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Description
Random peptide microarrays are a powerful tool for both the treatment and diagnostics of infectious diseases. On the treatment side, selected random peptides on the microarray have either binding or lytic potency against certain pathogens cells, thus they can be synthesized into new antimicrobial agents, denoted as synbodies (synthetic antibodies).

Random peptide microarrays are a powerful tool for both the treatment and diagnostics of infectious diseases. On the treatment side, selected random peptides on the microarray have either binding or lytic potency against certain pathogens cells, thus they can be synthesized into new antimicrobial agents, denoted as synbodies (synthetic antibodies). On the diagnostic side, serum containing specific infection-related antibodies create unique and distinct "pathogen-immunosignatures" on the random peptide microarray distinct from the healthy control serum, and this different mode of binding can be used as a more precise measurement than traditional ELISA tests. My thesis project is separated into these two parts: the first part falls into the treatment side and the second one focuses on the diagnostic side. My first chapter shows that a substitution amino acid peptide library helps to improve the activity of a recently reported synthetic antimicrobial peptide selected by the random peptide microarray. By substituting one or two amino acids of the original lead peptide, the new substitutes show changed hemolytic effects against mouse red blood cells and changed potency against two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. Two new substitutes are then combined together to form the synbody, which shows a significantly antimicrobial potency against Staphylococcus aureus (<0.5uM). In the second chapter, I explore the possibility of using the 10K Ver.2 random peptide microarray to monitor the humoral immune response of dengue. Over 2.5 billion people (40% of the world's population) live in dengue transmitting areas. However, currently there is no efficient dengue treatment or vaccine. Here, with limited dengue patient serum samples, we show that the immunosignature has the potential to not only distinguish the dengue infection from non-infected people, but also the primary dengue infection from the secondary dengue infections, dengue infection from West Nile Virus (WNV) infection, and even between different dengue serotypes. By further bioinformatic analysis, we demonstrate that the significant peptides selected to distinguish dengue infected and normal samples may indicate the epitopes responsible for the immune response.
ContributorsWang, Xiao (Author) / Johnston, Stephen Albert (Thesis advisor) / Blattman, Joseph (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2013
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Description
The object of this study is to charac terize the effect of focused ultrasound stimulation (FUS) on the rat ce rvix which has been observed to speed its ripening during pregnancy. Ce rvical ripening is required for successful fetal delivery. Timed-pregnant Sprague-Dawley rats (n=36) were used. On day 14 of

The object of this study is to charac terize the effect of focused ultrasound stimulation (FUS) on the rat ce rvix which has been observed to speed its ripening during pregnancy. Ce rvical ripening is required for successful fetal delivery. Timed-pregnant Sprague-Dawley rats (n=36) were used. On day 14 of gestation, the FUS system was placed on the body surface of the rat over the cervix and ultrasound energy was applied to cervix for variable times up to 1 hour in the control group, the FUS system was placed on rats but no energy was applied. Daily measurement of cervix light-induced florescence (LIF, photon counts of collagen x-bridge fluorescence) were made on days 16 of gestation and daily until spont-aneous delivery (day22) to estimate changes in cervical ripening. We found that pulses of 680 KHz ultrasound at 25 Hertz, 1 millisecond pulse duration at 1W/cm^2 applied for as little as 30 minutes would immediately afterwards show the cervix to hav e ripened to the degree seen just before delivery on day 22. Delivery times, fetal weights and viability were unaffected in the FUS-treated animals.
ContributorsLuo, Daishen (Author) / Towe, Bruce C (Thesis advisor) / Wang, Xiao (Committee member) / Caplan, Michael (Committee member) / Arizona State University (Publisher)
Created2012
Description
Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study of cellular morphology, a single cell three-dimensional (3D) imaging system

Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study of cellular morphology, a single cell three-dimensional (3D) imaging system can be used to diagnose fatal diseases, such as cancer, at an early stage. One proven method, CellCT, accomplishes 3D imaging by rotating a single cell around a fixed axis. However, some existing cell rotating mechanisms require either intricate microfabrication, and some fail to provide a suitable environment for living cells. This thesis develops a microvorterx chamber that allows living cells to be rotated by hydrodynamic alone while facilitating imaging access. In this thesis work, 1) the new chamber design was developed through numerical simulation. Simulations revealed that in order to form a microvortex in the side chamber, the ratio of the chamber opening to the channel width must be smaller than one. After comparing different chamber designs, the trapezoidal side chamber was selected because it demonstrated controllable circulation and met the imaging requirements. Microvortex properties were not sensitive to the chambers with interface angles ranging from 0.32 to 0.64. A similar trend was observed when chamber heights were larger than chamber opening. 2) Micro-particle image velocimetry was used to characterize microvortices and validate simulation results. Agreement between experimentation and simulation confirmed that numerical simulation was an effective method for chamber design. 3) Finally, cell rotation experiments were performed in the trapezoidal side chamber. The experimental results demonstrated cell rotational rates ranging from 12 to 29 rpm for regular cells. With a volumetric flow rate of 0.5 µL/s, an irregular cell rotated at a mean rate of 97 ± 3 rpm. Rotational rates can be changed by altering inlet flow rates.
ContributorsZhang, Wenjie (Author) / Frakes, David (Thesis advisor) / Meldrum, Deirdre (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2011
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Description
Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR systems have extreme versatility yet can induce off-target mutations and karyotypic destabilization. To address these constraints we developed an RNA-guided

Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR systems have extreme versatility yet can induce off-target mutations and karyotypic destabilization. To address these constraints we developed an RNA-guided recombinase protein by fusing a hyperactive mutant resolvase from transposon TN3 to catalytically inactive Cas9. We validated recombinase-Cas9 (rCas9) function in model eukaryote Saccharomyces cerevisiae using a chromosomally integrated fluorescent reporter. Moreover, we demonstrated cooperative targeting by CRISPR RNAs at spacings of 22 or 40bps is necessary for directing recombination. Using PCR and Sanger sequencing, we confirmed rCas9 targets DNA recombination. With further development we envision rCas9 becoming useful in the development of RNA-programmed genetic circuitry as well as high-specificity genome engineering.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis advisor) / Brafman, David A (Committee member) / Tian, Xiao-jun (Committee member) / Arizona State University (Publisher)
Created2018
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Description
Synthetic biology is a novel method that reengineers functional parts of natural genes of interest to build new biomolecular devices able to express as designed. There is increasing interest in synthetic biology due to wide potential applications in various fields such as clinics and fuel production. However, there are still

Synthetic biology is a novel method that reengineers functional parts of natural genes of interest to build new biomolecular devices able to express as designed. There is increasing interest in synthetic biology due to wide potential applications in various fields such as clinics and fuel production. However, there are still many challenges in synthetic biology. For example, many natural biological processes are poorly understood, and these could be more thoroughly studied through model synthetic gene networks. Additionally, since synthetic biology applications may have numerous design constraints, more inducer systems should be developed to satisfy different requirements for genetic design.

This thesis covers two topics. First, I attempt to generate stochastic resonance (SR) in a biological system. Synthetic bistable systems were chosen because the inducer range in which they exhibit bistability can satisfy one of the three requirements of SR: a weak periodic force is unable to make the transition between states happen. I synthesized several different bistable systems, including toggle switches and self-activators, to select systems matching another requirement: the system has a clear threshold between the two energy states. Their bistability was verified and characterized. At the same time, I attempted to figure out the third requirement for SR – an effective noise serving as the stochastic force – through one of the most widespread toggles, the mutual inhibition toggle, in both yeast and E. coli. A mathematic model for SR was written and adjusted.

Secondly, I began work on designing a new genetic system capable of responding to pulsed magnetic fields. The operators responding to pulsed magnetic stimuli in the rpoH promoter were extracted and reorganized. Different versions of the rpoH promoter were generated and tested, and their varying responsiveness to magnetic fields was recorded. In order to improve efficiency and produce better operators, a directed evolution method was applied with the help of a CRISPR-dCas9 nicking system. The best performing promoters thus far show a five-fold difference in gene expression between trials with and without the magnetic field.
ContributorsHu, Hao (Author) / Wang, Xiao (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2016
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Description
An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the

An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells.
ContributorsRaman, Sreedevi (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2017
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Description
Neurodegenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis are marked by the loss of different types of neurons and glial cells in the central nervous system (CNS). Human Pluripotent Stem Cell (hPSC)-derived Neural Progenitor Cells (hNPCs) have the ability to self-renew indefinitely and to differentiate into

Neurodegenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis are marked by the loss of different types of neurons and glial cells in the central nervous system (CNS). Human Pluripotent Stem Cell (hPSC)-derived Neural Progenitor Cells (hNPCs) have the ability to self-renew indefinitely and to differentiate into various cell types of the CNS. HNPCs can be used in cell based therapies and have the potential to reverse or arrest neurodegeneration and to replace lost neurons and glial cells. However, the lack of completely defined, scalable systems to culture these cells, limits their therapeutic and clinical applications. In a previous study, a completely defined, robust, synthetic peptide- a Vitronectin Derived Peptide (VDP) that supports the long term expansion and differentiation of various embryonic and induced pluripotent stem cell (hESC/hIPSC) derived hNPC lines on two dimensional (2D) tissue culture plates was identified. In this study, the culture of hNPCs was scaled up using VDP coated microcarriers (MC). VDP MC were able to support the long term expansion of hESC and hiPSC derived hNPCs over multiple passages and supported higher fold changes in cell densities, compared to VDP coated 2D surfaces. VDP MC also showed the ability to support the neuronal differentiation of hNPCs, and produced mature neurons expressing several neuronal, neurotransmitter and cortical markers. Additionally, alzheimer’s disease (AD) relevant phenotypes were studied in patient hIPSC derived hNPCs cultured on laminin MC to assess if the MC culture system could be used for disease modelling and drug screening. Finally, a microcarrier based bioreactor system was developed for the large scale expansion of hNPCs, exhibiting more than a five-fold change in cell density and supporting more than 100 million hNPCs in culture. Thus, the development of a xeno-free, scalable system allows hNPC culture under standard and reproducible conditions in quantities required for therapeutic and clinical applications.
ContributorsRajaram Srinivasan, Gayathri (Author) / Brafman, David (Thesis advisor) / Wang, Xiao (Committee member) / Haynes, Karmella (Committee member) / Arizona State University (Publisher)
Created2017
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Description
Alzheimer’s disease (AD), despite over a century of research, does not have a clearly defined pathogenesis for the sporadic form that makes up the majority of disease incidence. A variety of correlative risk factors have been identified, including the three isoforms of apolipoprotein E (ApoE), a cholesterol transport protein in

Alzheimer’s disease (AD), despite over a century of research, does not have a clearly defined pathogenesis for the sporadic form that makes up the majority of disease incidence. A variety of correlative risk factors have been identified, including the three isoforms of apolipoprotein E (ApoE), a cholesterol transport protein in the central nervous system. ApoE ε3 is the wild-type variant with no effect on risk. ApoE ε2, the protective and most rare variant, reduces risk of developing AD by 40%. ApoE ε4, the risk variant, increases risk by 3.2-fold and 14.9-fold for heterozygous and homozygous representation respectively. Study of these isoforms has been historically complex, but the advent of human induced pluripotent stem cells (hiPSC) provides the means for highly controlled, longitudinal in vitro study. The effect of ApoE variants can be further elucidated using this platform by generating isogenic hiPSC lines through precise genetic modification, the objective of this research. As the difference between alleles is determined by two cytosine-thymine polymorphisms, a specialized CRISPR/Cas9 system for direct base conversion was able to be successfully employed. The base conversion method for transitioning from the ε3 to ε2 allele was first verified using the HEK293 cell line as a model with delivery via electroporation. Following this verification, the transfection method was optimized using two hiPSC lines derived from ε4/ε4 patients, with a lipofection technique ultimately resulting in successful base conversion at the same site verified in the HEK293 model. Additional research performed included characterization of the pre-modification genotype with respect to likely off-target sites and methods of isolating clonal variants.
ContributorsLakers, Mary Frances (Author) / Brafman, David (Thesis advisor) / Haynes, Karmella (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2017
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Description
The blood-brain-barrier (BBB) is a significant obstacle for treating many neurological disorders. Bubble-assisted focused ultrasound (BAFUS) medicated BBB disruption is a promising technology that enables the delivery of large drug doses at targeted locations across the BBB. However, the current lack of an in vitro model of this process hinders

The blood-brain-barrier (BBB) is a significant obstacle for treating many neurological disorders. Bubble-assisted focused ultrasound (BAFUS) medicated BBB disruption is a promising technology that enables the delivery of large drug doses at targeted locations across the BBB. However, the current lack of an in vitro model of this process hinders the full understanding of BAFUS BBB disruption for better translation into clinics. In this work, a US-transparent organ-on-chip device has been fabricated that can be critical for the in vitro modeling of the BAFUS BBB disruption. The transparency of the device window to focused ultrasound (FUS) was calculated theoretically and demonstrated by experiments. Nanobubbles were fabricated, characterized by cryogenic transmission electron microscopy (cryo-TEM), and showed bubble cavitation under FUS. Human colorectal adenocarcinoma (Caco-2) cells were used to form a good cellular barrier for BAFUS barrier disruption, as suggested by the measured permeability and transepithelial electrical resistance (TEER). Finally, barrier disruption and recovery were observed in BAFUS disrupted US-transparent organ-on-chips with Caco-2 barriers, showing great promise of the platform for future modeling BAFUS BBB disruption in vitro.
ContributorsAkkad, Adam Rifat (Author) / Gu, Jian (Thesis advisor) / Nikkhah, Mehdi (Thesis advisor) / Belohlavek, Marek (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2022
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Description
Extrachromosomal circular DNA (eccDNA) has become an increasingly popular subject of study in eukaryotic cell biology due to its prevalence in human cancer. Though the literature reports a consensus regarding DNA break repair as a driver of eccDNA formation, there remains a lack of knowledge surrounding the exact mechanisms for

Extrachromosomal circular DNA (eccDNA) has become an increasingly popular subject of study in eukaryotic cell biology due to its prevalence in human cancer. Though the literature reports a consensus regarding DNA break repair as a driver of eccDNA formation, there remains a lack of knowledge surrounding the exact mechanisms for eccDNA formation and the selective dynamics that promote their retainment in a cell or population. A central issue to studying eccDNA is the inability to distinguish between linear and circular DNA of homologous sequence. The work presented here describes an adapted eccDNA enrichment and detection assay, specifically for investigating the effects of manipulating a known eccDNA-forming locus in the budding yeast Saccharomyces cerevisiae. First, a galactose inducible GFP reporter was integrated within the copper inducible CUP1 tandem repeat locus of yeast cells. The eccDNA enrichment and detection assay was first applied to wildtype yeast to demonstrate the presence of CUP1 eccDNA in copper induced cells by qPCR. Although subsequent sequencing analysis failed to validate this result, it indicated the presence of various other known and previously un-reported eccDNA species. Finally, application of the enrichment protocol and qPCR detection assay to CUP1-GFP reporter cells yielded inconclusive results, suggesting the assay requires further optimization to sensitively detect eccDNA from this altered locus. While more work is necessary to draw conclusions regarding the limits of eccDNA production at a manipulated eccDNA-forming locus, this knowledge will lend to the potential for therapeutically targeting eccDNA at the point of de novo formation.
ContributorsKeal, Tula (Author) / Wang, Xiao (Thesis advisor) / Tian, Xiaojun (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2022