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The diagnosis of bacterial infections based on phage multiplication has the potential for profound clinical implications, particularly for antibiotic-resistant strains and the slow-growing Mycobacterium tuberculosis. The possibility of hastening the diagnosis of antibiotic-resistant mycobacterial infections was accomplished via the study of Mycobacterium smegmatis, a generally non-pathogenic, comparatively fast growing microorganism to M. tuberculosis. These proof-of-concept studies established that after transduction of M. smegmatis cells with bacteriophages, MALDI-TOF MS could be used to detect increased amounts of phage proteins. Recording the growth of M. smegmatis over an 8-hour period, starting with very low OD600 measurements, simulated bacterial loads in clinical settings. For the purposes of MALDI-TOF MS, the procedure for the most effective lethal exposure for M. smegmatis was determined to be a 1-hour incubation in a 95°C water bath. Successful precipitation of the lytic mycobacteriophages D29 and Giles was performed using chloroform and methanol and overlaid with 1-2 μL of α-cyano-4-hydoxycinnaminic acid, which allowed for more distinct and repeatable MALDI-TOF MS spectra. Phage D29 was found to produce an m/z peak at 18.477 kDa, which may have indicated a 2+-charged ion of the 34.8 kDa minor tail protein. The Giles proteins that were identified with MALDI-TOF MS have not been directly compared to protein values reported in the scientific literature. However, the MALDI-TOF MS spectra suggested that distinct peaks existed between M. smegmatis mc2155 and mycobacteriophages, indicating that successful infection with lytic phage and replication thereafter may have occurred. The distinct peaks between M. smegmatis and the phage can be used as indicators of the presence of mycobacteria. At this point, the limits of detection of each phage must be elucidated in order for MALDI-TOF MS spectra to be successfully implemented as a mechanism to rapidly detect antibiotic-resistant mycobacteria.
ContributorsBarrett, Rachael Lauren (Author) / Haydel, Shelley (Thesis director) / Sandrin, Todd (Committee member) / Maarsingh, Jason (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The diagnosis of irritable bowel syndrome (IBS) is currently based on symptomatic criteria that exclude other conditions affecting the gastrointestinal tract, such as celiac disease, food allergies, and infections. The absence of appropriate diagnostic and therapeutic approaches for IBS places a significant burden on the patient and the health care system due to direct and indirect costs of care. Limitations associated with the application of symptomatic criteria include inappropriate use and/or intrinsic limitations such as the population to which these criteria are applied. The lack of biomarkers specific for IBS, non-specific abdominal symptoms, and considerable variability in the disease course creates additional uncertainty during diagnosis. This project involved screening tissue samples from patients with verified IBS to identify gene expression-based biomarkers associated with IBS. Through validation of microarray gene chip data on the tissue samples using PCR, it was determined that a number of genes within the diseased IBS patient tissue samples were differentially expressed in comparison to the healthy subjects. These findings could potentially lead to the diagnosis of IBS on the basis of a genetic "fingerprint".
ContributorsHockley, Maryam (Author) / Jurutka, Peter (Thesis director) / Sandrin, Todd (Committee member) / Zhang, Lin (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Natural Sciences (Contributor)
Created2013-12
Description
Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that afflicts more than 20% of the population in the United States. Symptoms include mild to severe abdominal discomfort accompanied by a change in stool character and form ranging from constipation to diarrhea. Additionally, IBS is associated with secondary effects including depression, anxiety, poor quality of life, insomnia and sexual dysfunction. Despite the known association of secondary effects, patients are often tested for potential illnesses that share similar pathological symptoms. This process can be costly and protracted and yet not deliver a completely accurate diagnosis. The aim of this research is to identify gene expression-based biological signatures and unique biomarkers for the detection of IBS. Through the use of quantitative polymerase chain reaction (qPCR), comparison of pooled samples of non-IBS patient-derived RNA were used to identify differentially expressed genes in patients with IBS. Data obtained from preliminary DNA microarray analysis demonstrated a degree of success in differentiating between IBS and asymptomatic patients. Additional comprehensive DNA microarray analyses have led to the identification of a series of 858 differentially expressed genes, including genes associated with serotonin metabolism, which may characterize the IBS pathological state. The microarray results were screened using a combination of gene ontological analysis and qPCR. Real-time PCR revealed repressed levels of tryptophan hydroxylase (TPH1), an enzyme involved in the rate- limiting step in serotonin biosynthesis, in IBS patients relative to controls. Lower concentrations of serum 25(OH)D were also observed among the IBS cohort relative to asymptomatic patients, especially among IBS-D subtype. Vitamin D was shown to modulate differentially expressed genes in IBS patients, suggesting that IBS pathophysiology may involve vitamin D insufficiency and/or an irregularity in serotonin metabolism. Additional qPCR analysis of 32 differentially expressed genes in IBS patients identified 7 putative genetic biomarkers proposed for a potential IBS diagnostic panel. Based on the quality of these results, we may be able to develop, test, and market a diagnostic kit for IBS.
ContributorsGrozic, Aleksandra (Author) / Jurutka, Peter (Thesis director) / Sandrin, Todd (Committee member) / Foxx-Orenstein, Amy (Committee member) / School of Mathematical and Natural Sciences (Contributor) / School of Social and Behavioral Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Proper developmental fidelity ensures uninterrupted progression towards sexual maturity and species longevity. However, early development, the time-frame spanning infancy through adolescence, is a fragile state since organisms have limited mobility and responsiveness towards their environment. Previous studies have shown that damage during development leads to an onset of developmental delay which is proportional to the extent of damage accrued by the organism. In contrast, damage sustained in older organisms does not delay development in response to tissue damage. In the fruit fly, Drosophila melanogaster, damage to wing precursor tissues is associated with developmental retardation if damage is sustained in young larvae. No developmental delay is observed when damage is inflicted closer to pupariation time. Here we use microarray analysis to characterize the genomic response to injury in Drosophila melanogaster in young and old larvae. We also begin to develop tools to examine in more detail, the role that the neurotransmitter dopamine might play in mediating injury-induced developmental delays.
ContributorsContreras Rodriguez, Jesus (Co-author) / Lupone, Teresa (Co-author) / Beckett, Chaz (Co-author) / Almajan, Ashley (Co-author) / Leek, Ty (Co-author) / Hussain, Sabahat (Co-author) / Marsh, Tyler (Co-author) / Broatch, Jennifer (Co-author) / Hackney Price, Jennifer (Thesis director) / Sandrin, Todd (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
With increasing urbanization, organisms face a myriad of novel ecological challenges. While the eco-evolutionary dynamics of urbanization are currently receiving a great deal of attention, the effect of urban disturbance on the microbiome of urban organisms is relatively unstudied. Indeed, studies of the microbiome may illuminate the mechanisms by which some species thrive after urbanization (pest implications), while other species go locally extinct (biodiversity implications). We investigated the gut microbiome of the Western black widow spider (Latrodectus hesperus). L. hesperus is an ideal model system as they are a pest species of medical importance in urban ecosystems, often forming dense urban infestations relative to the sparse populations found in their native Sonoran Desert. To gain insight into the composition of the microbiome in L. hesperus and its potential function, we sampled 4 urban, 4 desert, and 2 laboratory-reared spiders, and high-throughput sequencing of the 16S rRNA V4 region was used to investigate the diversity of gut microbiota. Dominant bacterial phyla across all samples were Firmicutes, Proteobacteria, and Actinobacteria. While desert widows showed more gut microbial diversity than urban widows, the difference was not statistically significant. The relative abundance of taxonomic classes Blastocatellia, Acidobacteriia, and Thermoleophilia detected in desert spiders was especially higher than those in urban and laboratory-reared spiders. However, urban spiders had a higher relative abundance of taxonomic class Actinomycetia. Differences in widow gut microbiome diversity improves our understanding of how features unique to a habitat, like prey diversity and soil microbes, may be shaping their microbiome. Additionally, this work further highlights the impact urbanization has on biodiversity loss, which indirectly develops a new biomarker for differentiating between urban and desert black widow spiders based on their gut microbiome.
ContributorsAsrari, Hasti (Author) / Johnson, Chad (Thesis director) / Sandrin, Todd (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Natural Sciences (Contributor) / School of Life Sciences (Contributor)
Created2022-12