Matching Items (48)
Description
Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated membrane filter that will sample, lyse, and extract the DNA from microorganisms in aqueous environments. An off-the-shelf membrane filter disc was embedded in a PDMS layer and sequentially bound with other PDMS channel layers. No leakage was observed during filtration. This device was validated by concentrating a large amount of cyanobacterium Synechocystis in simulated sample water with consistent performance across devices. After accumulating sufficient biomass on the filter, a sequential electrochemical lysing process was performed by applying 5VDC across the filter. This device was further evaluated by delivering several samples of differing concentrations of cyanobacterium Synechocystis then quantifying the DNA using real-time PCR. Lastly, an environmental sample was run through the device and the amount of photosynthetic microorganisms present in the water was determined. The major breakthroughs in this design are low energy demand, cheap materials, simple design, straightforward fabrication, and robust performance, together enabling wide-utility of similar chip-based devices for field-deployable operations in environmental micro-biotechnology.
ContributorsLecluse, Aurelie (Author) / Meldrum, Deirdre (Thesis advisor) / Chao, Joseph (Thesis advisor) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2011
Description
Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study of cellular morphology, a single cell three-dimensional (3D) imaging system can be used to diagnose fatal diseases, such as cancer, at an early stage. One proven method, CellCT, accomplishes 3D imaging by rotating a single cell around a fixed axis. However, some existing cell rotating mechanisms require either intricate microfabrication, and some fail to provide a suitable environment for living cells. This thesis develops a microvorterx chamber that allows living cells to be rotated by hydrodynamic alone while facilitating imaging access. In this thesis work, 1) the new chamber design was developed through numerical simulation. Simulations revealed that in order to form a microvortex in the side chamber, the ratio of the chamber opening to the channel width must be smaller than one. After comparing different chamber designs, the trapezoidal side chamber was selected because it demonstrated controllable circulation and met the imaging requirements. Microvortex properties were not sensitive to the chambers with interface angles ranging from 0.32 to 0.64. A similar trend was observed when chamber heights were larger than chamber opening. 2) Micro-particle image velocimetry was used to characterize microvortices and validate simulation results. Agreement between experimentation and simulation confirmed that numerical simulation was an effective method for chamber design. 3) Finally, cell rotation experiments were performed in the trapezoidal side chamber. The experimental results demonstrated cell rotational rates ranging from 12 to 29 rpm for regular cells. With a volumetric flow rate of 0.5 µL/s, an irregular cell rotated at a mean rate of 97 ± 3 rpm. Rotational rates can be changed by altering inlet flow rates.
ContributorsZhang, Wenjie (Author) / Frakes, David (Thesis advisor) / Meldrum, Deirdre (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2011
Description
Laboratory automation systems have seen a lot of technological advances in recent times. As a result, the software that is written for them are becoming increasingly sophisticated. Existing software architectures and standards are targeted to a wider domain of software development and need to be customized in order to use them for developing software for laboratory automation systems. This thesis proposes an architecture that is based on existing software architectural paradigms and is specifically tailored to developing software for a laboratory automation system. The architecture is based on fairly autonomous software components that can be distributed across multiple computers. The components in the architecture make use of asynchronous communication methodologies that are facilitated by passing messages between one another. The architecture can be used to develop software that is distributed, responsive and thread-safe. The thesis also proposes a framework that has been developed to implement the ideas proposed by the architecture. The framework is used to develop software that is scalable, distributed, responsive and thread-safe. The framework currently has components to control very commonly used laboratory automation devices such as mechanical stages, cameras, and also to do common laboratory automation functionalities such as imaging.
ContributorsKuppuswamy, Venkataramanan (Author) / Meldrum, Deirdre (Thesis advisor) / Collofello, James (Thesis advisor) / Sarjoughian, Hessam S. (Committee member) / Johnson, Roger (Committee member) / Arizona State University (Publisher)
Created2012
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
Clean water for drinking, food preparation, and bathing is essential for astronaut health and safety during long duration habitation of the International Space Station (ISS), including future missions to Mars. Despite stringent water treatment and recycling efforts on the ISS, it is impossible to completely prevent microbial contamination of onboard water supplies. In this work, we used a spaceflight analogue culture system to better understand how the microgravity environment can influence the pathogenesis-related characteristics of Burkholderia cepacia complex (Bcc), an opportunistic pathogen previously recovered from the ISS water system. The results of the present study suggest that there may be important differences in how this pathogen can respond and adapt to spaceflight and other low fluid shear environments encountered during their natural life cycles. Future studies are aimed at understanding the underlying mechanisms responsible for these phenotypes.
ContributorsKang, Bianca Younseon (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, Mark (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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
Spaceflight and spaceflight analogue culture enhance the virulence and pathogenesis-related stress resistance of the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). This is an alarming finding as it suggests that astronauts may have an increased risk of infection during spaceflight. This risk is further exacerbated as multiple studies indicate that spaceflight negatively impacts aspects of the immune system. In order to ensure astronaut safety during long term missions, it is important to study the phenotypic effects of the microgravity environment on a range of medically important microbial pathogens that might be encountered by the crew. This ground-based study uses the NASA-engineered Rotating Wall Vessel (RWV) bioreactor as a spaceflight analogue culture system to grow bacteria under low fluid shear forces relative to those encountered in microgravity, and interestingly, in the intestinal tract during infection. The culture environment in the RWV is commonly referred to as low shear modeled microgravity (LSMMG). In this study, we characterized the stationary phase stress response of the enteric pathogen, Salmonella enterica serovar Enteritidis (S. Enteritidis), to LSMMG culture. We showed that LSMMG enhanced the resistance of stationary phase cultures of S. Enteritidis to acid and thermal stressors, which differed from the LSSMG stationary phase response of the closely related pathovar, S. Typhimurium. Interestingly, LSMMG increased the ability of both S. Enteritidis and S. Typhimurium to adhere to, invade into, and survive within an in vitro 3-D intestinal co-culture model containing immune cells. Our results indicate that LSMMG regulates pathogenesis-related characteristics of S. Enteritidis in ways that may present an increased health risk to astronauts during spaceflight missions.
ContributorsKoroli, Sara (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, C. Mark (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The International Space Station (ISS) utilizes recycled water for consumption, cleaning and air humidity control. The Environmental Control and Life Support Systems (ECLSS) have been rigorously tested at the NASA Johnson Space Center. Despite the advanced engineering of the water recovery system, bacterial biofilms have been recovered from this potable water source. Microbial contamination of potable water poses a potential threat to crew members onboard the ISS. Because astronauts have been found to have compromised immune systems, bacterial strains that would not typically be considered a danger must be carefully studied to better understand the mechanisms enabling their survival, including polymicrobial interactions. The need for a more thorough understanding of the effect of spaceflight environment on polymicrobial interactions and potential impact on crew health and vehicle integrity is heightened since 1) several potential pathogens have been isolated from the ISS potable water system, 2) spaceflight has been shown to induce unexpected alterations in microbial responses, and 3) emergent phenotypes are often observed when multiple bacterial species are co- cultured together, as compared to pure cultures of single species. In order to address these concerns, suitable growth media are required that will not only support the isolation of these microbes but also the ability to distinguish between them when grown as mixed cultures. In this study, selective and/or differential media were developed for bacterial isolates collected from the ISS potable water supply. In addition to facilitating discrimination between bacteria, the ideal media for each strain was intended to have a 100% recovery rate compared to traditional R2A media. Antibiotic and reagent susceptibility and resistance tests were conducted for the purpose of developing each individual medium. To study a wide range of targets, 12 antibiotics were selected from seven major classes, including penicillin, cephalosporins, fluoroquinolones, aminoglycosides, glycopeptides/lipoglycopeptides, macrolides/lincosamides/streptogramins, tetracyclines, in addition to seven unclassified antibiotics and three reagents. Once developed, medium efficacy was determined by means of growth curve experiments. The development of these media is a critical step for further research into the mechanisms utilized by these strains to survive the harsh conditions of the ISS water system. Furthermore, with an understanding of the complex nature of these polymicrobial communities, specific contamination targeting and control can be conducted to reduce the risk to crew members. Understanding these microbial species and their susceptibilities has potential application for future NASA human explorations, including those to Mars.
ContributorsKing, Olivia Grace (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, Mark (Committee member) / School of Sustainability (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
One of the identified health risk areas for human spaceflight is infectious disease, particularly involving environmental microorganisms already found on the International Space Station (ISS). In particular, bacteria belonging to the Burkholderia cepacia complex (Bcc) which can cause human disease in those who are immunocompromised, have been identified in the ISS water supply. This present study characterized the effect of spaceflight analog culture conditions on Bcc to certain physiological stresses (acid and thermal as well as intracellular survival in U927 human macrophage cells). The NASA-designed Rotating Wall Vessel (RWV) bioreactor was used as the spaceflight analogue culture system in these studies to grow Bcc bacterial cells under Low Shear Modeled Microgravity (LSMMG) conditions. Results show that LSMMG culture increased the resistance of Bcc to both acid and thermal stressors, but did not alter phagocytic uptake in 2-D monolayers of human monocytes.
ContributorsVu, Christian-Alexander (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, Mark (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2023-05
Description
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative enteric pathogen that causes self-limiting gastroenteritis in healthy individuals and can cause systemic infections in those who are immunocompromised. During its natural lifecycle, S. Typhimurium encounters a wide variety of stresses it must sense and respond to in a dynamic and coordinated fashion to induce resistance and ensure survival. Salmonella is subjected to a series of stresses that include temperature shifts, pH variability, detergent-like bile salts, oxidative environments and changes in fluid shear levels. Previously, our lab showed that cultures of S. Typhimurium grown under physiological low fluid shear (LFS) conditions similar to those encountered in the intestinal tract during infection uniquely regulates the virulence, gene expression and pathogenesis-related stress responses of this pathogen during log phase. Interestingly, the log phase Salmonella mechanosensitive responses to LFS were independent of the master stress response sigma factor, RpoS, departing from our conventional understanding of RpoS regulation. Since RpoS is a growth phase dependent regulator with increased stability in stationary phase, the current study investigated the role of RpoS in mediating pathogenesis-related stress responses in stationary phase S. Typhimurium grown under LFS and control conditions. Specifically, stationary phase responses to acid, thermal, bile and oxidative stress were assayed. To our knowledge the results from the current study demonstrate the first report that the mechanical force of LFS globally alters the S. Typhimurium χ3339 stationary phase stress response independently of RpoS to acid and bile stressors but dependently on RpoS to oxidative and thermal stress. This indicates that fluid shear-dependent differences in acid and bile stress responses are regulated by alternative pathway(s) in S. Typhimurium, were the oxidative and thermal stress responses are regulated through RpoS in LFS conditions. Results from this study further highlight how bacterial mechanosensation may be important in promoting niche recognition and adaptation in the mammalian host during infection, and may lead to characterization of previously unidentified pathogenesis strategies.
ContributorsCrenshaw, Keith (Author) / Nickerson, Cheryl A. (Thesis advisor) / Barrila, Jennifer (Thesis advisor) / Ott, C. (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2016