Matching Items (175)
Description
V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.
ContributorsWang, Guannan (Author) / Chang, Yung (Thesis advisor) / Levitus, Marcia (Committee member) / Misra, Rajeev (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2012
Description
Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of these three proteins (and many of their homologs) are known, the exact mechanisms of interaction are still poorly understood. By mutational analysis of the TolC turn 1 residues, a drug hypersensitive mutant has been identified which is defective in functional interactions with AcrA and AcrB. Antibiotic resistant revertants carry alterations in both TolC and AcrA act by stabilizing functional complex assembly and opening of the TolC aperture, as monitored by stability of a labile TolC mutant and sensitivity to vancomycin, respectively. Alterations in the AcrB periplasmic hairpin loops lead to a similar antibiotic hypersensitivity phenotype and destabilized complex assembly. Likewise, alterations in TolC which constitutively open the aperture suppress this antibiotic sensitivity. Suppressor alterations in AcrA and AcrB partially restore antibiotic resistance by mediating stability of the complex. The AcrA suppressor alterations isolated in these studies map to the three crystallized domains and it is concluded they alter the AcrA conformation such that it is permanently fixed in an active state, which wild type only transiently goes through when activated by AcrB. Through this genetic evidence, a direct interaction between TolC and AcrB which is stabilized by AcrA has been proposed. In addition to stabilizing the interactions between TolC and AcrB, AcrA is also responsible for triggering opening of the TolC aperture by mediating energy flow from AcrB to TolC. By permanently altering the conformation of AcrA, suppressor mutants allow defective TolC or AcrB mutants to regain functional interactions lost by the initial mutations. The data provide the genetic proof for direct interaction between AcrB and that AcrA mediated opening of TolC requires AcrB as a scaffold.
ContributorsWeeks, Jon William (Author) / Misra, Rajeev (Thesis advisor) / Stout, Valerie (Committee member) / Shi, Yixin (Committee member) / Clark-Curtiss, Josephine (Committee member) / Arizona State University (Publisher)
Created2012
Description
The discovery and development of novel antibacterial agents is essential to address the rising health concern over antibiotic resistant bacteria. This research investigated the antibacterial activity of a natural clay deposit near Crater Lake, Oregon, that is effective at killing antibiotic resistant human pathogens. The primary rock types in the deposit are andesitic pyroclastic materials, which have been hydrothermally altered into argillic clay zones. High-sulfidation (acidic) alteration produced clay zones with elevated pyrite (18%), illite-smectite (I-S) (70% illite), elemental sulfur, kaolinite and carbonates. Low-sulfidation alteration at neutral pH generated clay zones with lower pyrite concentrations pyrite (4-6%), the mixed-layered I-S clay rectorite (R1, I-S) and quartz.
Antibacterial susceptibility testing reveals that hydrated clays containing pyrite and I-S are effective at killing (100%) of the model pathogens tested (E. coli and S. epidermidis) when pH (< 4.2) and Eh (> 450 mV) promote pyrite oxidation and mineral dissolution, releasing > 1 mM concentrations of Fe2+, Fe3+ and Al3+. However, certain oxidized clay zones containing no pyrite still inhibited bacterial growth. These clays buffered solutions to low pH (< 4.7) and oxidizing Eh (> 400 mV) conditions, releasing lower amounts (< 1 mM) of Fe and Al. The presence of carbonate in the clays eliminated antibacterial activity due to increases in pH, which lower pyrite oxidation and mineral dissolution rates.
The antibacterial mechanism of these natural clays was explored using metal toxicity and genetic assays, along with advanced bioimaging techniques. Antibacterial clays provide a continuous reservoir of Fe2+, Fe3+ and Al3+ that synergistically attack pathogens while generating hydrogen peroxide (H2O¬2). Results show that dissolved Fe2+ and Al3+ are adsorbed to bacterial envelopes, causing protein misfolding and oxidation in the outer membrane. Only Fe2+ is taken up by the cells, generating oxidative stress that damages DNA and proteins. Excess Fe2+ oxidizes inside the cell and precipitates Fe3+-oxides, marking the sites of hydroxyl radical (•OH) generation. Recognition of this novel geochemical antibacterial process should inform designs of new mineral based antibacterial agents and could provide a new economic industry for such clays.
Antibacterial susceptibility testing reveals that hydrated clays containing pyrite and I-S are effective at killing (100%) of the model pathogens tested (E. coli and S. epidermidis) when pH (< 4.2) and Eh (> 450 mV) promote pyrite oxidation and mineral dissolution, releasing > 1 mM concentrations of Fe2+, Fe3+ and Al3+. However, certain oxidized clay zones containing no pyrite still inhibited bacterial growth. These clays buffered solutions to low pH (< 4.7) and oxidizing Eh (> 400 mV) conditions, releasing lower amounts (< 1 mM) of Fe and Al. The presence of carbonate in the clays eliminated antibacterial activity due to increases in pH, which lower pyrite oxidation and mineral dissolution rates.
The antibacterial mechanism of these natural clays was explored using metal toxicity and genetic assays, along with advanced bioimaging techniques. Antibacterial clays provide a continuous reservoir of Fe2+, Fe3+ and Al3+ that synergistically attack pathogens while generating hydrogen peroxide (H2O¬2). Results show that dissolved Fe2+ and Al3+ are adsorbed to bacterial envelopes, causing protein misfolding and oxidation in the outer membrane. Only Fe2+ is taken up by the cells, generating oxidative stress that damages DNA and proteins. Excess Fe2+ oxidizes inside the cell and precipitates Fe3+-oxides, marking the sites of hydroxyl radical (•OH) generation. Recognition of this novel geochemical antibacterial process should inform designs of new mineral based antibacterial agents and could provide a new economic industry for such clays.
ContributorsMorrison, Keith D (Author) / Williams, Lynda B (Thesis advisor) / Williams, Stanley N (Thesis advisor) / Misra, Rajeev (Committee member) / Shock, Everett (Committee member) / Anbar, Ariel (Committee member) / Arizona State University (Publisher)
Created2015
Description
Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance.
ContributorsKurgan, Gavin (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Nannenga, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Description
The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic resistance becomes more and more prevalent it is necessary to think outside the box and do more than just increase the dosage of currently prescribed antibiotics. This study attempted to combat two forms of antibiotic resistance. The first is the AcrAB efflux pump which is able to pump antibiotics out of the cell. The second is the biofilms that E. coli can form. By using an inhibitor, the pump should be unable to rid itself of an antibiotic. On the other hand, using Tween allows for biofilm formation to either be disrupted or for the biofilm to be dissolved. By combining these two chemicals with an antibiotic that the efflux pump is known to expel, low concentrations of each chemical should result in an equivalent or greater effect on bacteria compared to any one chemical in higher concentrations. To test this hypothesis a 96 well plate BEC screen test was performed. A range of antibiotics were used at various concentrations and with varying concentrations of both Tween and the inhibitor to find a starting point. Following this, Erythromycin and Ciprofloxacin were picked as the best candidates and the optimum range of the antibiotic, Tween, and inhibitor were established. Finally, all three chemicals were combined to observe the effects they had together as opposed to individually or paired together. From the results of this experiment several conclusions were made. First, the inhibitor did in fact increase the effectiveness of the antibiotic as less antibiotic was needed if the inhibitor was present. Second, Tween showed an ability to prevent recovery in the MBEC reading, showing that it has the ability to disrupt or dissolve biofilms. However, Tween also showed a noticeable decrease in effectiveness in the overall treatment. This negative interaction was unable to be compensated for when using the inhibitor and so the hypothesis was proven false as combining the three chemicals led to a less effective treatment method.
ContributorsPetrovich Flynn, Chandler James (Author) / Misra, Rajeev (Thesis director) / Bean, Heather (Committee member) / Perkins, Kim (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Our lives are full of possibilities but also of self-imposed limitations. For some, finding their purpose in life and their place in the society is instinctive, but for many of us it requires a deliberate search. For the latter, self-discovery is a journey; an intentional path of many turns and twists, ups and downs, some surprises. It requires a person to slow down and assess oneself. What has been so far, what is right there on the surface, what is hidden deep down, and what is it that one strives for? Now, imagine narrowing down that path even more, and try to answer the question: Who am I as an artist?
ContributorsBlasko, Tanja (Author) / McMahon, Jeff (Thesis director) / Bowditch, Rachel (Committee member) / School of Film, Dance and Theatre (Contributor) / School of Art (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Horror as a genre is often dismissed on the basis of its reliance on jump scares and adrenaline highs, but history would suggest that society is just as well reflected in horror films as in any other genre. We only truly know our own experiences in real life, but film grants us the opportunity to see the world through another’s eyes, and what a more powerful way than through the experience of fear. In order to contextualize materials for the creation of a short horror film, the first part of this paper explores horror as a genre and the roles women tend to play in horror, both in the past and the present. It also considers how society perceives threats to women and how women perceive threats to themselves. It is important to define the foundation of women in horror and understand their current role in the genre to achieve the overarching goal of making a short horror film depicting a real issue that women face. A script, a storyboard, a timeline, and other materials for such a production are included as well.
ContributorsPeterschmidt, Bailey Willow (Author) / Mack, Robert L. (Thesis director) / Cheyne, Rebekah (Committee member) / School of Film, Dance and Theatre (Contributor) / Hugh Downs School of Human Communication (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
This project uses Kenneth Burke’s theory of dramatism and the pentad to analyze popular narrative films about human sex trafficking. It seeks to understand the relationship between a film’s dominant philosophy (as highlighted by utilizing Burke’s pentad), its inherently suggested solutions to trafficking, and the effect that the film has on viewers’ perception of trafficking. 20 narrative feature films about sex trafficking such as the 2008 film Taken were analyzed for this study. Three out of five of Burke’s philosophies were uncovered after analysis: idealism, mysticism, and materialism. Films that aligned with idealism were found to implicitly blame women for their own trafficking. Films that aligned with mysticism were found to rally audiences around violence and racism as opposed to women’s freedom. Films that aligned with materialism were found to be the most empathetic towards trafficked women. The conclusion of this paper is that films about sex trafficking have a high potential to be harmful to women who have exited trafficking. This paper asserts that the most valuable films about trafficking are those that are not simply based on a true story but are created by trafficking survivors themselves, such as the 2016 film Apartment 407.
ContributorsHamby, Hannah Mary (Co-author) / Raum, Brionna (Co-author) / Edson, Belle (Thesis director) / Zanin, Alaina (Committee member) / Dean, W.P. Carey School of Business (Contributor) / Hugh Downs School of Human Communication (Contributor) / School of Film, Dance and Theatre (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
This project uses Kenneth Burke’s theory of dramatism and the pentad to analyze popular narrative films about human sex trafficking. It seeks to understand the relationship between a film’s dominant philosophy (as highlighted by utilizing Burke’s pentad), its inherently suggested solutions to trafficking, and the effect that the film has on viewers’ perception of trafficking. 20 narrative feature films about sex trafficking such as the 2008 film Taken were analyzed for this study. Three out of five of Burke’s philosophies were uncovered after analysis: idealism, mysticism, and materialism. Films that aligned with idealism were found to implicitly blame women for their own trafficking. Films that aligned with mysticism were found to rally audiences around violence and racism as opposed to women’s freedom. Films that aligned with materialism were found to be the most empathetic towards trafficked women. The conclusion of this paper is that films about sex trafficking have a high potential to be harmful to women who have exited trafficking. This paper asserts that the most valuable films about trafficking are those that are not simply based on a true story but are created by trafficking survivors themselves, such as the 2016 film Apartment 407.
ContributorsRaum, Brionna L (Co-author) / Hamby, Hannah (Co-author) / Edson, Belle (Thesis director) / Zanin, Alaina (Committee member) / School of Film, Dance and Theatre (Contributor) / Hugh Downs School of Human Communication (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
South Korea possesses the only culture to successfully create a transnationality and hybridity formula that is not replicable. So why Korea and why now? The goal of this thesis creative project is to demonstrate the marketing and communications strategies used in the arts and culture industry to drive global awareness and interest in K-Pop. In order to achieve that goal, I created HellotoHallyu.com, a website designed for an audience of Millennials and Generation Z English speakers to increase their awareness of the growth and impact of the Korean Wave in a fun and engaging way. So those who may hear a song by K-Pop idol group BTS on a music awards show in the U.S. can get themselves up-to-speed before diving into the fast-paced world of K-culture gossip sites and forums. Hello to Hallyu delivers consumer-friendly, educational content easily understood by English speakers with no prior knowledge of Korean culture, while still piquing the interest of K-pop connoisseurs. It provides the background necessary for even the most dedicated fans to glean new knowledge of Korea's cultural industry and a new perspective on the content they consume. Hello to Hallyu is based on a combination of secondary and primary research conducted over four semesters beginning Spring 2017 and continuing through Spring 2018. This project is set up as an ever-expanding resource freely available to anyone with internet access. The research required to maintain the site will continue with the Wave. However, the content currently on the site is evergreen, a documentation of the history of the Wave as explained in peer-reviewed articles and by Dr. Ingyu Oh as well as a documentation of my personal experience with Hallyu while in Korea and as a Westerner living in the U.S. The site's goal is to demonstrate the marketing and communications strategies used in the industry to drive global awareness and interest. Through this means, Hello to Hallyu aims to provide fully developed multimedia content intended to increase English speakers' awareness of the growth and impact of the Korean Wave as shown through site visits, content views, and audience engagement.
ContributorsTravis, Lisa Anne (Author) / Hass, Mark (Thesis director) / Shewell, Justin (Committee member) / Walter Cronkite School of Journalism and Mass Communication (Contributor) / School of Film, Dance and Theatre (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05