. Then, in Camponotus floridanus, 3-methylheptacosane found on the queen cuticle and queen-laid eggs is verified as a releaser pheromone sufficient to disrupt normally occurring aggressive behavior toward foreign workers. Finally, the volatile and cuticular hydrocarbon pheromones present on the cuticle of Oecophylla smaragdina queens are shown to release strong attraction response by workers; when coupled with previous work, this result suggests that these chemicals may underly both the formation of a worker retinue around the queen as well as egg-located mechanisms of reproductive regulation in distant satellite nests. Whereas most previous studies have focused on the short-range role of hydrocarbons on the cuticle of the queen, these studies demonstrate that eusocial insects may employ longer range regulatory mechanisms. Both queen volatiles and distributed brood can extend the range of queen fertility signaling, and the use of larvae for fertility signaling suggest that feeding itself may be a non-chemical mechanism for reproductive regulation. Although trail laying in mass-recruiting ants is often used as an example of complex communication, reproductive regulation in ants may be a similarly complex example of insect communication, especially in the case of large, polydomous ant colonies.
ContributorsEbie, Jessica (Author) / Liebig, Jürgen (Thesis advisor) / Hölldobler, Bert (Thesis advisor) / Pratt, Stephen (Committee member) / Smith, Brian (Committee member) / Rutowski, Ronald (Committee member) / Arizona State University (Publisher)
Created2020