Matching Items (59)

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Synthesis of Hybrid DNA-Protein Nanostructures

Description

While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the

While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the protein will have to undergo site-specific modification, such as the incorporation of an unnatural amino, p-azidophenylalanine (AzF), via Shultz amber codon suppression method, which can then participate in click chemistry with modified DNA. These newly synthesized structures will then be able to self-assemble into higher order structures. Thus far, a surface exposed residue on the aldolase protein has been mutated into an amber stop codon. The next steps are to express the protein with the unnatural amino acid, allow it to participate in click chemistry, and visualize the hybrid structure. If the structure is correct, it will be able to self-assemble.

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  • 2017-05

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The Simian Barrier Dilemma: Improving Gene Vaccines for the Future

Description

Since its inception in the early 1990s, the concept of gene vaccines, particularly DNA vaccines, has enticed researchers across the board due to its simple design, flexible modification, and overall

Since its inception in the early 1990s, the concept of gene vaccines, particularly DNA vaccines, has enticed researchers across the board due to its simple design, flexible modification, and overall inexpensive cost of manufacturing. However, the past three decades have proven to be less fruitful than anticipated as scientists have yet to tackle the issue of inducing a strong enough response in humans and non-human primates to protect against foreign pathogens, an issue that has since been coined as the “simian barrier.” This appears to be a human/primate barrier as protective vaccines have been produced for other mammals. Despite millions of dollars in research along with some of the world’s brightest minds chipping in to resolve this, there has yet to be any truly viable solution to overcoming this barrier. With current research illustrating effective applications of RNA vaccines in humans, these studies may be uncovering the solution to the largely unsolved simian barrier dilemma. If vaccines using RNA, the transcribed version of DNA, are effective in humans, the problem may be inefficient transcription of the DNA. This may be attributable to a DNA promoter that has insufficient activity in primates. Additionally, with DNA vaccines being even cheaper and easier to manufacture than RNA vaccines, along with having no required cold chain for distribution, this concept remains more promising than RNA vaccines that are further along in clinical trials.

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Date Created
  • 2020-12

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Exploration of Enzymatic Efficiency in Double-Stranded DNA by Uracil-DNA Glycosylase and Optimization of Glycosylation Reaction of DNA Precursor

Description

The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and

The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.

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  • 2016-05

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Possibility VS Practicality; A Study of the Sequential Processing of Fired Cartridge Casings

Description

There are unrealistic expectations of the forensic science discipline by the public today. More specifically about the types of evidence that can be recovered from a fired cartridge casing. The

There are unrealistic expectations of the forensic science discipline by the public today. More specifically about the types of evidence that can be recovered from a fired cartridge casing. The common misconception with the evidence that can be recovered from a cartridge casing is that all three types of evidence: DNA, latent prints, and firearms can be recovered from the same cartridge casing. However, just because some analyses are possible does not mean that they are practical. The definition of possibility is that an event can happen. However, the definition of practicality is not only that it can happen, but that the event should occur to optimize the efficiency of a given task. Through literature review of previous studies as well as experimental data, each discipline (DNA, latent prints, and firearms and toolmark analysis) were evaluated. For the experimental trials, three total experiments were carried out. Experiment one focused on the possibility aspect, so in experiment one the best conditions were simulated to receive a positive result. Experiment two focused on creating conditions that would occur at a crime scene, and experiment three refined those variables to serve as middle ground. After evaluation, each discipline was classified as possible and/or practical. These results were then used to determine practical sequential processing for a fired cartridge casing. After both experimentation and review, it was determined that the best possible sequential processing path for a cartridge casing collected at the crime scene to get the quickest information back is as follows: Firearms, DNA, Latent Prints.

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Date Created
  • 2018-05

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Quantifying Intragenomic Variability in the 18S Gene of Trichonympha from Zootermopsis

Description

The 18S ribosomal RNA gene is ubiquitous across eukaryotes as it encodes the RNA component of the ribosomal small subunit. It is the most commonly used marker in molecular studies

The 18S ribosomal RNA gene is ubiquitous across eukaryotes as it encodes the RNA component of the ribosomal small subunit. It is the most commonly used marker in molecular studies of unicellular eukaryotes (protists) due to its species specificity and high copy number in the protist genome. Recent studies have revealed the widespread occurrence of intragenomic (intra-individual) polymorphism in many protists, an understudied phenomenon which contradicts the assumed homogeneity of the 18S throughout an individual genome. This thesis quantifies and analyzes the level of intragenomic and intraspecific 18S sequence variability in three Trichonympha species (T. campanula, T. collaris, T. postcylindrica) from Zootermopsis termites. Single-cell DNA extractions, PCR, cloning, and sequencing were performed to obtain 18S rRNA sequence reads, which were then analyzed to determine levels of sequence divergence among individuals and among species. Intragenomic variability was encountered in all three species. However, excluding singleton mutations, sequence divergence was less than 1% in 53 of the 56 compared individuals. T. collaris exhibited the most substantial intragenomic variability, with sequence divergence ranging from 0 to 3.4%. Further studies with more clones per cell are needed to elucidate the true extent of intragenomic variability in Trichonympha.

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Date Created
  • 2020-05

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A Determination of the Sequence-Dependent Kinetic Constants of Uracil-DNA Glycosylase

Description

Mutations in the DNA of somatic cells, resulting from inaccuracies in DNA<br/>replication or exposure to harsh conditions (ionizing radiation, carcinogens), may be<br/>loss-of-function mutations, and the compounding of these mutations can

Mutations in the DNA of somatic cells, resulting from inaccuracies in DNA<br/>replication or exposure to harsh conditions (ionizing radiation, carcinogens), may be<br/>loss-of-function mutations, and the compounding of these mutations can lead to cancer.<br/>Such mutations can come in the form of thymine dimers, N-𝛽 glycosyl bond hydrolysis,<br/>oxidation by hydrogen peroxide or other radicals, and deamination of cytosine to uracil.<br/>However, many cells possess the machinery to counteract the deleterious effects of<br/>such mutations. While eukaryotic DNA repair enzymes decrease the incidence of<br/>mutations from 1 mistake per 10^7 nucleotides to 1 mistake per 10^9 nucleotides, these<br/>mutations, however sparse, are problematic. Of particular interest is a mutation in which<br/>uracil is incorporated into DNA, either by spontaneous deamination of cysteine or<br/>misincorporation. Such mutations occur about one in every 107 cytidine residues in 24<br/>hours. DNA uracil glycosylase (UDG) recognizes these mutations and cleaves the<br/>glycosidic bond, creating an abasic site. However, the rate of this form of DNA repair<br/>varies, depending on the nucleotides that surround the uracil. Most enzyme-DNA<br/>interactions depend on the sequence of DNA (which may change the duplex twist),<br/>even if they only bind to the sugar-phosphate backbone. In the mechanism of uracil<br/>excision, UDG flips the uracil out of the DNA double helix, and this step may be<br/>impaired by base pairs that neighbor the uracil. The deformability of certain regions of<br/>DNA may facilitate this step in the mechanism, causing these regions to be less<br/>mutable. In DNA, base stacking, a form of van der Waals forces between the aromatic<br/>nucleic bases, may make these uracil inclusions more difficult to excise. These regions,<br/>stabilized by base stacking interactions, may be less susceptible to repair by<br/>glycosylases such as UDG, and thus, more prone to mutation.

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Date Created
  • 2021-05

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Novel DNA Extraction Methods for Mollusks and the History and Significance of Bermuda Land Snails

Description

Bermuda Land Snails make up a genus called Poecilozonites that is endemic to Bermuda and is extensively present in its fossil record. These snails were also integral to the creation

Bermuda Land Snails make up a genus called Poecilozonites that is endemic to Bermuda and is extensively present in its fossil record. These snails were also integral to the creation of the theory of punctuated equilibrium. The DNA of mollusks is difficult to sequence because of a class of proteins called mucopolysaccharides that are present in high concentrations in mollusk tissue, and are not removed with standard DNA extraction methods. They inhibit Polymerase Chain Reactions (PCRs) and interfere with Next Generation Sequencing methods. This paper will discuss the DNA extraction methods that were designed to remove the inhibitory proteins that were tested on another gastropod species (Pomacea canaliculata). These were chosen because they are invasive and while they are not pulmonates, they are similar enough to Bermuda Land Snails to reliably test extraction methods. The methods that were tested included two commercially available kits: the Qiagen Blood and Tissue Kit and the Omega Biotek Mollusc Extraction Kit, and one Hexadecyltrimethylammonium Bromide (CTAB) Extraction method that was modified for use on mollusk tissue. The Blood and Tissue kit produced some DNA, the mollusk kit produced almost none, and the CTAB Extraction Method produced the highest concentrations on average, and may prove to be the most viable option for future extractions. PCRs attempted with the extracted DNA have all failed, though it is likely due to an issue with reagents. Further spectrographic analysis of the DNA from the test extractions has shown that they were successful at removing mucopolysaccharides. When the protocol is optimized, it will be used to extract DNA from the tissue from six individuals from each of the two extant species of Bermuda Land Snails. This DNA will be used in several experiments involving Next Generation Sequencing, with the goal of assembling a variety of genome data. These data will then be used to a construct reference genome for Bermuda Land Snails. The genomes generated by this project will be used in population genetic analyses between individuals of the same species, and between individuals of different species. These analyses will then be used to aid in conservation efforts for the species.

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  • 2021-05

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From Report to Prosecution: A Comprehensive Approach to Sexual Assault Response in Arizona

Description

Every minute and a half, an American is sexually assaulted (Department of Justice, 2017). After an instance of sexual assault, some victims are given the choice of having a

Every minute and a half, an American is sexually assaulted (Department of Justice, 2017). After an instance of sexual assault, some victims are given the choice of having a sexual assault evidence kit (SAK) collected. These kits are designed to collect DNA evidence that will, in the best case scenario, result in the identification of the perpetrator. If the perpetrator cannot be located, the DNA profile can still be submitted to the FBI’s CODIS databank, which houses hundreds of thousands of DNA profiles from criminal cases, and may still lead to apprehension of the rapist. Unfortunately, some SAKs experience long delays, decades even, before being tested. To date, there are hundreds of thousands of untested SAKs that remain in police custody awaiting to be submitted for forensic profiling across the country. Here, we completed a holistic investigation of sexual assault response and SAK processing in Arizona. It is important to notice that the focus of our study not only includes SAK processing and the backlog but sexual assault prevention and improving victim reporting in an effort to understand the SAK “pipeline,” from assault to prosecution.
We identified problems in three major categories that negatively impact the SAK pipeline: historical inertia, legislative and institutional limitations, and community awareness. We found that a large number of SAKs in Arizona have remained untested due insufficient funding and staffing for public crime labs making it difficult for state labs to alleviate the SAK backlog while simultaneously responding to incoming cases (“Why the Backlog Exists,” n.d.). However, surveys of ASU undergraduate students revealed a significant interest in campus assault and the SAK backlog. Based on our findings, we suggest harnessing the interest of undergraduate students and recruiting them to specialized SAK-oriented forensic technician and sexual assault nurse examiner (SANE) training at ASU with the goal of creating a workforce that will alleviate the absence of trained professionals within the country. We also explore the possibility of the creation of a private crime laboratory at ASU devoted the processing of SAKs in Arizona as a measure of alleviating the demand on local public laboratories and providing a more economic alternative to commercial laboratories. The creation of an SAK laboratory at ASU would provide undergraduates the opportunity to learn more about real forensic analysis on campus, provide a pipeline for students to become technicians themselves, and help reduce and prevent a future SAK backlog in Arizona.

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Date Created
  • 2019-05

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The "CSI Effect" on Potential Juries: Correlations between Age, Gender, and Program-Watching Habits on Evidence Expectations

Description

The effects that forensic-themed programs such as CSI: Crime Scene Investigation has on the public's understanding and expectations of the criminal justice system has been a main focus of study

The effects that forensic-themed programs such as CSI: Crime Scene Investigation has on the public's understanding and expectations of the criminal justice system has been a main focus of study in recent years. This phenomenon was coined by the media and termed the "CSI Effect." This study aimed to research the correlations between age, gender, and program-watching habits on potential juries' evidence expectations in court. To do so, 70 people were surveyed and asked a series of demographic questions, as well as questions about how often they watch forensic-themed shows and their experience with the criminal justice system. They were given a mock crime scene scenario and asked about their scientific and non-scientific evidence expectations in this particular case. The most notable results showed that a longer exposure time to forensic-themed programs correlated to high evidence expectations. However, how often viewers watch forensic-themed programs did not seem to affect their evidence expectations. It was concluded that the higher evidence expectations by modern jurors may be due to a combination of the "CSI Effect" and the newly hypothesized "Tech Effect," instead of just being the consequence of the watching too much forensic-themed television.

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Date Created
  • 2014-05

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Molecular Engineering of Novel Polymeric Agents for Targeted Cancer Gene Therapy

Description

Abstract Molecular Engineering of Novel Polymeric Agents for Targeted Cancer Gene Therapy Dana Matthews Cancer gene cell therapy is a strategy that involves the administration of genes for correcting the

Abstract Molecular Engineering of Novel Polymeric Agents for Targeted Cancer Gene Therapy Dana Matthews Cancer gene cell therapy is a strategy that involves the administration of genes for correcting the effect of mutated cancer cells in order to induce tumor cell death. In particular, genes that encode for pro-apoptotic proteins can result in death of tumor cells. Prostate cancer is a very common cancer among males in America, and as highly destructive chemotherapy and radiation are generally the only treatments available once the cancer has metastasized, there is a need for the development of treatments that can specifically target and kill prostate cancer cells, while demonstrating low toxicity to other tissue. This experiment will attempt to create such a treatment through gene therapy techniques. The parallel synthesis and DNA binding affinity assay utilized in these experiments have produced a polymer that surpasses pEI-25, a gene delivery polymer standard, in both transfection efficacy and low cytotoxicity and trafficking of polyplexes in the cell, and finding methods to increase the transfection efficacy and specificity of polyplexes for PC3-PSMA cells.

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Date Created
  • 2008-12