Matching Items (106)
Description
Dire wolves have recently risen to fame as a result of the popular television program Game of Thrones, and thus many viewers know dire wolves as the sigil and loyal companions of the Stark house. Far fewer recognize dire wolves by their scientific name, Canis dirus, or understand the population history of this ‘fearsome wolf’ species that roamed the Americas until the megafaunal mass extinction event of the Late Pleistocene. Although numerous studies have examined the species using morphological and geographical methods, thus far their results have been either inconclusive or contradictory. Remaining questions include the relationships dire wolves share with other members of the Canis genus and the internal structure of their populations. Advancements in ancient DNA recovery methods may make it possible to study dire wolf specimens at the molecular level for the first time and may therefore prove useful in clarifying the answers to these questions. Eighteen dire wolf specimens were collected from across the United States and subjected to ancient DNA extraction, library preparation, amplification and purification, bait preparation and capture, and next-generation sequencing. There was an average of 76.9 unique reads and 5.73% coverage when mapped to the Canis familiaris reference genome in ultraconserved regions of the mitochondrial genome. The results indicate that endogenous ancient DNA was not successfully recovered and perhaps ancient DNA recovery methods have not advanced to the point of retrieving informative amounts of DNA from particularly old, thermally degraded specimens. Nevertheless, the ever-changing nature of ancient DNA research makes it vital to continually test the limitations of the field and suggests that ancient DNA recovery methods will prove useful in illuminating dire wolf population history at some point in the future.
ContributorsSkerry, Katherine Marie (Author) / Stone, Anne (Thesis director) / Amdam, Gro (Committee member) / Larson, Greger (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Nutrition and Health Promotion (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Biomarkers are the cornerstone of modern-day medicine. They are defined as any biological substance in or outside the body that gives insight to the body's condition. Doctors and researchers can measure specific biomarkers to diagnose and treat patients, such as the concentration of hemoglobin Alc and its connection to diabetes. There are a variety of methods, or assays, to detect biomarkers, but the most common assay is enzyme-linked immunosorbent assay (ELISA). A new-generation assay termed mass spectrometric immunoassay (MSIA) can measure proteoforms, the different chemical variations of proteins, and their relative abundance. ELISA on the other hand measures the overall concentration of protein in the sample. Measuring each of the proteoforms of a protein is important because only one or two variations could be biologically significant and/or cause diseases. However, running MSIA is expensive. For this reason, an alternative plate-based MSIA technique was tested for its ability to detect the proteoforms of a protein called apolipoprotein C-III (ApoC-III). This technique combines the protein capturing procedure of ELISA to isolate the protein with detection in a mass spectrometer. A larger amount of ApoC-III present in the body indicates a considerable risk for coronary heart disease. The precision of the assay is determined on the coefficient of variation (CV). A CV value is the ratio of standard deviation in relation to the mean, represented as a percentage. The smaller the percentage, the less variation the assay has, and therefore the more ability it has to detect subtle changes in the biomarker. An accepted CV would be less than 10% for single-day tests (intra-day) and less than 15% for multi-day tests (inter-day). The plate-based MSIA was started by first coating a 96-well round bottom plate with 2.5 micrograms of ApoC-III antibody. Next, a series of steps were conducted: a buffer wash, then the sample incubation, followed by another buffer wash and two consecutive water washes. After the final wash, the wells were filled with a MALDI matrix, then spotted onto a gold plate to dry. The dry gold target was then placed into a MALDI-TOF mass spectrometer to produce mass spectra for each spot. The mass spectra were calibrated and the area underneath each of the four peaks representing the ApoC-III proteoforms was exported as an Excel file. The intra-day CV values were found by dividing the standard deviation by the average relative abundance of each peak. After repeating the same procedure for three more days, the inter-day CVs were found using the same method. After completing the experiment, the CV values were all within the acceptable guidelines. Therefore, the plate-based MSIA is a viable alternative for finding proteoforms than the more expensive MSIA tips. To further validate this, additional tests will need to be conducted with different proteins and number of samples to determine assay flexibility.
ContributorsTieu, Luc (Author) / Borges, Chad (Thesis director) / Nedelkov, Dobrin (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Brown adipose tissue (BAT) is thought to be important in combating obesity as it can expend energy in the form of heat, e.g. thermogenesis. The goal of this study was to study the effect of injected norepinephrine (NE) on the activation of BAT in rats that were fed a high fat diet (HFD). A dose of 0.25 mg/kg NE was used to elicit a temperature response that was measured using transponders inserted subcutaneously over the BAT and lower back and intraperitoneally to measure the core temperature. The results found that the thermic effect of the BAT increased after the transition from low fat diet to a high fat diet (LFD) yet, after prolonged exposure to the HFD, the effects resembled levels found with the LFD. This suggests that while a HFD may stimulate the effect of BAT, long term exposure may have adverse effects on BAT activity. This may be due to internal factors that will need to be examined further.
ContributorsSion, Paul William (Author) / Herman, Richard (Thesis director) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Vitellogenin (vg) is a precursor protein of egg yolk in honeybees, but it is also known to have immunological functions. The purpose of this experiment was to determine the effect of vg on the viral load of deformed wing virus (DWV) in worker honey bees (Apis mellifera). I hypothesized that a reduction in vg expression would lead to an increase in the viral load. I collected 180 worker bees and split them into four groups: half the bees were subjected to a vg gene knockdown by injections of double stranded vg RNA, and the rest were injected with green fluorescent protein (gfp) double stranded RNA. Half of each group was thereafter injected with DWV, and half given a sham injection. The rate of mortality in all four groups was higher than expected, leaving only 17 bees total. I dissected these bees' fat bodies and extracted their RNA to test for vg and DWV. PCR results showed that, out of the small group of remaining bees, the levels of vg were not statistically different. Furthermore, both groups of virus-injected bees showed similar viral loads. Because of the high mortality rate bees and the lack of differing levels of vg transcript between experimental and control groups, I could not draw conclusions from these results. The high mortality could be caused by several factors: temperature-induced stress, repeated stress from the two injections, and stress from viral infection. In addition, it is possible that the vg dsRNA batch I used was faulty. This thesis exemplifies that information cannot safely be extracted when loss of sampling units result in a small datasets that do not represent the original sampling population.
ContributorsCrable, Emma Lewis (Author) / Amdam, Gro (Thesis director) / Wang, Ying (Committee member) / Dahan, Romain (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Almost every form of cancer deregulates the expression and activity of anabolic glycosyltransferase (GT) enzymes, which incorporate particular monosaccharides in a donor acceptor as well as linkage- and anomer-specific manner to assemble complex and diverse glycans that significantly affect numerous cellular events, including tumorigenesis and metastasis. Because glycosylation is not template-driven, GT deregulation yields heterogeneous arrays of aberrant intact glycan products, some in undetectable quantities in clinical bio-fluids (e.g., blood plasma). Numerous glycan features (e.g., 6 sialylation, β-1,6-branching, and core fucosylation) stem from approximately 25 glycan “nodes:” unique linkage specific monosaccharides at particular glycan branch points that collectively confer distinguishing features upon glycan products. For each node, changes in normalized abundance (Figure 1) may serve as nearly 1:1 surrogate measure of activity for culpable GTs and may correlate with particular stages of carcinogenesis. Complementary to traditional top down glycomics, the novel bottom-up technique applied herein condenses each glycan node and feature into a single analytical signal, quantified by two GC-MS instruments: GCT (time-of-flight analyzer) and GCMSD (transmission quadrupole analyzers). Bottom-up analysis of stage 3 and 4 breast cancer cases revealed better overall precision for GCMSD yet comparable clinical performance of both GC MS instruments and identified two downregulated glycan nodes as excellent breast cancer biomarker candidates: t-Gal and 4,6-GlcNAc (ROC AUC ≈ 0.80, p < 0.05). Resulting from the activity of multiple GTs, t-Gal had the highest ROC AUC (0.88) and lowest ROC p‑value (0.001) among all analyzed nodes. Representing core-fucosylation, glycan node 4,6-GlcNAc is a nearly 1:1 molecular surrogate for the activity of α-(1,6)-fucosyltransferase—a potential target for cancer therapy. To validate these results, future projects can analyze larger sample sets, find correlations between breast cancer stage and changes in t-Gal and 4,6-GlcNAc levels, gauge the specificity of these nodes for breast cancer and their potential role in other cancer types, and develop clinical tests for reliable breast cancer diagnosis and treatment monitoring based on t-Gal and 4,6-GlcNAc.
ContributorsZaare, Sahba (Author) / Borges, Chad (Thesis director) / LaBaer, Joshua (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Aberrant glycosylation has been shown to be linked to specific cancers, and using this idea, it was proposed that the levels of glycans in the blood could predict stage I adenocarcinoma. To track this glycosylation, glycan were broken down into glycan nodes via methylation analysis. This analysis utilized information from N-, O-, and lipid linked glycans detected from gas chromatography-mass spectrometry. The resulting glycan node-ratios represent the initial quantitative data that were used in this experiment.
For this experiment, two Sets of 50 µl blood plasma samples were provided by NYU Medical School. These samples were then analyzed by Dr. Borges’s lab so that they contained normalized biomarker levels from patients with stage 1 adenocarcinoma and control patients with matched age, smoking status, and gender were examined. An ROC curve was constructed under individual and paired conditions and AUC calculated in Wolfram Mathematica 10.2. Methods such as increasing size of training set, using hard vs. soft margins, and processing biomarkers together and individually were used in order to increase the AUC. Using a soft margin for this particular data set was proved to be most useful compared to the initial set hard margin, raising the AUC from 0.6013 to 0.6585. In regards to which biomarkers yielded the better value, 6-Glc/6-Man and 3,6-Gal glycan node ratios had the best with 0.7687 AUC and a sensitivity of .7684 and specificity of .6051. While this is not enough accuracy to become a primary diagnostic tool for diagnosing stage I adenocarcinoma, the methods examined in the paper should be evaluated further. . By comparison, the current clinical standard blood test for prostate cancer that has an AUC of only 0.67.
For this experiment, two Sets of 50 µl blood plasma samples were provided by NYU Medical School. These samples were then analyzed by Dr. Borges’s lab so that they contained normalized biomarker levels from patients with stage 1 adenocarcinoma and control patients with matched age, smoking status, and gender were examined. An ROC curve was constructed under individual and paired conditions and AUC calculated in Wolfram Mathematica 10.2. Methods such as increasing size of training set, using hard vs. soft margins, and processing biomarkers together and individually were used in order to increase the AUC. Using a soft margin for this particular data set was proved to be most useful compared to the initial set hard margin, raising the AUC from 0.6013 to 0.6585. In regards to which biomarkers yielded the better value, 6-Glc/6-Man and 3,6-Gal glycan node ratios had the best with 0.7687 AUC and a sensitivity of .7684 and specificity of .6051. While this is not enough accuracy to become a primary diagnostic tool for diagnosing stage I adenocarcinoma, the methods examined in the paper should be evaluated further. . By comparison, the current clinical standard blood test for prostate cancer that has an AUC of only 0.67.
ContributorsDe Jesus, Celine Spicer (Author) / Taylor, Thomas (Thesis director) / Borges, Chad (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
In Apis mellifera, gustatory responsiveness to sucrose is a good indicator of learning ability \u2014 in that individuals with high sucrose responsiveness will typically form faster, longer-lasting associations with conditioned stimulus than individuals with a low sucrose responsiveness. The purpose of this study was to determine whether experience with olfactory conditioning had lasting effects on gustatory responsiveness. Groups were placed in an environment that would facilitate association of an odor to a sucrose reward, tested for retention, then tested for gustatory responsiveness. Control groups underwent the same testing schedule, but were not exposed to odor in the first environment. There was no significant difference in gustatory responsiveness between the two groups. Mann-Whitney tests were used to analyze the results, and though the mean GRS score was lower among the treatment group there was no significant trend, possibly due to small sample sizes.
ContributorsSeemann, J. H. (Author) / Amdam, Gro (Thesis director) / Smith, Brian (Committee member) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Genetic counseling is a medical field that was established in the 1970s, but whose demand is now growing exponentially due to modern genetic technology. We now have the ability to look into the human genetic code, detect the genotype of individuals, and use this knowledge to our benefit. However, Genetic testing results in a need for new ethical boundaries to be drawn. The idea of the "best possible conditions" of conceiving a child and whether this child has a right to not know are the two major ethical issues that will be focused on in order to analyze the ethical boundary that needs to be drawn for genetic counseling. In order to analyze these ethical issues, a focus group of Arizona State University students was organized. After producing results for the focus group, there are no true conclusions that can be drawn that applied to all of society. The focus group sample size was too small to produce a broad range of results and the participants were all Arizona State University Undergraduate students. However, it did become apparent that knowledge on these ethical issues is crucial in order to ensure they do not hinder the field of genetic counseling. It is predicted that in order to have the best outcome for the field of genetic counseling, genetic counselors themselves need to draw the ethical boundaries for the issues studied.
ContributorsBarker, Samantha (Author) / Amdam, Gro (Thesis director) / Bang, Christofer (Committee member) / Wang, Ying (Committee member) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Disturbances in the protein interactome often play a large role in cancer progression. Investigation of protein-protein interactions (PPI) can increase our understanding of cancer pathways and will disclose unknown targets involved in cancer disease biology. Although numerous methods are available to study protein interactions, most platforms suffer from drawbacks including high false positive rates, low throughput, and lack of quantification. Moreover, most methods are not compatible for use in a clinical setting. To address these limitations, we have developed a multiplexed, in-solution protein microarray (MISPA) platform with broad applications in proteomics. MISPA can be used to quantitatively profile PPIs and as a robust technology for early detection of cancers. This method utilizes unique DNA barcoding of individual proteins coupled with next generation sequencing to quantitatively assess interactions via barcode enrichment. We have tested the feasibility of this technology in the detection of patient immune responses to oropharyngeal carcinomas and in the discovery of novel PPIs in the B-cell receptor (BCR) pathway. To achieve this goal, 96 human papillomavirus (HPV) antigen genes were cloned into pJFT7-cHalo (99% success) and pJFT7-n3xFlag-Halo (100% success) expression vectors. These libraries were expressed via a cell-free in vitro transcription-translation system with 93% and 96% success, respectively. A small-scale study of patient serum interactions with barcoded HPV16 antigens was performed and a HPV proteome-wide study will follow using additional patient samples. In addition, 15 query proteins were cloned into pJFT7_nGST expression vectors, expressed, and purified with 93% success to probe a library of 100 BCR pathway proteins and detect novel PPIs.
ContributorsRinaldi, Capria Lakshmi (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Microfluidic platforms have been exploited extensively as a tool for the separation of particles by electric field manipulation. Microfluidic devices can facilitate the manipulation of particles by dielectrophoresis. Separation of particles by size and type has been demonstrated by insulator-based dielectrophoresis in a microfluidic device. Thus, manipulating particles by size has been widely studied throughout the years. It has been shown that size-heterogeneity in organelles has been linked to multiple diseases from abnormal organelle size. Here, a mixture of two sizes of polystyrene beads (0.28 and 0.87 μm) was separated by a ratchet migration mechanism under a continuous flow (20 nL/min). Furthermore, to achieve high-throughput separation, different ratchet devices were designed to achieve high-volume separation. Recently, enormous efforts have been made to manipulate small size DNA and proteins. Here, a microfluidic device comprising of multiple valves acting as insulating constrictions when a potential is applied is presented. The tunability of the electric field gradient is evaluated by a COMSOL model, indicating that high electric field gradients can be reached by deflecting the valve at a certain distance. Experimentally, the tunability of the dynamic constriction was demonstrated by conducting a pressure study to estimate the gap distance between the valve and the substrate at different applied pressures. Finally, as a proof of principle, 0.87 μm polystyrene beads were manipulated by dielectrophoresis. These microfluidic platforms will aid in the understanding of size-heterogeneity of organelles for biomolecular assessment and achieve separation of nanometer-size DNA and proteins by dielectrophoresis.
ContributorsOrtiz, Ricardo (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2021