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In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the

In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment.
ContributorsBley, Christopher James (Author) / Chen, Julian (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
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Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group,

Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group, filamentous fungi, is presented. Based on phylogenetic analysis of 69 TER sequences and mutagenesis analysis of in vitro reconstituted Neurospora telomerase, we discovered a conserved functional core in filamentous fungal TERs sharing homologous structural features with vertebrate TERs. This core contains the template-pseudoknot and P6/P6.1 domains, essential for enzymatic activity, which retain function in trans. The in vitro reconstituted Neurospora telomerase is highly processive, synthesizing canonical TTAGGG repeats. Similar to Schizosaccharomycetes pombe, filamentous fungal TERs utilize the spliceosomal splicing machinery for 3' processing. Neurospora telomerase, while associating with the Est1 protein in vivo, does not bind homologous Ku or Sm proteins found in both budding and fission yeast telomerase holoenzyme, suggesting a unique biogenesis pathway. The development of Neurospora as a model organism to study telomeres and telomerase may shed light upon the evolution of the canonical TTAGGG telomeric repeat and telomerase processivity within fungal species.
ContributorsQi, Xiaodong (Author) / Chen, Julian (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2011
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Cardiovascular disease is one of the most deadly outcomes of end stage renal disease. Bioelectrical impedance is a intriguing, yet unproven method of measuring fluid buildup in the heart, and is marketed as a early diagnostic tool for onset of cardiovascular disease. In this study, selenium supplements were given to

Cardiovascular disease is one of the most deadly outcomes of end stage renal disease. Bioelectrical impedance is a intriguing, yet unproven method of measuring fluid buildup in the heart, and is marketed as a early diagnostic tool for onset of cardiovascular disease. In this study, selenium supplements were given to a cohort of dialysis patients in the Phoenix metro area and their fluid tolerance was measured with thoracic biolectrical impedance. BNP was used as a correlate to see if bioelectrical impedance was correlated with heart disease. The study found no correlation between BNP and bioelectrical impedance and thus was not an accurate diagnostic tool in a medical setting.
ContributorsBrown, Patrick Michael (Author) / Johnston, Carol (Thesis director) / Orchinik, Miles (Committee member) / Tingey, Michael (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Historical, Philosophical and Religious Studies (Contributor)
Created2013-05
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Ephemeral and intermittent streams are valuable sources of surface water support in the arid ecosystems of the Southwestern United States. These streams account for over 80% of the streams in the American Southwest and their importance has been indicated in many studies. Ephemeral and intermittent streams support a wide range

Ephemeral and intermittent streams are valuable sources of surface water support in the arid ecosystems of the Southwestern United States. These streams account for over 80% of the streams in the American Southwest and their importance has been indicated in many studies. Ephemeral and intermittent streams support a wide range of plant and animal species in both continuous and episodic fashions. This study aimed to gain a better understanding of the relationship between streamflow permanence and patterns of biomass and secondary production of the riparian fauna these ecosystems support. This was accomplished through a yearlong survey in the Huachuca Mountains of Southeastern, Arizona where macroinvertebrates were collected at various sites along a gradient of streamflow permanence before, during, and after the three month monsoon season that supplies most of the annual rainfall in this region. The results of my surveys indicate that 1) Sites characterized by low streamflow permanence were more responsive to changes in precipitation than sites characterized by relatively high streamflow permanence 2) In ephemeral streams, there is a significant peak in terrestrial macroinvertebrate production and biomass both during and after the monsoon season 3) streamflow permanence may convey consistent but not exceptional secondary production whereas seasonality in rainfall may convey exceptional but episodic secondary production—more so in sites where streamflow is not consistent.
ContributorsMcCartin, Michael Patrick (Author) / Sabo, John (Thesis director) / Stromberg, Juliet (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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We examined the evolutionary morphological responses of Drosophila melanogaster that had evolved at constant cold (16°), constant hot (25°C), and fluctuating (16° and 25°C). Flies that were exposed to the constant low mean temperature developed larger thorax, wing, and cell sizes than those exposed to constant high mean temperatures. Males

We examined the evolutionary morphological responses of Drosophila melanogaster that had evolved at constant cold (16°), constant hot (25°C), and fluctuating (16° and 25°C). Flies that were exposed to the constant low mean temperature developed larger thorax, wing, and cell sizes than those exposed to constant high mean temperatures. Males and females both responded similarly to thermal treatments in average wing and cell size. The resulting cell area for a given wing size in thermal fluctuating populations remains unclear and remains a subject for future research.
ContributorsAdrian, Gregory John (Author) / Angilletta, Michael (Thesis director) / Harrison, Jon (Committee member) / Rusch, Travis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Studies of animal contests often focus solely on a single static measurement of fighting ability, such as the size or the strength of the individual. However, recent studies have highlighted the importance of individual variation in the dynamic behaviors used during a fight, such as, assessment strategies, decision making, and

Studies of animal contests often focus solely on a single static measurement of fighting ability, such as the size or the strength of the individual. However, recent studies have highlighted the importance of individual variation in the dynamic behaviors used during a fight, such as, assessment strategies, decision making, and fine motor control, as being strong predictors of the outcome of aggression. Here, I combined morphological and behavioral data to discover how these features interact during aggressing interactions in male virile crayfish, Faxonius virilis. I predicted that individual variation in behavioral skill for decision making (i.e., number of strikes thrown), would determine the outcome of contest success in addition to morphological measurements (e.g. body size, relative claw size). To evaluate this prediction, I filmed staged territorial interactions between male F. virilis and later analyzed trial behaviors (e.g. strike, pinches, and bout time) and aggressive outcomes. I found very little support for skill to predict win/loss outcome in trials. Instead, I found that larger crayfish engaged in aggression for longer compared to smaller crayfish, but that larger crayfish did not engage in a greater number of claw strikes or pinches when controlling for encounter duration. Future studies should continue to investigate the role of skill, by using finer-scale techniques such as 3D tracking software, which could track advanced measurements (e.g. speed, angle, and movement efficiency). Such studies would provide a more comprehensive understanding of the relative influence of fighting skill technique on territorial contests.

ContributorsNguyen, Phillip Huy (Author) / Angilletta, Michael (Thesis director) / McGraw, Kevin (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules

The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution.
ContributorsYu, Hanyang (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
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The discovery of DNA helical structure opened the door of modern molecular biology. Ned Seeman utilized DNA as building block to construct different nanoscale materials, and introduced a new field, know as DNA nanotechnology. After several decades of development, different DNA structures had been created, with different dimension, different morphology

The discovery of DNA helical structure opened the door of modern molecular biology. Ned Seeman utilized DNA as building block to construct different nanoscale materials, and introduced a new field, know as DNA nanotechnology. After several decades of development, different DNA structures had been created, with different dimension, different morphology and even with complex curvatures. In addition, after construction of enough amounts DNA structure candidates, DNA structure template, with excellent spatial addressability, had been used to direct the assembly of different nanomaterials, including nanoparticles and proteins, to produce different functional nanomaterials. However there are still many challenges to fabricate functional DNA nanostructures. The first difficulty is that the present finite sized template dimension is still very small, usually smaller than 100nm, which will limit the application for large amount of nanomaterials assembly or large sized nanomaterials assembly. Here we tried to solve this problem through developing a new method, superorigami, to construct finite sized DNA structure with much larger dimension, which can be as large as 500nm. The second problem will be explored the ability of DNA structure to assemble inorganic nanomaterials for novel photonic or electronic properties. Here we tried to utilize DNA Origami method to assemble AuNPs with controlled 3D spacial position for possible chiral photonic complex. We also tried to assemble SWNT with discrete length for possible field effect transistor device. In addition, we tried to mimic in vivo compartment with DNA structure to study internalized enzyme behavior. From our results, constructed DNA cage origami can protect encapsulated enzyme from degradation, and internalized enzyme activity can be boosted for up to 10 folds. In summary, DNA structure can serve as an ideal template for construction of functional nanomaterials with lots of possibilities to be explored.
ContributorsZhao, Zhao (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Seo, Dong-Kyun (Committee member) / Arizona State University (Publisher)
Created2013
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DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles

DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles and proteins. New protein-DNA bio-conjugation chemistries make it possible to precisely position proteins and other biomolecules on underlying DNA scaffolds, generating multi-biomolecule pathways with the ability to modulate inter-molecular interactions and the local environment. This dissertation focuses on studying the application of using DNA nanostructure to direct the self-assembly of other biomolecular networks to translate biochemical pathways to non-cellular environments. Presented here are a series of studies toward this application. First, a novel strategy utilized DNA origami as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multi-component systems from biological scaffolds using the power of rationally engineered DNA nanostructures. Next, discrete glucose oxidase (GOx)/ horseradish peroxidase (HRP) enzyme pairs were organized on DNA origami tiles with controlled interenzyme spacing and position. This study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Finally, a tweezer-like DNA nanodevice was designed and constructed to actuate the activity of an enzyme/cofactor pair. Using this approach, several cycles of externally controlled enzyme inhibition and activation were successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.
ContributorsLiu, Minghui (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2013
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Since Darwin popularized the evolution theory in 1895, it has been completed and studied through the years. Starting in 1990s, evolution at molecular level has been used to discover functional molecules while studying the origin of functional molecules in nature by mimicing the natural selection process in laboratory. Along this

Since Darwin popularized the evolution theory in 1895, it has been completed and studied through the years. Starting in 1990s, evolution at molecular level has been used to discover functional molecules while studying the origin of functional molecules in nature by mimicing the natural selection process in laboratory. Along this line, my Ph.D. dissertation focuses on the in vitro selection of two important biomolecules, deoxynucleotide acid (DNA) and protein with binding properties. Chapter two focuses on in vitro selection of DNA. Aptamers are single-stranded nucleic acids that generated from a random pool and fold into stable three-dimensional structures with ligand binding sites that are complementary in shape and charge to a desired target. While aptamers have been selected to bind a wide range of targets, it is generally thought that these molecules are incapable of discriminating strongly alkaline proteins due to the attractive forces that govern oppositely charged polymers. By employing negative selection step to eliminate aptamers that bind with off-target through charge unselectively, an aptamer that binds with histone H4 protein with high specificity (>100 fold)was generated. Chapter four focuses on another functional molecule: protein. It is long believed that complex molecules with different function originated from simple progenitor proteins, but very little is known about this process. By employing a previously selected protein that binds and catalyzes ATP, which is the first and only protein that was evolved completely from random pool and has a unique α/β-fold protein scaffold, I fused random library to the C-terminus of this protein and evolved a multi-domain protein with decent properties. Also, in chapter 3, a unique bivalent molecule was generated by conjugating peptides that bind different sites on the protein with nucleic acids. By using the ligand interactions by nucleotide conjugates technique, off-the shelf peptide was transferred into high affinity protein capture reagents that mimic the recognition properties of natural antibodies. The designer synthetic antibody amplifies the binding affinity of the individual peptides by ∼1000-fold to bind Grb2 with a Kd of 2 nM, and functions with high selectivity in conventional pull-down assays from HeLa cell lysates.
ContributorsJiang, Bing (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2013