Matching Items (86)
Description
Single cell analysis has become increasingly important in understanding disease onset, progression, treatment and prognosis, especially when applied to cancer where cellular responses are highly heterogeneous. Through the advent of single cell computerized tomography (Cell-CT), researchers and clinicians now have the ability to obtain high resolution three-dimensional (3D) reconstructions of single cells. Yet to date, no live-cell compatible version of the technology exists. In this thesis, a microfluidic chip with the ability to rotate live single cells in hydrodynamic microvortices about an axis parallel to the optical focal plane has been demonstrated. The chip utilizes a novel 3D microchamber design arranged beneath a main channel creating flow detachment into the chamber, producing recirculating flow conditions. Single cells are flowed through the main channel, held in the center of the microvortex by an optical trap, and rotated by the forces induced by the recirculating fluid flow. Computational fluid dynamics (CFD) was employed to optimize the geometry of the microchamber. Two methods for the fabrication of the 3D microchamber were devised: anisotropic etching of silicon and backside diffuser photolithography (BDPL). First, the optimization of the silicon etching conditions was demonstrated through design of experiment (DOE). In addition, a non-conventional method of soft-lithography was demonstrated which incorporates the use of two positive molds, one of the main channel and the other of the microchambers, compressed together during replication to produce a single ultra-thin (<200 µm) negative used for device assembly. Second, methods for using thick negative photoresists such as SU-8 with BDPL have been developed which include a new simple and effective method for promoting the adhesion of SU-8 to glass. An assembly method that bonds two individual ultra-thin (<100 µm) replications of the channel and the microfeatures has also been demonstrated. Finally, a pressure driven pumping system with nanoliter per minute flow rate regulation, sub-second response times, and < 3% flow variability has been designed and characterized. The fabrication and assembly of this device is inexpensive and utilizes simple variants of conventional microfluidic fabrication techniques, making it easily accessible to the single cell analysis community.
ContributorsMyers, Jakrey R (Author) / Meldrum, Deirdre (Thesis advisor) / Johnson, Roger (Committee member) / Frakes, David (Committee member) / Arizona State University (Publisher)
Created2012
Description
Inflammation is part of the body’s response to invading pathogens, injury, and a wide range of diseases. Although inflammation is paramount to maintain a healthy immune system, unregulated inflammation can aggravate chronic conditions or cause severe, acute pathologies. Pyroptosis, a caspase-1-dependent, pro-inflammatory cell death that results in the release of IL-1β and IL-18, has been implicated in propagating an inflammatory response in the body. Pyroptosis has been shown to result from the activation of the NLRP3 inflammasome. Furthermore, multiple reports have demonstrated that intracellular potassium efflux and spleen tyrosine kinase (Syk) activity are both essential for facilitating the assembly of the NLRP3 inflammasome and proper processing and release of IL-1β and IL-18. The focus of this thesis was to determine the relationship between intracellular potassium efflux and Syk during key regulatory events in the activation of the NLRP3 inflammasome by identifying their effect on pro-inflammatory cytokine release, inflammasome assembly, mitochondrial reactive oxygen species (mROS) generation, and cell death. Both inhibiting potassium efflux from occurring and deactivating Syk significantly reduced the amount of pro-inflammatory cytokine released (70-100% reduction), the number of inflammasomes assembled (60-80% reduction), the amount of mROS generation, and the quantity of cell death (50-90% reduction). Moreover, it was discovered that potassium efflux was required for Syk activation, but Syk activation had no effect on potassium efflux. Their relationship proved to be unidirectional. This study provides the first demonstration of ion flux-dependent regulation of kinase activation in the NLRP3 inflammasome pathway and provides support for targeting ion regulation mechanisms and Syk kinase activity to manipulate macrophage-mediate inflammatory processes.
ContributorsRao, Mounica Yarlagadda (Author) / Meldrum, Deirdre R. (Thesis director) / Ankeny, Casey (Committee member) / Glenn, Honor (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2015-05
Description
Thirty six percent of Americans are obese and thirty three percent are overweight; obesity has become a known killer in the U.S. yet its prevalence has maintained a firm grasp on the U.S. population and continues to spread across the globe as other countries slowly adopt the American lifestyle. A survey was compiled collecting demographic and body mass index (BMI) information, as well as Tanofsky-Kraff’s (2009) “Assess Eating in the Absence of Hunger” survey questions. The survey used for this study was emailed out to Arizona State University students in Barrett, The Honors College, and the ASU School of Nutrition and Health Promotion listservs. A total of 457 participants completed the survey, 72 males and 385 females (mean age, 24.5±7.7 y; average body mass index (BMI), 23.4 ± 4.8 [a BMI of 25-29.9 is classified as overweight]). When comparing BMI with the living situation, 71% of obese students were living at home with family versus off campus with friends or alone. For comparison, 45% of normal weight students lived at home with family. These data could help structure prevention plans targeting college students by focusing on weight gain prevention at the family level. Results from the Tanofsky-Kraff (2009) survey revealed there was not a significant relationship between external or physical cues and BMI in men or women, but there was a significant positive correlation between emotional cues and BMI in women only. Anger and sadness were the emotional cues in women related to initiating consumption past satiation and consumption following several hours of fasting. Although BMI was inversely related to physical activity in this sample (r = -0.132; p=0.005), controlling for physical activity did not impact the significant associations of BMI with anger or sadness (P>0.05). This information is important in targeting prevention programs to address behavioral change and cognitive awareness of the effects of emotion on over-consumption.
ContributorsGarza, Andrea Marie (Author) / Johnston, Carol (Thesis director) / Jacobs, Mark (Committee member) / Coletta, Dawn (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
New-onset diabetes after kidney transplantation (NODAT) occurs in 20% of kidney transplant patients. In 5 patients who are at risk for new-onset diabetes after kidney transplantation, skeletal muscle gene expression profiling was performed both before and after kidney transplant. The differences in gene expression before and after transplant were compared in order to identify specific genes that could be linked to developing NODAT. These findings could open new avenues for future research.
ContributorsLowery, Clint Curtis (Author) / Coletta, Dawn (Thesis director) / Katsanos, Christos (Committee member) / Willis, Wayne (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / W. P. Carey School of Business (Contributor)
Created2014-05
Description
DNA methylation, a subset of epigenetics, has been found to be a significant marker associated with variations in gene expression and activity across the entire human genome. As of now, however, there is little to no information about how DNA methylation varies between different tissues inside a singular person's body. By using research data from a preliminary study of lean and obese clinical subjects, this study attempts to put together a profile of the differences in DNA methylation that can be observed between two particular body tissues from this subject group: blood and skeletal muscle. This study allows us to start describing the changes that occur at the epigenetic level that influence how differently these two tissues operate, along with seeing how these tissues change between individuals of different weight classes, especially in the context of the development of symptoms of Type 2 Diabetes.
ContributorsRappazzo, Micah Gabriel (Author) / Coletta, Dawn (Thesis director) / Katsanos, Christos (Committee member) / Dinu, Valentin (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / Department of Psychology (Contributor)
Created2013-12
Description
Some cyanobacteria, referred to as boring or euendolithic, are capable of excavating tunnels into calcareous substrates, both mineral and biogenic. The erosive activity of these cyanobacteria results in the destruction of coastal limestones and dead corals, the reworking of carbonate sands, and the cementation of microbialites. They thus link the biological and mineral parts of the global carbon cycle directly. They are also relevant for marine aquaculture as pests of mollusk populations. In spite of their importance, the mechanism by which these cyanobacteria bore remains unknown. In fact, boring by phototrophs is geochemically paradoxical, in that they should promote precipitation of carbonates, not dissolution. To approach this paradox experimentally, I developed an empirical model based on a newly isolated euendolith, which I characterized physiologically, ultrastructurally and phylogenetically (Mastigocoleus testarum BC008); it bores on pure calcite in the laboratory under controlled conditions. Mechanistic hypotheses suggesting the aid of accompanying heterotrophic bacteria, or the spatial/temporal separation of photosynthesis and boring could be readily rejected. Real-time Ca2+ mapping by laser scanning confocal microscopy of boring BC008 cells showed that boring resulted in undersaturation at the boring front and supersaturation in and around boreholes. This is consistent with a process of uptake of Ca2+ from the boring front, trans-cellular mobilization, and extrusion at the distal end of the filaments (borehole entrance). Ca2+ disequilibrium could be inhibited by ceasing illumination, preventing ATP generation, and, more specifically, by blocking P-type Ca2+ ATPase transporters. This demonstrates that BC008 bores by promoting calcite dissolution locally at the boring front through Ca2+ uptake, an unprecedented capacity among living organisms. Parallel studies using mixed microbial assemblages of euendoliths boring into Caribbean, Mediterranean, North and South Pacific marine carbonates, demonstrate that the mechanism operating in BC008 is widespread, but perhaps not universal.
ContributorsRamírez-Reinat, Edgardo L (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Chandler, Douglas (Committee member) / Farmer, Jack (Committee member) / Neuer, Susanne (Committee member) / Arizona State University (Publisher)
Created2010
Description
Background
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.
Methodology
We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.
Principal Findings
We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.
Conclusions
Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.
Methodology
We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.
Principal Findings
We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.
Conclusions
Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.
ContributorsNandakumar, Vivek (Author) / Kelbauskas, Laimonas (Author) / Hernandez, Kathryn (Author) / Lintecum, Kelly (Author) / Senechal, Patti (Author) / Bussey, Kimberly (Author) / Davies, Paul (Author) / Johnson, Roger (Author) / Meldrum, Deirdre (Author) / Ira A. Fulton Schools of Engineering (Contributor) / School of Electrical, Computer and Energy Engineering (Contributor) / Biodesign Institute (Contributor) / Center for Biosignatures Discovery Automation (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Department of Physics (Contributor)
Created2012-01-05
Description
The experiments conducted in this report supported previous evidence (Bethany et al., 2019) that a newly identified predatory bacterium causes a higher rate of mortality in the biological soil crust cyanobacterium M. vaginatus when in hot soils than in cold soils. I predicted that the extracellular propagules of this predatory bacterium were inactivated at seasonally low temperatures, rendering them non-viable when introduced to M. vaginatus at room temperature. However, I found that the predatory bacterium became only transiently inactive at low temperatures, recovering its pathogenicity when later exposed to warmer temperatures. By contrast, inactivation of infectivity was complete by exposure in both liquid and dry conditions for five days at 40 °C. I also expected that its infectivity towards M. vaginatus was temperature dependent. Indeed, infection was hampered and did not cause high mortality when predator and prey were incubated at or below 10 °C, which could have been due to slowed metabolisms of M. vaginatus or to an inability of the predatory bacterium to attack in cold conditions. Above 10 °C, when M. vaginatus grew faster, time to full death of predator/prey incubations correlated with the rate of growth of healthy cultures.
The experiments in this study observed a correlation between the growth rate of uninfected cultures and the decay rate of infected cultures, meaning that temperatures that cultures that displayed a higher growth rate for uninfected M. vaginatus would die faster when infected with the predatory bacterium. Infected cultures that were incubated at temperatures 4 and 10 °C did not display death and this could have been due to lower activity of M. vaginatus at lower temperatures or the inability for the predatory bacterium to attack at lower temperatures.
The experiments in this study observed a correlation between the growth rate of uninfected cultures and the decay rate of infected cultures, meaning that temperatures that cultures that displayed a higher growth rate for uninfected M. vaginatus would die faster when infected with the predatory bacterium. Infected cultures that were incubated at temperatures 4 and 10 °C did not display death and this could have been due to lower activity of M. vaginatus at lower temperatures or the inability for the predatory bacterium to attack at lower temperatures.
ContributorsAhamed, Anisa Nour (Author) / Garcia-Pichel, Ferran (Thesis director) / Giraldo Silva, Ana Maria (Committee member) / Bethany Rakes, Julie (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
An aim of fundamental immunology is quantifying the diversity of the T cell receptor (TCR) repertoire to elucidate the vast recognition by T cells for protection against pathogen and cancer. The utilization of DNA origami nanostructures engineered to capture single cell paired TCR mRNA sequences has transformed the financial and time requirements of repertoire establishment. To further support this protocol, confocal laser scanning microscopy was implemented following transfection to visualize the stability of the DNA origami within primary immune lymphocytes.
ContributorsReed, Abigail Elizabeth (Author) / Blattman, Joseph (Thesis director) / Glenn, Honor (Committee member) / Schoettle, Louis (Committee member) / School of Life Sciences (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Obesity and related health disparities including type 2 diabetes disproportionately impact Latino youth. These health disparities may be the result of gene-environment interactions, but limited research has examined these interactions in the pediatric age group. Lifestyle intervention is the cornerstone for preventing diabetes among high-risk populations and epigenetic and genetic factors may help explain the biological mechanisms underlying diabetes risk reduction following lifestyle changes. MicroRNAs (miRNAs) are small, non-coding RNA’s that regulate gene expression and have emerged as potential biomarkers for predicting type 2 diabetes risk in adults but have yet to be applied to youth. Therefore, the purpose of this study was to identify changes in miRNA expression among Latino youth with prediabetes (4 female/2 male, ages 14-16, BMI percentile 99 ±.2) who participated in a 12-week lifestyle intervention focused on increasing physical activity and improving nutrition-related behaviors.
ContributorsKarch, Jamie (Co-author) / Day, Samantha (Co-author) / Shaibi, Gabriel (Thesis director) / Coletta, Dawn (Committee member) / Arizona State University. College of Nursing & Healthcare Innovation (Contributor) / College of Integrative Sciences and Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05