Matching Items (10)
Filtering by

Clear all filters

152348-Thumbnail Image.png
Description
Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e.,

Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections.
ContributorsLeonetti, Cori (Author) / Shi, Yixin (Thesis advisor) / Stout, Valerie (Committee member) / Nickerson, Cheryl (Committee member) / Sandrin, Todd (Committee member) / Arizona State University (Publisher)
Created2013
150387-Thumbnail Image.png
Description
The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need

The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need to understand vaccination from vaccine design to prediction of vaccine efficacy using mathematical models. Post-exposure vaccination with VACV has been suggested to be effective if administered within four days of smallpox exposure although this has not been definitively studied in humans. The first and second chapters analyze post-exposure prophylaxis of VACV in an animal model using v50ΔB13RMγ, a recombinant VACV expressing murine interferon gamma (IFN-γ) also known as type II IFN. While untreated animals infected with wild type VACV die by 10 days post-infection (dpi), animals treated with v50ΔB13RMγ 1 dpi had decreased morbidity and 100% survival. Despite these differences, the viral load was similar in both groups suggesting that v50ΔB13RMγ acts as an immunoregulator rather than as an antiviral. One of the main characteristics of VACV is its resistance to type I IFN, an effect primarily mediated by the E3L protein, which has a Z-DNA binding domain and a double-stranded RNA (dsRNA) binding domain. In the third chapter a VACV that independently expresses both domains of E3L was engineered and compared to wild type in cells in culture. The dual expression virus was unable to replicate in the JC murine cell line where both domains are needed together for replication. Moreover, phosphorylation of the dsRNA dependent protein kinase (PKR) was observed at late times post-infection which indicates that both domains need to be linked together in order to block the IFN response. Because smallpox has already been eradicated, the utility of mathematical modeling as a tool for predicting disease spread and vaccine efficacy was explored in the last chapter using dengue as a disease model. Current modeling approaches were reviewed and the 2000-2001 dengue outbreak in a Peruvian region was analyzed. This last section highlights the importance of interdisciplinary collaboration and how it benefits research on infectious diseases.
ContributorsHolechek, Susan A (Author) / Jacobs, Bertram L (Thesis advisor) / Castillo-Chavez, Carlos (Committee member) / Frasch, Wayne (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
149931-Thumbnail Image.png
Description
HIV/AIDS is the sixth leading cause of death worldwide and the leading cause of death among women of reproductive age living in low-income countries. Clinicians in industrialized nations monitor the efficacy of antiretroviral drugs and HIV disease progression with the HIV-1 viral load assay, which measures the copy number of

HIV/AIDS is the sixth leading cause of death worldwide and the leading cause of death among women of reproductive age living in low-income countries. Clinicians in industrialized nations monitor the efficacy of antiretroviral drugs and HIV disease progression with the HIV-1 viral load assay, which measures the copy number of HIV-1 RNA in blood. However, viral load assays are not widely available in sub-Saharan Africa and cost between 50-$139 USD per test on average where available. To address this problem, a mixed-methods approach was undertaken to design a novel and inexpensive viral load diagnostic for HIV-1 and to evaluate barriers to its adoption in a developing country. The assay was produced based on loop-mediated isothermal amplification (LAMP). Blood samples from twenty-one individuals were spiked with varying concentrations of HIV-1 RNA to evaluate the sensitivity and specificity of LAMP. Under isothermal conditions, LAMP was performed with an initial reverse-transcription step (RT-LAMP) and primers designed for HIV-1 subtype C. Each reaction generated up to a few billion copies of target DNA within an hour. Presence of target was detected through naked-eye observation of a fluorescent indicator and verified by DNA gel electrophoresis and real-time fluorescence. The assay successfully detected the presence of HIV in samples with a broad range of HIV RNA concentration, from over 120,000 copies/reaction to 120 copies/reaction. In order to better understand barriers to adoption of LAMP in developing countries, a feasibility study was undertaken in Tanzania, a low-income country facing significant problems in healthcare. Medical professionals in Northern Tanzania were surveyed for feedback regarding perspectives of current HIV assays, patient treatment strategies, availability of treatment, treatment priorities, HIV transmission, and barriers to adoption of the HIV-1 LAMP assay. The majority of medical providers surveyed indicated that the proposed LAMP assay is too expensive for their patient populations. Significant gender differences were observed in response to some survey questions. Female medical providers were more likely to cite stigma as a source problem of the HIV epidemic than male medical providers while males were more likely to cite lack of education as a source problem than female medical providers.
ContributorsSalamone, Damien Thomas (Author) / Jacobs, Bertram L (Thesis advisor) / Marsiglia, Flavio (Committee member) / Stout, Valerie (Committee member) / Johnson, Crista (Committee member) / Arizona State University (Publisher)
Created2011
156067-Thumbnail Image.png
Description
Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene.

Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene. While genetically modified bacteria have historically been used for producing useful biopharmaceuticals like human insulin, plants can assemble much more complicated proteins, like human antibodies, that bacterial systems cannot. As plants do not harbor human pathogens, they are also safer alternatives than animal cell cultures. Additionally, plants can be grown very cheaply, in massive quantities.

In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
ContributorsDiamos, Andy (Author) / Mason, Hugh S (Thesis advisor) / Mor, Tsafrir (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2017
137550-Thumbnail Image.png
Description
This report provides information concerning qualities of methylcellulose and how those properties affect further experimentation within the biomedical world. Utilizing the compound’s biocompatibility many issues, ranging from surgical to cosmetic, can be solved. As of recent, studies indicate, methylcellulose has been used as a physically cross-linked gel, which

This report provides information concerning qualities of methylcellulose and how those properties affect further experimentation within the biomedical world. Utilizing the compound’s biocompatibility many issues, ranging from surgical to cosmetic, can be solved. As of recent, studies indicate, methylcellulose has been used as a physically cross-linked gel, which cannot sustain a solid form within the body. Therefore, this report will ultimately explore the means of creating a non-degradable, injectable, chemically cross-linking methylcellulose- based hydrogel. Methylcellulose will be evaluated and altered in experiments conducted within this report and a chemical cross-linker, developed from Jeffamine ED 2003 (O,O′-Bis(2-aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol), will be created. Experimentation with these elements is outlined here, and will ultimately prompt future revisions and analysis.
ContributorsBundalo, Zoran Luka (Author) / Vernon, Brent (Thesis director) / LaBelle, Jeffrey (Committee member) / Overstreet, Derek (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2013-05
153589-Thumbnail Image.png
Description
Invasive salmonellosis caused by Salmonella enterica serovar Typhimurium ST313 is a major health crisis in sub-Saharan Africa, with multidrug resistance and atypical clinical presentation challenging current treatment regimens and resulting in high mortality. Moreover, the increased risk of spreading ST313 pathovars worldwide is of major concern, given global public transportation

Invasive salmonellosis caused by Salmonella enterica serovar Typhimurium ST313 is a major health crisis in sub-Saharan Africa, with multidrug resistance and atypical clinical presentation challenging current treatment regimens and resulting in high mortality. Moreover, the increased risk of spreading ST313 pathovars worldwide is of major concern, given global public transportation networks and increased populations of immunocompromised individuals (as a result of HIV infection, drug use, cancer therapy, aging, etc). While it is unclear as to how Salmonella ST313 strains cause invasive disease in humans, it is intriguing that the genomic profile of some of these pathovars indicates key differences between classic Typhimurium (broad host range), but similarities to human-specific typhoidal Salmonella Typhi and Paratyphi. In an effort to advance fundamental understanding of the pathogenesis mechanisms of ST313 in humans, I report characterization of the molecular genetic, phenotypic and virulence profiles of D23580 (a representative ST313 strain). Preliminary studies to characterize D23580 virulence, baseline stress responses, and biochemical profiles, and in vitro infection profiles in human surrogate 3-D tissue culture models were done using conventional bacterial culture conditions; while subsequent studies integrated a range of incrementally increasing fluid shear levels relevant to those naturally encountered by D23580 in the infected host to understand the impact of biomechanical forces in altering these characteristics. In response to culture of D23580 under these conditions, distinct differences in transcriptional biosignatures, pathogenesis-related stress responses, in vitro infection profiles and in vivo virulence in mice were observed as compared to those of classic Salmonella pathovars tested.

Collectively, this work represents the first characterization of in vivo virulence and in vitro pathogenesis properties of D23580, the latter using advanced human surrogate models that mimic key aspects of the parental tissue. Results from these studies highlight the importance of studying infectious diseases using an integrated approach that combines actions of biological and physical networks that mimic the host-pathogen microenvironment and regulate pathogen responses.
ContributorsYang, Jiseon (Author) / Nickerson, Cheryl A. (Thesis advisor) / Chang, Yung (Committee member) / Stout, Valerie (Committee member) / Ott, C Mark (Committee member) / Roland, Kenneth (Committee member) / Barrila, Jennifer (Committee member) / Arizona State University (Publisher)
Created2015
154930-Thumbnail Image.png
Description
Viral protein U (Vpu) is a type-III integral membrane protein encoded by the Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays vital roles in down-regulation of CD4 receptors in T cells and also in the budding of virions. But, there remain key structure/function questions

Viral protein U (Vpu) is a type-III integral membrane protein encoded by the Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays vital roles in down-regulation of CD4 receptors in T cells and also in the budding of virions. But, there remain key structure/function questions regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis and thus, it makes for an attractive target to study the structural attributes of this protein by elucidating a structural model with X-ray crystallography. This study describes a multi-pronged approach of heterologous over-expression of Vpu. The strategies of purification and biophysical/ biochemical characterization of the different versions of the protein to evaluate their potential for crystallization are also detailed. Furthermore, various strategies employed for the crystallization of Vpu by both in surfo and in cubo techniques, and the challenges faced towards the structural studies of this membrane protein by characterization with solution Nuclear magnetic resonance (NMR) spectroscopy are also described.
ContributorsDeb, Arpan (Author) / Leket-Mor, Tsafrir S (Thesis advisor) / Fromme, Petra (Committee member) / Mason, Hugh (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2016
155123-Thumbnail Image.png
Description
Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death,

Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches.
ContributorsAbdelgalel, Rowida (Author) / Reyes del Valle, Jorge (Thesis advisor) / Mason, Hugh (Thesis advisor) / Lake, Douglas (Committee member) / Stout, Valerie (Committee member) / Frasch, Wayne (Committee member) / Arizona State University (Publisher)
Created2016
151797-Thumbnail Image.png
Description
The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve

The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.
ContributorsWeatherspoon-Griffin, Natasha (Author) / Shi, Yixin (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Misra, Rajeev (Committee member) / Nickerson, Cheryl (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013
Description
The concentration necessary to kill bacterial biofilms with antimicrobials is the minimum biofilm eradication concentration (MBEC). This is usually determined using an in vitro approach and will vary within different strains of bacteria. Biomedical implants produce biofilm-related infections presenting a unique challenge due to the combination of subpopulations of the

The concentration necessary to kill bacterial biofilms with antimicrobials is the minimum biofilm eradication concentration (MBEC). This is usually determined using an in vitro approach and will vary within different strains of bacteria. Biomedical implants produce biofilm-related infections presenting a unique challenge due to the combination of subpopulations of the bacterial community and the polysaccharide matrix presented by biofilms. The purpose of this investigation is to determine how exposure times in the order of weeks to months affect the MBEC. Using an in vitro approach, Staphylococcus aureus (UAMS-1) and methicillin-resistant Staphylococcus aureus (MRSA) biofilms were produced with a 24 hour growth time and exposed to two antimicrobials, tobramycin and vancomycin, and one combination treatment that consisted of 1:1 tobramycin: vancomycin by weight. Crystal violet screening was used in order to ensure the integrity of the biofilm matrix throughout the full time of exposure. It was determined that UAMS-1 MBECs were lowered after 56 days of exposure than after 5 days for all three treatment groups. MRSA MBECs after 5 days of exposure decreased only with in vancomycin treatment group.
ContributorsSteinhauff, Douglas Busch (Author) / Caplan, Michael (Thesis director) / Overstreet, Derek (Committee member) / Castaneda, Paulo (Committee member) / Materials Science and Engineering Program (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05