Matching Items (41)
Description
This dissertation investigates the condition of skeletal muscle insulin resistance using bioinformatics and computational biology approaches. Drawing from several studies and numerous data sources, I have attempted to uncover molecular mechanisms at multiple levels. From the detailed atomistic simulations of a single protein, to datamining approaches applied at the systems biology level, I provide new targets to explore for the research community. Furthermore I present a new online web resource that unifies various bioinformatics databases to enable discovery of relevant features in 3D protein structures.
ContributorsMielke, Clinton (Author) / Mandarino, Lawrence (Committee member) / LaBaer, Joshua (Committee member) / Magee, D. Mitchell (Committee member) / Dinu, Valentin (Committee member) / Willis, Wayne (Committee member) / Arizona State University (Publisher)
Created2013
Description
While exercising mammalian muscle increasingly relies on carbohydrates for fuel as aerobic exercise intensity rises above the moderate range, flying birds are extraordinary endurance athletes and fuel flight, a moderate-high intensity exercise, almost exclusively with lipid. In addition, Aves have long lifespans compared to weight-matched mammals. As skeletal muscle mitochondria account for the majority of oxygen consumption during aerobic exercise, the primary goal was to investigate differences in isolated muscle mitochondria between these species and to examine to what extent factors intrinsic to mitochondria may account for the behavior observed in the intact tissue and whole organism. First, maximal enzyme activities were assessed in sparrow and rat mitochondria. Citrate synthase and aspartate aminotransferase activity were higher in sparrow compared to rat mitochondria, while glutamate dehydrogenase activity was lower. Sparrow mitochondrial NAD-linked isocitrate dehydrogenase activity was dependent on phosphate, unlike the mammalian enzyme. Next, the rate of oxygen consumption (JO), electron transport chain (ETC) activity, and reactive oxygen species (ROS) production were assessed in intact mitochondria. Maximal rates of fat oxidation were lower than for carbohydrate in rat but not sparrow mitochondria. ETC activity was higher in sparrows, but no differences were found in ROS production between species. Finally, fuel selection and control of respiration at three rates between rest and maximum were assessed. Mitochondrial fuel oxidation and selection mirrored that of the whole body; in rat mitochondria the reliance on carbohydrate increased as the rate of oxygen consumption increased, whereas fat dominated under all conditions in the sparrow. These data indicate fuel selection, at least in part, can be modulated at the level of the mitochondrial matrix when multiple substrates are present at saturating levels. As an increase in matrix oxidation-reduction potential has been linked to a suppression of fat oxidation and high ROS production, the high ETC activity relative to dehydrogenase activity in avian compared to mammalian mitochondria may result in lower matrix oxidation-reduction potential, allowing fatty acid oxidation to proceed while also resulting in low ROS production in vivo.
ContributorsKuzmiak, Sarah (Author) / Willis, Wayne T (Thesis advisor) / Mandarino, Lawrence (Committee member) / Sweazea, Karen (Committee member) / Harrison, Jon (Committee member) / Gadau, Juergen (Committee member) / Arizona State University (Publisher)
Created2012
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
It is important to consider factors that contribute to successful fertilization and the development of viable offspring. Better understanding the factors that contribute to infertility can be used to assist in the development of viable offspring, especially for human beings looking to successfully reproduce. Identifying paternal effect genes, genes that come from the father, introduces more targets that can be manipulated to produce specific reproductive effects. Use of Drosophila melanogaster as a model to study reproduction has increased, in part, due to the use of the GAL4 system. In this system, the GAL4 gene encodes an 881 amino acid protein that binds to the 4-site Upstream Activating Sequence (UAS) to induce transcription of the gene of interest. These sequences constitute the two components of the system: the driver (GAL4) and the responder (gene of interest) \u2014 each of which is maintained as a separate parental line. Effects of the GAL4 driver line "driving" transcription of the responder can be assessed by examining the offspring. One of the more common uses of the GAL4 system involves analyzing phenotypic effects of reducing or eliminating expression of a target gene through the induction of RNAi transcription, which often results in toxicity, lethality, or reduced viability. Utilizing these principles, we strove to demonstrate the effect of knocking down the expression of testis-specific sperm-leucyl-aminopeptidases gene CG13340 on progeny by inducing expression of RNAi with two distinct GAL4 driver lines - one with a nonspecific actin-binding activation sequence and the other with a testis-specific activation sequence. Comparison of both GAL4 driver lines to crosses using N01 wild type ("wt") flies verify that inducing RNAi transcription using the GAL4 system results in reduction of proper offspring development. Further studies using D. melanogaster and the GAL4 system can improve knowledge of factors contributing to male fertility and also be applied to better understand mammalian, specifically human, fertility.
ContributorsEvans, Donna Marie (Author) / Karr, Timothy L. (Thesis director) / Roland, Kenneth (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of English (Contributor)
Created2014-05
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
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection.
ContributorsMaarsingh, Jason (Author) / Haydel, Shelley E (Thesis advisor) / Roland, Kenneth (Committee member) / Sandrin, Todd (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2019
Description
The emergence of invasive non-Typhoidal Salmonella (iNTS) infections belonging to sequence type (ST) 313 are associated with severe bacteremia and high mortality in sub-Saharan Africa. Distinct features of ST313 strains include resistance to multiple antibiotics, extensive genomic degradation, and atypical clinical diagnosis including bloodstream infections, respiratory symptoms, and fever. Herein, I report the use of dynamic bioreactor technology to profile the impact of physiological fluid shear levels on the pathogenesis-related responses of ST313 pathovar, 5579. I show that culture of 5579 under these conditions induces profoundly different pathogenesis-related phenotypes than those normally observed when cultures are grown conventionally. Surprisingly, in response to physiological fluid shear, 5579 exhibited positive swimming motility, which was unexpected, since this strain was initially thought to be non-motile. Moreover, fluid shear altered the resistance of 5579 to acid, oxidative and bile stress, as well as its ability to colonize human colonic epithelial cells. This work leverages from and advances studies over the past 16 years in the Nickerson lab, which are at the forefront of bacterial mechanosensation and further demonstrates that bacterial pathogens are “hardwired” to respond to the force of fluid shear in ways that are not observed during conventional culture, and stresses the importance of mimicking the dynamic physical force microenvironment when studying host-pathogen interactions. The results from this study lay the foundation for future work to determine the underlying mechanisms operative in 5579 that are responsible for these phenotypic observations.
ContributorsCastro, Christian (Author) / Nickerson, Cheryl A. (Thesis advisor) / Ott, C. Mark (Committee member) / Roland, Kenneth (Committee member) / Barrila, Jennifer (Committee member) / Arizona State University (Publisher)
Created2016
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 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.
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
Description
Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by progressive autoimmune destruction of insulin-producing pancreatic β-cells. Genetic, immunological and environmental factors contribute to T1D development. The focus of this dissertation is to track the humoral immune response in T1D by profiling autoantibodies (AAbs) and anti-viral antibodies using an innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA).
AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
ContributorsBian, Xiaofang (Author) / LaBaer, Joshua (Thesis advisor) / Mandarino, Lawrence (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2015
Description
In sub-Saharan Africa, an invasive form of nontyphoidal Salmonella (iNTS) belonging to sequence type (ST)313 has emerged as a major public health concern causing widespread bacteremia and mortality in children with malaria and adults with HIV. Clinically, ST313 pathovars are characterized by the absence of gastroenteritis, which is commonly found in “classical” nontyphoidal Salmonella (NTS), along with multidrug resistance, pseudogene formation, and chromosome degradation. There is an urgent need to understand the biological and physical factors that regulate the disease causing properties of ST313 strains. Previous studies from our lab using dynamic Rotating Wall Vessel (RWV) bioreactor technology and “classical” NTS strain χ3339 showed that physiological fluid shear regulates gene expression, stress responses and virulence in unexpected ways that are not observed using conventional shake and static flask conditions, and in a very different manner as compared to ST313 strain D23580. Leveraging from these findings, the current study was the first to report the effect of fluid shear on the pathogenesis-related stress responses of S. Typhimurium ST313 strain A130, which evolved earlier than D23580 within the ST313 clade. A130 displayed enhanced resistance to acid, oxidative and bile stresses when cultured in the high fluid shear (HFS) control condition relative to the low fluid shear (LFS) condition in stationary phase using Lennox Broth (LB) as the culture medium. The greatest magnitude of the survival benefit conferred by high fluid shear was observed in response to oxidative and acid stresses. No differences were observed for thermal and osmotic stresses. Based on previous findings from our laboratory, we also assessed how the addition of phosphate or magnesium ions to the culture medium altered the acid or oxidative stress responses of A130 grown in the RWV. Addition of either
phosphate or magnesium to the culture medium abrogated the fluid shear-related differences observed for A130 in LB medium for the acid or oxidative stress responses, respectively. Collectively, these findings indicate that like other Salmonella strains assessed thus far by our team, A130 responds to differences in physiological fluid shear, and that ion concentrations can modulate those responses.
phosphate or magnesium to the culture medium abrogated the fluid shear-related differences observed for A130 in LB medium for the acid or oxidative stress responses, respectively. Collectively, these findings indicate that like other Salmonella strains assessed thus far by our team, A130 responds to differences in physiological fluid shear, and that ion concentrations can modulate those responses.
ContributorsGutierrez-Jensen, Ami Dave (Author) / Nickerson, Cheryl A. (Thesis advisor) / Barrila, Jennifer (Thesis advisor) / Ott, C. M. (Committee member) / Roland, Kenneth (Committee member) / Arizona State University (Publisher)
Created2017