Matching Items (95)
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., 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
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 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
Salmonella enterica is a gastrointestinal (GI) pathogen that can cause systemic diseases. It invades the host through the GI tract and can induce powerful immune responses in addition to disease. Thus, it is considered as a promising candidate to use as oral live vaccine vectors. Scientists have been making great efforts to get a properly attenuated Salmonella vaccine strain for a long time, but could not achieve a balance between attenuation and immunogenicity. So the regulated delayed attenuation/lysis Salmonella vaccine vectors were proposed as a design to seek this balance. The research work is progressing steadily, but more improvements need to be made. As one of the possible improvements, the cyclic adenosine monophosphate (cAMP) -independent cAMP receptor protein (Crp*) is expected to protect the Crp-dependent crucial regulator, araC PBAD, in these vaccine designs from interference by glucose, which decreases synthesis of cAMP, and enhance the colonizing ability by and immunogenicity of the vaccine strains. In this study, the cAMP-independent crp gene mutation, crp-70, with or without araC PBAD promoter cassette, was introduced into existing Salmonella vaccine strains. Then the plasmid stability, growth rate, resistance to catabolite repression, colonizing ability, immunogenicity and protection to challenge of these new strains were compared with wild-type crp or araC PBAD crp strains using western blots, enzyme-linked immunosorbent assays (ELISA) and animal studies, so as to evaluate the effects of the crp-70 mutation on the vaccine strains. The performances of the crp-70 strains in some aspects were closed to or even exceeded the crp+ strains, but generally they did not exhibit the expected advantages compared to their wild-type parents. Crp-70 rescued the expression of araC PBAD fur from catabolite repression. The strain harboring araC PBAD crp-70 was severely affected by its slow growth, and its colonizing ability and immunogenicity was much weaker than the other strains. The Pcrp crp-70 strain showed relatively good ability in colonization and immune stimulation. Both the araC PBAD crp-70 and the Pcrp crp-70 strains could provide certain levels of protection against the challenge with virulent pneumococci, which were a little lower than for the crp+ strains.
ContributorsShao, Shihuan (Author) / Curtiss, Roy (Thesis advisor) / Arizona State University (Publisher)
Created2012
Description
Hepatitis C virus (HCV) is a globally prevalent infection which is a main contributor to the global burden of liver disease. Due to its ability to establish a chronic infection, and the lack of usefulness of traditional neutralizing antibody vaccine design in producing a protective immune response, a preventative vaccine has been notoriously difficult to produce. To overcome this, a vaccine using non-structural protein 3 (NS3) as a target to elicit a T cell specific immune response is thought to be a possible strategy for eliciting a protective immune response against hepatitis C infection. In this paper, a recombinant strain of measles virus (MV) that expresses HCV NS3 protein was analyzed. The replication fitness of this recombinant virus also indicates that this construct replicates at a higher rate than parental measles strain. It is also demonstrated through western blot analysis of protein expression and immunofluorescence that this recombinant virus expresses both the inserted HCV NS3 protein, as well as native measles proteins.
ContributorsWoell, Dana Marie (Author) / Reyes del Valle, Jorge (Thesis director) / Nickerson, Cheryl (Committee member) / Julik, Emily (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05
Description
This thesis explores and analyzes the emergence of for-profit stem cell clinics in the United States, specifically in the Phoenix metropolitan area. Stem cell therapy is an emerging field that has great potential in preventing or treating a number of diseases. Certain companies are currently researching the application of stem cells as therapeutics. At present the FDA has only approved one stem cell-based product; however, there are a number of companies currently offering stem cell therapies. In the past five years, most news articles discussing these companies offering stem cell treatments talk of clinics in other countries. Recently, there seems to be a number of stem cell clinics appearing in the United States. Using a web search engine, fourteen stem cell clinics were identified and analyzed in the Phoenix metropolitan area. Each clinic was analyzed by their four key characteristics: business operations, stem cell types, stem cell isolation methods, and their position with the FDA. Based off my analysis, most of the identified clinics are located in Scottsdale or Phoenix. Some of these clinics even share the same location as another medical practice. Each of the fourteen clinics treat more than one type of health condition. The stem clinics make use of four stem cell types and three different isolation methods to obtain the stem cells. The doctors running these clinics almost always treat health conditions outside of their expertise. Some of these clinics even claim they are not subject to FDA regulation.
ContributorsAmrelia, Divya Vikas (Author) / Brafman, David (Thesis director) / Frow, Emma (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Clean water for drinking, food preparation, and bathing is essential for astronaut health and safety during long duration habitation of the International Space Station (ISS), including future missions to Mars. Despite stringent water treatment and recycling efforts on the ISS, it is impossible to completely prevent microbial contamination of onboard water supplies. In this work, we used a spaceflight analogue culture system to better understand how the microgravity environment can influence the pathogenesis-related characteristics of Burkholderia cepacia complex (Bcc), an opportunistic pathogen previously recovered from the ISS water system. The results of the present study suggest that there may be important differences in how this pathogen can respond and adapt to spaceflight and other low fluid shear environments encountered during their natural life cycles. Future studies are aimed at understanding the underlying mechanisms responsible for these phenotypes.
ContributorsKang, Bianca Younseon (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, Mark (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
DescriptionA novel and unconventional approach for delivering a eukaryotic apoptosis factor, TNF-related apoptosis-inducing ligand (TRAIL), to cancer cells within and around necrotizing tumors by utilizing a S. Typhimurium purine requiring auxotroph as a biological vector to develop two anticancer therapies with multiple modality and broad economic feasibility.
ContributorsKoons, Andrew (Author) / Curtiss, Roy (Thesis director) / Lake, Douglas (Committee member) / Janthakahalli, Nagaraj Vinay (Committee member) / Barrett, The Honors College (Contributor)
Created2013-12
Description
As life expectancy increases worldwide, age related diseases are becoming greater health concerns. One of the most prevalent age-related diseases in the United States is dementia, with Alzheimer’s disease (AD) being the most common form, accounting for 60-80% of cases. Genetics plays a large role in a person’s risk of developing AD. Familial AD, which makes up less than 1% of all AD cases, is caused by autosomal dominant gene mutations and has almost 100% penetrance. Genetic risk factors are believed to make up about 49%-79% of the risk in sporadic cases. Many different genetic risk factors for both familial and sporadic AD have been identified, but there is still much work to be done in the field of AD, especially in non-Caucasian populations. This review summarizes the three major genes responsible for familial AD, namely APP, PSEN1 and PSEN2. Also discussed are seven identified genetic risk factors for sporadic AD, single nucleotide polymorphisms in the APOE, ABCA7, NEDD9, CASS4, PTK2B, CLU, and PICALM genes. An overview of the main function of the proteins associated with the genes is given, along with the supposed connection to AD pathology.
ContributorsRichey, Alexandra Emmeline (Author) / Brafman, David (Thesis director) / Raman, Sreedevi (Committee member) / School of International Letters and Cultures (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Cell viability is an important assessment in cell culture to characterize the health of the cell population and confirm if cells are alive. Morphology or end-line assays are used to determine cell viability of entire populations. Intracellular pO2 levels is indicative of cell health and metabolism that can be used as a factor to asses cell viability in an in-line assay. Siloxane based pO2 sensing nanoprobes present a modality to visualize intracellular pO2. Using fluorescent lifetime imaging microscopy (FLIM), pO2 levels can be mapped intracellular as a highly functional in-line assay for cell viability. FLIM is an imaging modality that reconstructs an image based of its fluorescent lifetime. Nanoprobes were synthesized in different manufacturing/storage conditions. The nanoprobes for both long- and short-term storage were characterized in a cell free environment testing for changes in fluorescent intensity, average and maximum nanoprobe diameter. The nanoprobes were validated in two different culture systems, 2D and microcarrier culture systems, for human derived neural progenitor cells (NPCs) and neurons. Long- and short-term storage nanoprobes were used to label different neuronal based culture systems to asses labeling efficiency through fluorescent microscopy and flow cytometry. NPCs and neurons in each culture system was tested to see if nanoprobe labeling effected cellular phenotype for traits such as: cell proliferation, gene expression, and calcium imaging. Long-term and short-term storage nanoprobes were successfully validated for both NPCs and neurons in all culture systems. Assessments of the pO2 sensing nanoprobes will be further developed to create a highly functional and efficient in-line test for cell viability.
ContributorsLeyasi, Salma (Author) / Brafman, David (Thesis director) / Kodibagkar, Vikram (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Over 5.8 million people are currently living with Alzheimer’s disease (AD), with the sixth highest mortality rate in the United States. No known cure or substantially life-extending treatment exists. With the growing aging population these numbers are only expected to increase to about 13.8 million by the year 2050. Alzheimer’s is a multifactorial disease, giving rise to two main types: familial AD (FAD) and sporadic AD (SAD). Although there are different factors associated with each type of the disease, both FAD and SAD result in neuronal and synaptic loss and remain difficult to model in-vitro and treat overall.
Current advances in cellular models of neurodegenerative diseases overcome a variety of limitations possessed in animal and post-mortem human models. Human-induced pluripotent stem cells (hiPSCs) provide a platform with cells that can self-renew and differentiate into mature and functional cell types. HiPSCs are at the forefront of neurodegenerative disease research because of their ability to differentiate into neural cell types. Apolipoprotein E (ApoE) is a protein encoded by the APOE gene found on chromosome 19 of the human genome. There are three common polymorphisms in the APOE gene, resulting from a single amino acid change in the protein. The presence of these polymorphisms are studied as associated risk factors of developing AD. Different combinations of these alleles closely relate to the risk a patient has in developing Alzheimer’s disease. The risk associated effects of this gene are primarily investigated, however the protective effects are not examined to the same extent.
This research aims to overcome the existing limitations in cell differentiations and improve cell population purity that limits the variables present in the culture. To do this, this study optimized a differentiation protocol by separating and purifying neuronal cell populations to study the potential protective effects associated with ApoE, a risk factor seen in SAD. This platform aims to use a purified cell population to effectively analyze cell type specific affects of the ApoE risk factor, specifically in neurons.
Current advances in cellular models of neurodegenerative diseases overcome a variety of limitations possessed in animal and post-mortem human models. Human-induced pluripotent stem cells (hiPSCs) provide a platform with cells that can self-renew and differentiate into mature and functional cell types. HiPSCs are at the forefront of neurodegenerative disease research because of their ability to differentiate into neural cell types. Apolipoprotein E (ApoE) is a protein encoded by the APOE gene found on chromosome 19 of the human genome. There are three common polymorphisms in the APOE gene, resulting from a single amino acid change in the protein. The presence of these polymorphisms are studied as associated risk factors of developing AD. Different combinations of these alleles closely relate to the risk a patient has in developing Alzheimer’s disease. The risk associated effects of this gene are primarily investigated, however the protective effects are not examined to the same extent.
This research aims to overcome the existing limitations in cell differentiations and improve cell population purity that limits the variables present in the culture. To do this, this study optimized a differentiation protocol by separating and purifying neuronal cell populations to study the potential protective effects associated with ApoE, a risk factor seen in SAD. This platform aims to use a purified cell population to effectively analyze cell type specific affects of the ApoE risk factor, specifically in neurons.
ContributorsFrisch, Carlye Arin (Author) / Brafman, David (Thesis director) / Tian, Xiaojun (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05