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Description
Coccidioidomycosis, also known as Valley Fever, is a disease caused by the dimorphic soil-dwelling fungus, Coccidioides sp. Coccidioidomycosis is difficult to diagnose because symptoms are similar to community-acquired pneumonia. Current diagnostic tests rely on antibody responses, but immune responses can be delayed and aberrant, resulting in false negative diagnoses. Unlike serology, detection of coccidioidal proteins or other fungal components in blood could distinguish valley fever from other pulmonary infections and provide a definitive diagnosis. Using mass spectrometry (LC-MS/MS) we examined the plasma peptidome from patients with serologically confirmed coccidioidomycosis. Mass spectra were searched using the protein database from the Coccidioides species, generated and annotated by the Broad Institute. 15 of 20 patients with serologically confirmed coccidioidomycosis demonstrated the presence of a peptide in plasma, "PGLDSKSLACTFSQV" (PGLD). The peptide is derived from an open reading frame from a "conserved hypothetical protein" annotated with 2 exons, and to date, found only in the C. posadasii strain Silviera RMSCC 3488 genomic sequence. In this thesis work, cDNA sequence analysis from polyadenylated RNA confirms the peptide sequence and genomic location of the peptide, but does not indicate that the intron in the gene prediction of C. posadasii strain Silviera RMSCC 3488 is present. A monoclonal antibody generated against the peptide bound to a 16kDa protein in T27K coccidioidal lysate. Detecting components of the fungus plasma could be a useful diagnostic tool, especially when serology does not provide a definitive diagnosis.
ContributorsDuffy, Stacy Leigh (Author) / Lake, Douglas (Thesis advisor) / Magee, Dewey Mitch (Committee member) / Antwi, Kwasi (Committee member) / Arizona State University (Publisher)
Created2013
Description
Infertility has become an increasing problem in developed countries and in many cases can be attributed to compromised sperm quality. Assessment of male fertility typically utilizes semen analysis which mainly examines sperm morphology, however many males whose sperm appear normal are sub- or infertile, suggesting that sperm from these males may be deficient in a protein or suite of proteins. To date, very little is known about the composition of sperm or the complex maturation process that confers motility and fertilization competency to sperm. Chapter 1 discusses the use of whole cell mass spectrometry to identify 1247 proteins comprising the Rhesus macaque (Macaca mulatta) sperm proteome, a commonly used model of human reproduction. This study provides a more robust proxy of human sperm composition than was previously available and facilitates studies of sperm using the rhesus macaque as a model. Chapters 2 & 3 provide a systems level overview of changes in sperm proteome composition that occurs during epididymal transit. Chapter 2 reports the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. Chapter 3 reports the sperm proteome from four distinct segments of the Rhesus macaque epididymis, including the caput, proximal corpus, distal corpus and cauda, identifying 1951, 2014, 1764 and 1423 proteins respectively. These studies identify a number of proteins that are added and removed from sperm during epididymal transit which likely play an important role in the sperm maturation process. To date no comparative evolutionary studies of sperm proteomes have been undertaken. Chapter 4 compares four mammalian sperm proteomes including the human, macaque, mouse and rat. This study identified 98 proteins common to all four sperm proteomes, 82 primate and 90 rodent lineage-specific proteins and 494, 467, 566, and 193 species specific proteins in the human, macaque, mouse and rat sperm proteomes respectively and discusses how differences in sperm composition may ultimately lead to functional differences across species. Finally, chapter 5 uses sperm proteome data to inform the preliminary design of a rodent contraceptive vaccine delivered orally using recombinant attenuated Salmonella vaccine vectors.
ContributorsSkerget, Sheri Jo (Author) / Karr, Timothy L. (Thesis advisor) / Lake, Douglas (Committee member) / Petritis, Konstantinos (Committee member) / Arizona State University (Publisher)
Created2013
Description
The majority of non-small cell lung cancer (NSCLC) patients (70%) are diagnosed with adenocarcinoma versus other histological subtypes. These patients often present with advanced, metastatic disease and frequently relapse after treatment. The tumor suppressor, Liver Kinase B1, is frequently inactivated in adenocarcinomas and loss of function is associated with a highly aggressive, metastatic tumor (1). Identification of the mechanisms deregulated with LKB1 inactivation could yield targeted therapeutic options for adenocarcinoma patients. Re-purposing the immune system to support tumor growth and aid in metastasis has been shown to be a feature in cancer progression (2). Tumor associated macrophages (TAMs) differentiate from monocytes, which are recruited to the tumor microenvironment via secretion of chemotaxic factors by cancer cells. We find that NSCLC cells deficient in LKB1 display increased secretion of C-C motif ligand 2 (CCL2), a chemokine involved in monocyte recruitment. To elucidate the molecular pathway regulating CCL2 up-regulation, we investigated inhibitors of substrates downstream of LKB1 signaling in A549, H23, H2030 and H838 cell lines. Noticeably, BAY-11-7082 (NF-κB inhibitor) reduced CCL2 secretion by an average 92%. We further demonstrate that a CCR2 antagonist and neutralizing CCL2 antibody substantially reduce monocyte migration to NSCLC (H23) cell line conditioned media. Using an in vivo model of NSCLC, we find that LKB1 deleted tumors demonstrate a discernible increase in CCL2 levels compared to normal lung. Moreover, tumors display an increase in the M2:M1 macrophage ratio and increase in tumor associated neutrophil (TAN) infiltrate compared to normal lung. This M2 shift was significantly reduced in mice treated with anti-CCL2 or a CCR2 antagonist and the TAN infiltrate was significantly reduced with the CCR2 antagonist. These data suggest that deregulation of the CCL2/CCR2 signaling axis could play a role in cancer progression in LKB1 deficient tumors.
ContributorsFriel, Jacqueline (Author) / Inge, Landon (Thesis advisor) / Lake, Douglas (Thesis advisor) / Blattman, Joseph (Committee member) / Arizona State University (Publisher)
Created2015
Description
Antibodies are naturally occurring proteins that protect a host during infection through direct neutralization and/or recruitment of the innate immune system. Unfortunately, in some infections, antibodies present unique hurdles that must be overcome for a safer and more efficacious antibody-based therapeutic (e.g., antibody dependent viral enhancement (ADE) and inflammatory pathology). This dissertation describes the utilization of plant expression systems to produce N-glycan specific antibody-based therapeutics for Dengue Virus (DENV) and Chikungunya Virus (CHIKV). The Fc region of an antibody interacts with Fcγ Receptors (FcγRs) on immune cells and components of the innate immune system. Each class of immune cells has a distinct action of neutralization (e.g., antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP)). Therefore, structural alteration of the Fc region results in novel immune pathways of protection. One approach is to modulate the N-glycosylation in the Fc region of the antibody. Of scientific significance, is the plant’s capacity to express human antibodies with homogenous plant and humanized N-glycosylation (WT and GnGn, respectively). This allows to study how specific glycovariants interact with other components of the immune system to clear an infection, producing a tailor-made antibody for distinct diseases. In the first section, plant-produced glycovariants were explored for reduced interactions with specific FcγRs for the overall reduction in ADE for DENV infections. The results demonstrate a reduction in ADE of our plant-produced monoclonal antibodies in in vitro experiments, which led to a greater survival in vivo of immunodeficient mice challenged with lethal doses of DENV and a sub-lethal dose of DENV in ADE conditions. In the second section, plant-produced glycovariants were explored for increased interaction with specific FcγRs to improve ADCC in the treatment of the highly inflammatory CHIKV. The results demonstrate an increase ADCC activity in in vitro experiments and a reduction in CHIKV-associated inflammation in in vivo mouse models. Overall, the significance of this dissertation is that it can provide a treatment for DENV and CHIKV; but equally importantly, give insight to the role of N-glycosylation in antibody effector functions, which has a broader implication for therapeutic development for other viral infections.
ContributorsHurtado, Jonathan (Author) / Chen, Qiang (Thesis advisor) / Arntzen, Charles (Committee member) / Borges, Chad (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2019
Description
The modern web presents an opportunity for educators and researchers to create tools that are highly accessible. Because of the near-ubiquity of modern web browsers, developers who hope to create educational and analytical tools can reach a large au- dience by creating web applications. Using JavaScript, HTML, and other modern web development technologies, Genie was developed as a simulator to help educators in biology, genetics, and evolution classrooms teach their students about population genetics. Because Genie was designed for the modern web, it is highly accessible to both educators and students, who can access the web application using any modern web browser on virtually any device. Genie demonstrates the efficacy of web devel- opment technologies for demonstrating and simulating complex processes, and it will be a unique educational tool for educators who teach population genetics.
ContributorsRoos, Benjamin Hirsch (Author) / Cartwright, Reed (Thesis director) / Wilson Sayres, Melissa (Committee member) / Mayron, Liam (Committee member) / Barrett, The Honors College (Contributor) / Computer Science and Engineering Program (Contributor)
Created2015-05
Description
Lactase persistence is the ability of adults to digest lactose in milk (Segurel & Bon, 2017). Mammals are generally distinguished by their mammary glands which gives females the ability to produce milk and feed their newborn children. The new born therefore requires the ability to breakdown the lactose in the milk to ensure its proper digestion (Segurel & Bon, 2017). Generally, humans lose the expression of lactase after weaning, which prevents them being able to breakdown lactose from dairy (Flatz, 1987).
My research is focused on the people of Turkana, a human pastoral population inhabiting Northwest Kenya. The people of Turkana are Nilotic people that are native to the Turkana district. There are currently no conclusive studies done on evidence for genetic lactase persistence in Turkana. Therefore, my research will be on the evolution of lactase persistence in the people of Turkana. The goal of this project is to investigate the evolutionary history of two genes with known involvement in lactase persistence, LCT and MCM6, in the Turkana. Variants in these genes have previously been identified to result in the ability to digest lactose post-weaning age. Furthermore, an additional study found that a closely related population to the Turkana, the Massai, showed stronger signals of recent selection for lactase persistence than Europeans in these genes. My goal is to characterize known variants associated with lactase persistence by calculating their allele frequencies in the Turkana and conduct selection scans to determine if LCT/MCM6 show signatures of positive selection. In doing this, we conducted a pilot study consisting of 10 female Turkana individuals and 10 females from four different populations from the 1000 genomes project namely: the Yoruba in Ibadan, Nigeria (YRI); Luhya in Webuye, Kenya; Utah Residents with Northern and Western European Ancestry (CEU); and the Southern Han Chinese. The allele frequency calculation suggested that the CEU (Utah Residents with Northern and Western European Ancestry) population had a higher lactase persistence associated allele frequency than all the other populations analyzed here, including the Turkana population. Our Tajima’s D calculations and analysis suggested that both the Turkana population and the four haplotype map populations shows signatures of positive selection in the same region. The iHS selection scans we conducted to detect signatures of positive selection on all five populations showed that the Southern Han Chinese (CHS), the LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations had stronger signatures of positive selection than the Turkana population. The LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations showed the strongest signatures of positive selection in this region. This project serves as a first step in the investigation of lactase persistence in the Turkana population and its evolution over time.
My research is focused on the people of Turkana, a human pastoral population inhabiting Northwest Kenya. The people of Turkana are Nilotic people that are native to the Turkana district. There are currently no conclusive studies done on evidence for genetic lactase persistence in Turkana. Therefore, my research will be on the evolution of lactase persistence in the people of Turkana. The goal of this project is to investigate the evolutionary history of two genes with known involvement in lactase persistence, LCT and MCM6, in the Turkana. Variants in these genes have previously been identified to result in the ability to digest lactose post-weaning age. Furthermore, an additional study found that a closely related population to the Turkana, the Massai, showed stronger signals of recent selection for lactase persistence than Europeans in these genes. My goal is to characterize known variants associated with lactase persistence by calculating their allele frequencies in the Turkana and conduct selection scans to determine if LCT/MCM6 show signatures of positive selection. In doing this, we conducted a pilot study consisting of 10 female Turkana individuals and 10 females from four different populations from the 1000 genomes project namely: the Yoruba in Ibadan, Nigeria (YRI); Luhya in Webuye, Kenya; Utah Residents with Northern and Western European Ancestry (CEU); and the Southern Han Chinese. The allele frequency calculation suggested that the CEU (Utah Residents with Northern and Western European Ancestry) population had a higher lactase persistence associated allele frequency than all the other populations analyzed here, including the Turkana population. Our Tajima’s D calculations and analysis suggested that both the Turkana population and the four haplotype map populations shows signatures of positive selection in the same region. The iHS selection scans we conducted to detect signatures of positive selection on all five populations showed that the Southern Han Chinese (CHS), the LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations had stronger signatures of positive selection than the Turkana population. The LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations showed the strongest signatures of positive selection in this region. This project serves as a first step in the investigation of lactase persistence in the Turkana population and its evolution over time.
ContributorsJobe, Ndey Bassin (Author) / Wilson Sayres, Melissa (Thesis director) / Paaijmans, Krijn (Committee member) / Taravella, Angela (Committee member) / School of Earth and Space Exploration (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Unlike the autosomes, recombination on the sex chromosomes is limited to the pseudoautosomal regions (PARs) at each end of the chromosome. PAR1 spans approximately 2.7 Mb from the tip of the proximal arm of each sex chromosome, and a pseudoautosomal boundary between the PAR1 and non-PAR region is thought to have evolved from a Y-specific inversion that suppressed recombination across the boundary. In addition to the two PARs, there is also a human-specific X-transposed region (XTR) that was duplicated from the X to the Y chromosome. Genetic diversity is expected to be higher in recombining than nonrecombining regions, particularly because recombination reduces the effects of linked selection, allowing neutral variation to accumulate. We previously showed that diversity decreases linearly across the previously defined pseudoautosomal boundary (rather than drop suddenly at the boundary), suggesting that the pseudoautosomal boundary may not be as strict as previously thought. In this study, we analyzed data from 1271 genetic females to explore the extent to which the pseudoautosomal boundary varies among human populations (broadly, African, European, South Asian, East Asian, and the Americas). We found that, in all populations, genetic diversity was significantly higher in the PAR1 and XTR than in the non-PAR regions, and that diversity decreased linearly from the PAR1 to finally reach a non-PAR value well past the pseudoautosomal boundary in all populations. However, we also found that the location at which diversity changes from reflecting the higher PAR1 diversity to the lower nonPAR diversity varied by as much as 500 kb among populations. The lack of genetic evidence for a strict pseudoautosomal boundary and the variability in patterns of diversity across the pseudoautosomal boundary are consistent with two potential explanations: (1) the boundary itself may vary across populations, or (2) that population-specific demographic histories have shaped diversity across the pseudoautosomal boundary.
ContributorsCotter, Daniel Juetten (Author) / Wilson Sayres, Melissa (Thesis director) / Stone, Anne (Committee member) / Webster, Timothy (Committee member) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Measles is a contagious, vaccine-preventable disease that continues to be the leading
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
ContributorsMushtaq, Zuena (Author) / Anderson, Karen (Thesis advisor) / Blattman, Joseph (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2016
Description
This work examines one dimension of the effect that complex human transport systems have on the spread of Chikungunya Virus (CHIKV) in the Caribbean from 2013 to 2015. CHIKV is transmitted by mosquitos and its novel spread through the Caribbean islands provided a chance to examine disease transmission through complex human transportation systems. Previous work by Cauchemez et al. had shown a simple distance-based model successfully predict CHIKV spread in the Caribbean using Markov chain Monte Carlo (MCMC) statistical methods. A MCMC simulation is used to evaluate different transportation methods (air travel, cruise ships, and local maritime traffic) for the primary transmission patterns through linear regression. Other metrics including population density to account for island size variation and dengue fever incidence rates as a proxy for vector control and health spending were included. Air travel and cruise travel were gathered from monthly passenger arrivals by island. Local maritime traffic is approximated with a gravity model proxy incorporating GDP-per-capita and distance and historic dengue rates were used for determine existing vector control measures for the islands. The Caribbean represents the largest cruise passenger market in the world, cruise ship arrivals were expected to show the strongest signal; however, the gravity model representing local traffic was the best predictor of infection routes. The early infected islands (<30 days) showed a heavy trend towards an alternate primary transmission but our consensus model able to predict the time until initial infection reporting with 94.5% accuracy for islands 30 days post initial reporting. This result can assist public health entities in enacting measures to mitigate future epidemics and provide a modelling basis for determining transmission modes in future CHIKV outbreaks.
ContributorsFries, Brendan F (Author) / Perrings, Charles (Thesis director) / Wilson Sayres, Melissa (Committee member) / Morin, Ben (Committee member) / School of Life Sciences (Contributor) / Department of Military Science (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T cell line is a challenge. The CD4+ Jurkat T cell line expresses endogenous TCRs that compete for space, accessory proteins, and proliferative signaling, and there is the potential for mixed dimer formation between the α and β chains of the endogenous receptor and that of the synthetic cancer-specific TCRs. To prevent hybridization between the receptors and to ensure the binding affinity measured with flow cytometry analysis is between the tetramer and the TCR construct, a CRISPR-Cas9 gene editing pipeline was developed. The guide RNAs (gRNAs) within the complex were designed to target the constant region of the α and β chains, as they are conserved between TCR clonotypes. To minimize further interference and confer cytotoxic capabilities, gRNAs were designed to target the CD4 coreceptor, and the CD8 coreceptor was delivered in a mammalian expression vector. Further, Golden Gate cloning methods were validated in integrating the gRNAs into a CRISPR-compatible mammalian expression vector. These constructs were transfected via electroporation into CD4+ Jurkat T cells to create a CD8+ knockout TCR Jurkat cell line for broadly applicable uses in T cell immunotherapies.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis advisor) / Mason, Hugh (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2020