Matching Items (7)

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In vitro selection of aptamers and protein

Description

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

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.

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Created

Date Created
  • 2013

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Exploring the Efficacy of Using Augmented Reality to Alleviate Common Misconceptions about Natural Selection

Description

Evidence suggests that Augmented Reality (AR) may be a powerful tool for

alleviating certain, lightly held scientific misconceptions. However, many

misconceptions surrounding the theory of evolution are deeply held

Evidence suggests that Augmented Reality (AR) may be a powerful tool for

alleviating certain, lightly held scientific misconceptions. However, many

misconceptions surrounding the theory of evolution are deeply held and resistant to

change. This study examines whether AR can serve as an effective tool for alleviating

these misconceptions by comparing the change in the number of misconceptions

expressed by users of a tablet-based version of a well-established classroom simulation to

the change in the number of misconceptions expressed by users of AR versions of the

simulation.

The use of realistic representations of objects is common for many AR

developers. However, this contradicts well-tested practices of multimedia design that

argue against the addition of unnecessary elements. This study also compared the use of

representational visualizations in AR, in this case, models of ladybug beetles, to symbolic

representations, in this case, colored circles.

To address both research questions, a one-factor, between-subjects experiment

was conducted with 189 participants randomly assigned to one of three conditions: non

AR, symbolic AR, and representational AR. Measures of change in the number and types

of misconceptions expressed, motivation, and time on task were examined using a pair of

planned orthogonal contrasts designed to test the study’s two research questions.

Participants in the AR-based condition showed a significantly smaller change in

the number of total misconceptions expressed after the treatment as well as in the number

of misconceptions related to intentionality; none of the other misconceptions examined

showed a significant difference. No significant differences were found in the total

number of misconceptions expressed between participants in the representative and

symbolic AR-based conditions, or on motivation. Contrary to the expectation that the

simulation would alleviate misconceptions, the average change in the number of

misconceptions expressed by participants increased. This is theorized to be due to the

juxtaposition of virtual and real-world entities resulting in a reduction in assumed

intentionality.

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Created

Date Created
  • 2019

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Advancing the causal theory of natural selection

Description

The Modern Synthesis embodies a theory of natural selection where selection is to be fundamentally understood in terms of measures of fitness and the covariance of reproductive success and trait

The Modern Synthesis embodies a theory of natural selection where selection is to be fundamentally understood in terms of measures of fitness and the covariance of reproductive success and trait or character variables. Whether made explicit or left implicit, the notion that selection requires that some trait variable cause reproductive success has been deemphasized in our modern understanding of exactly what selection amounts to. The dissertation seeks to advance a theory of natural selection that is fundamentally causal. By focusing on the causal nature of natural selection (rather than on fitness or statistical formulae), certain conceptual and methodological problems are seen in a new, clarifying light and avenues toward new, interesting solutions to those problems are illustrated. First, the dissertation offers an update to explicitly causal theories of when exactly a trait counts as an adaptation upon fixation in a population and draws out theoretical and practical implications for evolutionary biology. Second, I examine a case of a novel character that evolves by niche construction and argue that it evolves by selection for it and consider implications for understanding adaptations and drift. The third contribution of the dissertation is an argument for the importance of defining group selection causally and an argument against model pluralism in the levels of selection debate. Fourth, the dissertation makes a methodological contribution. I offer the first steps toward an explicitly causal methodology for inferring the causes of selection—something often required in addition to inferring the causes of reproductive success. The concluding chapter summarizes the work and discusses potential paths for future work.

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Date Created
  • 2016

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Identification and characterization of functional biomolecules by in vitro selection

Description

In vitro selection technologies allow for the identification of novel biomolecules endowed with desired functions. Successful selection methodologies share the same fundamental requirements. First, they must establish a strong link

In vitro selection technologies allow for the identification of novel biomolecules endowed with desired functions. Successful selection methodologies share the same fundamental requirements. First, they must establish a strong link between the enzymatic function being selected (phenotype) and the genetic information responsible for the function (genotype). Second, they must enable partitioning of active from inactive variants, often capturing only a small number of positive hits from a large population of variants. These principles have been applied to the selection of natural, modified, and even unnatural nucleic acids, peptides, and proteins. The ability to select for and characterize new functional molecules has significant implications for all aspects of research spanning the basic understanding of biomolecules to the development of new therapeutics. Presented here are four projects that highlight the ability to select for and characterize functional biomolecules through in vitro selection.

Chapter one outlines the development of a new characterization tool for in vitro selected binding peptides. The approach enables rapid screening of peptide candidates in small sample volumes using cell-free translated peptides. This strategy has the potential to accelerate the pace of peptide characterization and help advance the development of peptide-based affinity reagents.

Chapter two details an in vitro selection strategy for searching entire genomes for RNA sequences that enhance cap-independent initiation of translation. A pool of sequences derived from the human genome was enriched for members that function to enhance the translation of a downstream coding region. Thousands of translation enhancing elements from the human genome are identified and the function of a subset is validated in vitro and in cells.

Chapter three discusses the characterization of a translation enhancing element that promotes rapid and high transgene expression in mammalian cells. Using this ribonucleic acid sequence, a series of full length human proteins is expressed in a matter of only hours. This advance provides a versatile platform for protein synthesis and is espcially useful in situations where prokaryotic and cell-free systems fail to produce protein or when post-translationally modified protein is essential for biological analysis.

Chapter four outlines a new selection strategy for the identification of novel polymerases using emulsion droplet microfluidics technology. With the aid of a fluorescence-based activity assay, libraries of polymerase variants are assayed in picoliter sized droplets to select for variants with improved function. Using this strategy a variant of the 9°N DNA polymerase is identified that displays an enhanced ability to synthesize threose nucleic acid polymers.

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Created

Date Created
  • 2015

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Differences that make a difference: a study In collaborative learning

Description

Collaborative learning is a common teaching strategy in classrooms across age groups and content areas. It is important to measure and understand the cognitive process involved during collaboration to improve

Collaborative learning is a common teaching strategy in classrooms across age groups and content areas. It is important to measure and understand the cognitive process involved during collaboration to improve teaching methods involving interactive activities. This research attempted to answer the question: why do students learn more in collaborative settings? Using three measurement tools, 142 participants from seven different biology courses at a community college and at a university were tested before and after collaborating about the biological process of natural selection. Three factors were analyzed to measure their effect on learning at the individual level and the group level. The three factors were: difference in prior knowledge, sex and religious beliefs. Gender and religious beliefs both had a significant effect on post-test scores.

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Date Created
  • 2012

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A practical and theoretical approach to understanding the selective mechanisms behind genetic caste determination in Pogonomyrmex rugosus and Pogonomyrmex barbatus

Description

Gene-centric theories of evolution by natural selection have been popularized and remain generally accepted in both scientific and public paradigms. While gene-centrism is certainly parsimonious, its explanations fall short of

Gene-centric theories of evolution by natural selection have been popularized and remain generally accepted in both scientific and public paradigms. While gene-centrism is certainly parsimonious, its explanations fall short of describing two patterns of evolutionary and social phenomena: the evolution of sex and the evolution of social altruism. I review and analyze current theories on the evolution of sex. I then introduce the conflict presented to gene-centric evolution by social phenomena such as altruism and caste sterility in eusocial insects. I review gene-centric models of inclusive fitness and kin selection proposed by Hamilton and Maynard Smith. Based their assumptions, that relatedness should be equal between sterile workers and reproductives, I present several empirical examples that conflict with their models. Following that, I introduce a unique system of genetic caste determination (GCD) observed in hybrid populations of two sister-species of seed harvester ants, Pogonomyrmex rugosus and Pogonomyrmex barbatus. I review the evidence for GCD in those species, followed by a critique of the current gene-centric models used to explain it. In chapter two I present my own theoretical model that is both simple and extricable in nature to explain the origin, evolution, and maintenance of GCD in Pogonomyrmex. Furthermore, I use that model to fill in the gaps left behind by the contributing authors of the other GCD models. As both populations in my study system formed from inter-specific hybridization, I review modern discussions of heterosis (also called hybrid vigor) and use those to help explain the ecological competitiveness of GCD. I empirically address the inbreeding depression the lineages of GCD must overcome in order to remain ecologically stable, demonstrating that as a result of their unique system of caste determination, GCD lineages have elevated recombination frequencies. I summarize and conclude with an argument for why GCD evolved under selective mechanisms which cannot be considered gene-centric, providing evidence that natural selection can effectively operate on non-heritable genotypes appearing in groups and other social contexts.

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Date Created
  • 2012

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The effects of natural selection and random genetic drift in structured populations

Description

Building mathematical models and examining the compatibility of their theoretical predictions with empirical data are important for our understanding of evolution. The rapidly increasing amounts of genomic data on polymorphisms

Building mathematical models and examining the compatibility of their theoretical predictions with empirical data are important for our understanding of evolution. The rapidly increasing amounts of genomic data on polymorphisms greatly motivate evolutionary biologists to find targets of positive selection. Although intensive mathematical and statistical studies for characterizing signatures of positive selection have been conducted to identify targets of positive selection, relatively little is known about the effects of other evolutionary forces on signatures of positive selection. In this dissertation, I investigate the effects of various evolutionary factors, including purifying selection and population demography, on signatures of positive selection. Specifically, the effects on two highly used methods for detecting positive selection, one by Wright's Fst and its analogues and the other by footprints of genetic hitchhiking, are investigated. In Chapters 2 and 3, the effect of purifying selection on Fst is studied. The results show that purifying selection intensity greatly affects Fst by modulating allele frequencies across populations. The footprints of genetic hitchhiking in a geographically structured population are studied in Chapter 4. The results demonstrate that footprints of genetic hitchhiking are significantly influenced by geographic structure, which may help scientists to infer the origin and spread of the beneficial allele. In Chapter 5, the stochastic dynamics of a hitchhiking allele are studied using the diffusion process of genetic hitchhiking conditioned on the fixation of the beneficial allele. Explicit formulae for the conditioned two-locus diffusion process of genetic hitchhiking are derived and stochastic aspects of genetic hitchhiking are investigated. The results in this dissertation show that it is essential to model the interaction of neutral and selective forces for correct identification of the targets of positive selection.

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Created

Date Created
  • 2011