Matching Items (14)
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Description
Despite significant advances in digital pathology and automation sciences, current diagnostic practice for cancer detection primarily relies on a qualitative manual inspection of tissue architecture and cell and nuclear morphology in stained biopsies using low-magnification, two-dimensional (2D) brightfield microscopy. The efficacy of this process is limited by inter-operator variations in

Despite significant advances in digital pathology and automation sciences, current diagnostic practice for cancer detection primarily relies on a qualitative manual inspection of tissue architecture and cell and nuclear morphology in stained biopsies using low-magnification, two-dimensional (2D) brightfield microscopy. The efficacy of this process is limited by inter-operator variations in sample preparation and imaging, and by inter-observer variability in assessment. Over the past few decades, the predictive value quantitative morphology measurements derived from computerized analysis of micrographs has been compromised by the inability of 2D microscopy to capture information in the third dimension, and by the anisotropic spatial resolution inherent to conventional microscopy techniques that generate volumetric images by stacking 2D optical sections to approximate 3D. To gain insight into the analytical 3D nature of cells, this dissertation explores the application of a new technology for single-cell optical computed tomography (optical cell CT) that is a promising 3D tomographic imaging technique which uses visible light absorption to image stained cells individually with sub-micron, isotropic spatial resolution. This dissertation provides a scalable analytical framework to perform fully-automated 3D morphological analysis from transmission-mode optical cell CT images of hematoxylin-stained cells. The developed framework performs rapid and accurate quantification of 3D cell and nuclear morphology, facilitates assessment of morphological heterogeneity, and generates shape- and texture-based biosignatures predictive of the cell state. Custom 3D image segmentation methods were developed to precisely delineate volumes of interest (VOIs) from reconstructed cell images. Comparison with user-defined ground truth assessments yielded an average agreement (DICE coefficient) of 94% for the cell and its nucleus. Seventy nine biologically relevant morphological descriptors (features) were computed from the segmented VOIs, and statistical classification methods were implemented to determine the subset of features that best predicted cell health. The efficacy of our proposed framework was demonstrated on an in vitro model of multistep carcinogenesis in human Barrett's esophagus (BE) and classifier performance using our 3D morphometric analysis was compared against computerized analysis of 2D image slices that reflected conventional cytological observation. Our results enable sensitive and specific nuclear grade classification for early cancer diagnosis and underline the value of the approach as an objective adjunctive tool to better understand morphological changes associated with malignant transformation.
ContributorsNandakumar, Vivek (Author) / Meldrum, Deirdre R (Thesis advisor) / Nelson, Alan C. (Committee member) / Karam, Lina J (Committee member) / Ye, Jieping (Committee member) / Johnson, Roger H (Committee member) / Bussey, Kimberly J (Committee member) / Arizona State University (Publisher)
Created2013
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Description
Cell-cell interactions in a microenvironment under stress conditions play a critical role in pathogenesis and pre-malignant progression. Hypoxia is a central factor in carcinogenesis, which induces selective pressure in this process. Understanding the role of intercellular communications and cellular adaptation to hypoxia can help discover new cancer biosignatures and more

Cell-cell interactions in a microenvironment under stress conditions play a critical role in pathogenesis and pre-malignant progression. Hypoxia is a central factor in carcinogenesis, which induces selective pressure in this process. Understanding the role of intercellular communications and cellular adaptation to hypoxia can help discover new cancer biosignatures and more effective diagnostic and therapeutic strategies. This dissertation presents a study on transcriptomic and metabolic profiling of pre-malignant progression of Barrett's esophagus. It encompasses two methodology developments and experimental findings of two related studies. To integrate phenotype and genotype measurements, a minimally invasive method was developed for selectively retrieving single adherent cells from cell cultures. Selected single cells can be harvested by a combination of mechanical force and biochemical treatment after phenotype measurements and used for end-point assays. Furthermore, a method was developed for analyzing expression levels of ten genes in individual mammalian cells with high sensitivity and reproducibility without the need of pre-amplifying cDNA. It is inexpensive and compatible with most of commercially available RT-qPCR systems, which warrants a wide applicability of the method to gene expression analysis in single cells. In the first study, the effect of intercellular interactions was investigated between normal esophageal epithelial and dysplastic Barrett's esophagus cells on gene expression levels and cellular functions. As a result, gene expression levels in dysplastic cells were found to be affected to a significantly larger extent than in the normal esophageal epithelial cells. These differentially expressed genes are enriched in cellular movement, TGFβ and EGF signaling networks. Heterotypic interactions between normal and dysplastic cells can change cellular motility and inhibit proliferation in both normal and dysplastic cells. In the second study, alterations in gene transcription levels and metabolic phenotypes between hypoxia-adapted cells and age-matched normoxic controls representing four different stages of pre-malignant progression in Barrett's esophagus were investigated. Through differential gene expression analysis and mitochondrial membrane potential measurements, evidence of clonal evolution induced by hypoxia selection pressure in metaplastic and high-grade dysplastic cells was found. These discoveries on cell-cell interactions and hypoxia adaptations provide a deeper insight into the dynamic evolutionary process in pre-malignant progression of Barrett's esophagus.
ContributorsZeng, Jia (Author) / Meldrum, Deirdre R (Thesis advisor) / Kelbauskas, Laimonas (Committee member) / Barrett, Michael T (Committee member) / Bussey, Kimberly J (Committee member) / Zhang, Weiwen (Committee member) / Arizona State University (Publisher)
Created2014
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Description
Within the last decade there has been remarkable interest in single-cell metabolic analysis as a key technology for understanding cellular heterogeneity, disease initiation, progression, and drug resistance. Technologies have been developed for oxygen consumption rate (OCR) measurements using various configurations of microfluidic devices. The technical challenges of current approaches include:

Within the last decade there has been remarkable interest in single-cell metabolic analysis as a key technology for understanding cellular heterogeneity, disease initiation, progression, and drug resistance. Technologies have been developed for oxygen consumption rate (OCR) measurements using various configurations of microfluidic devices. The technical challenges of current approaches include: (1) deposition of multiple sensors for multi-parameter metabolic measurements, e.g. oxygen, pH, etc.; (2) tedious and labor-intensive microwell array fabrication processes; (3) low yield of hermetic sealing between two rigid fused silica parts, even with a compliance layer of PDMS or Parylene-C. In this thesis, several improved microfabrication technologies are developed and demonstrated for analyzing multiple metabolic parameters from single cells, including (1) a modified "lid-on-top" configuration with a multiple sensor trapping (MST) lid which spatially confines multiple sensors to micro-pockets enclosed by lips for hermetic sealing of wells; (2) a multiple step photo-polymerization method for patterning three optical sensors (oxygen, pH and reference) on fused silica and on a polyethylene terephthalate (PET) surface; (3) a photo-polymerization method for patterning tri-color (oxygen, pH and reference) optical sensors on both fused silica and on the PET surface; (4) improved KMPR/SU-8 microfabrication protocols for fabricating microwell arrays that can withstand cell culture conditions. Implementation of these improved microfabrication methods should address the aforementioned challenges and provide a high throughput and multi-parameter single cell metabolic analysis platform.
ContributorsSong, Ganquan (Author) / Meldrum, Deirdre R (Thesis advisor) / Goryll, Michael (Committee member) / Wang, Hong (Committee member) / Tian, Yanqing (Committee member) / Arizona State University (Publisher)
Created2014
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Description
The ocean is vital to the health of our planet but remains virtually unexplored. Many researchers seek to understand a wide range of geological and biological phenomena by developing technologies which enable exploration of the deep-sea. The task of developing a technology which can withstand extreme pressure and

The ocean is vital to the health of our planet but remains virtually unexplored. Many researchers seek to understand a wide range of geological and biological phenomena by developing technologies which enable exploration of the deep-sea. The task of developing a technology which can withstand extreme pressure and temperature gradients in the deep ocean is not trivial. Of these technologies, underwater vehicles were developed to study the deep ocean, but remain large and expensive to manufacture. I am proposing the development of cost efficient miniaturized underwater vehicle (mUV) with propulsion systems to carry small measurement devices and enable deep-sea exploration. These mUV's overall size is optimized based on the vehicle parameters such as energy density, desired velocity, swimming time and propulsion performance. However, there are limitations associated with the size of the mUV which leads to certain challenges. For example, 2000 m below the sea level, the pressure is as high as 3000 psi. Therefore, certain underwater vehicle modules, such as the propulsion system, will require pressure housing to ensure the functionality of the thrust generation. In the case of a mUV swimming against the deep-sea current, a thrust magnitude is required to enable the vehicle to overcome the ocean current speed and move forward. Therefore, the size of the mUV is limited by the energy density and the propeller size. An equation is derived to miniaturize underwater vehicle while performing with a certain specifications. An inrunner three-phase permanent magnet brushless DC motor is designed and fabricated with a specific size to fit inside the mUV's core. The motor is composed of stator winding in a pressure housing and an open to water ring-propeller rotor magnet. Several ring-propellers are 3D printed and tested experimentally to determine their performances and efficiencies. A planer motion optimal trajectory for the mUV is determined to minimize the energy usage. Those studies enable the design of size optimized underwater vehicle with propulsion to carry small measurement sensors and enable underwater exploration. Developing mUV's will enable ocean exploration that can lead to significant scientific discoveries and breakthroughs that will solve current world health and environmental problems.
ContributorsMerza, Saeed A (Author) / Meldrum, Deirdre R (Thesis advisor) / Chao, Shih-hui (Committee member) / Shankar, Praveen (Committee member) / Saripalli, Srikanth (Committee member) / Berman, Spring Melody (Committee member) / Arizona State University (Publisher)
Created2014
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Description
The production of monomer compounds for synthesizing plastics has to date been largely restricted to the petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economic feasibility. Cyanobacteria have, however, become attractive microbial factories to produce renewable fuels and chemicals directly from sunlight and CO2. To explore the

The production of monomer compounds for synthesizing plastics has to date been largely restricted to the petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economic feasibility. Cyanobacteria have, however, become attractive microbial factories to produce renewable fuels and chemicals directly from sunlight and CO2. To explore the feasibility of photosynthetic production of (S)- and (R)-3-hydroxybutyrate (3HB), building-block monomers for synthesizing the biodegradable plastics polyhydroxyalkanoates and precursors to fine chemicals, synthetic metabolic pathways have been constructed, characterized and optimized in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803). Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing PHB biosynthesis pathway further promoted the 3HB production. Analysis of the intracellular acetyl-CoA and anion concentrations in the culture media indicated that the phosphate consumption during the photoautotrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. Fine-tuning of the gene expression levels via strategies, including tuning gene copy numbers, promoter engineering and ribosome binding site optimization, proved critical to mitigating metabolic bottlenecks and thus improving the 3HB production. One of the engineered Synechocystis strains, namely R168, was able to produce (R)-3HB to a cumulative titer of ~1600 mg/L, with a peak daily productivity of ~200 mg/L, using light and CO2 as the sole energy and carbon sources, respectively. Additionally, in order to establish a high-efficiency transformation protocol in cyanobacterium Synechocystis 6803, methyltransferase-encoding genes were cloned and expressed to pre-methylate the exogenous DNA before Synechocystis transformation. Eventually, the transformation efficiency was increased by two orders of magnitude in Synechocystis. This research has demonstrated the use of cyanobacteria as cell factories to produce 3HB directly from light and CO2, and developed new synthetic biology tools for cyanobacteria.
ContributorsWang, Bo (Author) / Meldrum, Deirdre R (Thesis advisor) / Zhang, Weiwen (Committee member) / Sandrin, Todd R. (Committee member) / Nielsen, David R (Committee member) / Arizona State University (Publisher)
Created2014
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Description
The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is essential for the innate immune response to danger signals. Importantly, the NLRP3 inflammasome responds to structurally and functionally dissimilar stimuli. It is currently unknown how the NLRP3 inflammasome responds to such diverse triggers. This dissertation investigates the role of ion flux

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is essential for the innate immune response to danger signals. Importantly, the NLRP3 inflammasome responds to structurally and functionally dissimilar stimuli. It is currently unknown how the NLRP3 inflammasome responds to such diverse triggers. This dissertation investigates the role of ion flux in regulating the NLRP3 inflammasome. Project 1 explores the relationship between potassium efflux and Syk tyrosine kinase. The results reveal that Syk activity is upstream of mitochondrial oxidative signaling and is crucial for inflammasome assembly, pro-inflammatory cytokine processing, and caspase-1-dependent pyroptotic cell death. Dynamic potassium imaging and molecular analysis revealed that Syk is downstream of, and regulated by, potassium efflux. Project 1 reveals the first identified intermediate regulator of inflammasome activity regulated by potassium efflux. Project 2 focuses on P2X7 purinergic receptor-dependent ion flux in regulating the inflammasome. Dynamic potassium imaging revealed an ATP dose-dependent efflux of potassium driven by P2X7. Surprisingly, ATP induced mitochondrial potassium mobilization, suggesting a mitochondrial detection of purinergic ion flux. ATP-induced potassium and calcium flux was found to regulate mitochondrial oxidative signaling upstream of inflammasome assembly. First-ever multiplexed imaging of potassium and calcium dynamics revealed that potassium efflux is necessary for calcium influx. These results suggest that ATP-induced potassium efflux regulates the inflammasome by calcium influx-dependent mitochondrial oxidative signaling. Project 2 defines a coordinated cation flux dependent on the efflux of potassium and upstream of mitochondrial oxidative signaling in inflammasome regulation. Lastly, this dissertation contributes two methods that will be useful for investigating inflammasome biology: an optimized pipeline for single cell transcriptional analysis, and a mouse macrophage cell line expressing a genetically encoded intracellular ATP sensor. This dissertation contributes to understanding the fundamental role of ion flux in regulation of the NLRP3 inflammasome and identifies potassium flux and Syk as potential targets to modulate inflammation.
ContributorsYaron, Jordan Robin (Author) / Meldrum, Deirdre R (Thesis advisor) / Blattman, Joseph N (Committee member) / Glenn, Honor L (Committee member) / Arizona State University (Publisher)
Created2015
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Description
Due to heterogeneity at the cellular level, single cell analysis (SCA) has become a necessity to study cellomics for the early detection of diseases like cancer. Development of single cell manipulation systems is very critical for performing SCA. In this thesis, electrorotation (ROT) chips to trap and rotate single cells

Due to heterogeneity at the cellular level, single cell analysis (SCA) has become a necessity to study cellomics for the early detection of diseases like cancer. Development of single cell manipulation systems is very critical for performing SCA. In this thesis, electrorotation (ROT) chips to trap and rotate single cells using electrokinetic forces have been developed. The ROT chip mainly consists of a set of closely spaced metal electrodes (60µm interspacing between opposite electrodes) that forms a closed electric field cage (electrocage) when driven with high frequency AC voltages. Cells were flowed through a microchannel to the electrocage where they could be precisely trapped, levitated and rotated in 3-D along the axis of interest. The dielectrophoresis based ROT chip design and relevant electrokinetic effects have been simulated using COMSOL 3.4 to optimize the design parameters. Also, various semiconductor technology fabrication process steps have been developed and optimized for better yield and repeatability in the manufacture of the ROT chip. The ROT chip thus fabricated was used to characterize rotation of single cells with respect to the control parameters namely excitation voltage, frequency and cell line. The longevity of cell rotation under electric fields has been probed. Also, the Joule heating inside the ROT chip due to applied voltage has been characterized to know the thermal stress on the cells. The major advantages of the ROT chip developed are precise electrorotation of cells, simple design and straight forward fabrication process.
ContributorsSoundappa Elango, Iniyan (Author) / Meldrum, Deirdre R (Thesis advisor) / Christen, Jennifer Blain (Committee member) / Johnson, Roger H (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Continuous underwater observation is a challenging engineering task that could be accomplished by development and deployment of a sensor array that can survive harsh underwater conditions. One approach to this challenge is a swarm of micro underwater robots, known as Sensorbots, that are equipped with biogeochemical sensors that can relay

Continuous underwater observation is a challenging engineering task that could be accomplished by development and deployment of a sensor array that can survive harsh underwater conditions. One approach to this challenge is a swarm of micro underwater robots, known as Sensorbots, that are equipped with biogeochemical sensors that can relay information among themselves in real-time. This innovative method for underwater exploration can contribute to a more comprehensive understanding of the ocean by not limiting sampling to a single point and time. In this thesis, Sensorbot Beta, a low-cost fully enclosed Sensorbot prototype for bench-top characterization and short-term field testing, is presented in a modular format that provides flexibility and the potential for rapid design. Sensorbot Beta is designed around a microcontroller driven platform comprised of commercial off-the-shelf components for all hardware to reduce cost and development time. The primary sensor incorporated into Sensorbot Beta is an in situ fluorescent pH sensor. Design considerations have been made for easy adoption of other fluorescent or phosphorescent sensors, such as dissolved oxygen or temperature. Optical components are designed in a format that enables additional sensors. A real-time data acquisition system, utilizing Bluetooth, allows for characterization of the sensor in bench top experiments. The Sensorbot Beta demonstrates rapid calibration and future work will include deployment for large scale experiments in a lake or ocean.
ContributorsJohansen, John (Civil engineer) (Author) / Meldrum, Deirdre R (Thesis advisor) / Chao, Shih-hui (Committee member) / Papandreou-Suppappola, Antonia (Committee member) / Arizona State University (Publisher)
Created2012
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Description
A single cell is the very fundamental element in an organism; however, it contains the most complicated and stochastic information, such as DNA, RNA, and protein expression. Thus, it is a necessity to study stochastic gene expression in order to discover the biosignatures at the single-cell level. The heterogeneous gene

A single cell is the very fundamental element in an organism; however, it contains the most complicated and stochastic information, such as DNA, RNA, and protein expression. Thus, it is a necessity to study stochastic gene expression in order to discover the biosignatures at the single-cell level. The heterogeneous gene expression of single cells from an isogenic cell population has already been studied for years. Yet to date, single-cell studies have been confined in a fashion of analyzing isolated single cells or a dilution of cells from the bulk-cell populations. These techniques or devices are limited by either the mechanism of cell lysis or the difficulties to target specific cells without harming neighboring cells.

This dissertation presents the development of a laser lysis chip combined with a two-photon laser system to perform single-cell lysis of single cells in situ from three-dimensional (3D) cell spheroids followed by analysis of the cell lysate with two-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The 3D spheroids were trapped in a well in the custom-designed laser lysis chip. Next, each single cell of interest in the 3D spheroid was identified and lysed one at a time utilizing a two-photon excited laser. After each cell lysis, the contents inside the target cell were released to the surrounding media and carried out to the lysate collector. Finally, the gene expression of each individual cell was measured by two-step RT-qPCR then spatially mapped back to its original location in the spheroids to construct a 3D gene expression map.

This novel technology and approach enables multiple gene expression measurements in single cells of multicellular organisms as well as cell-to-cell heterogeneous responses to the environment with spatial recognition. Furthermore, this method can be applied to study precancerous tissues for a better understanding of cancer progression and for identifying early tumor development.
ContributorsWang, Guozhen (Author) / Meldrum, Deirdre R (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Hong (Committee member) / Goryll, Michael (Committee member) / Arizona State University (Publisher)
Created2016
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Description
Many environmental microorganisms such as marine microbes are un-culturable; hence, they should be analyzed in situ. Even though a few in situ ocean observing instruments have been available to oceanographers, their applications are limited, because these instruments are expensive and power hungry.

In this dissertation project, an inexpensive, portable, low-energy consuming,

Many environmental microorganisms such as marine microbes are un-culturable; hence, they should be analyzed in situ. Even though a few in situ ocean observing instruments have been available to oceanographers, their applications are limited, because these instruments are expensive and power hungry.

In this dissertation project, an inexpensive, portable, low-energy consuming, and highly quantitative microbiological genomic sensor has been developed for in situ ocean-observing systems. A novel real-time colorimetric loop-mediated isothermal amplification (LAMP) technology has been developed for quantitative detection of microbial nucleic acids. This technology was implemented on a chip-level device with an embedded inexpensive imaging device and temperature controller to achieve quantitative detection within one hour. A bubble-free liquid handling approach was introduced to avoid bubble trapping during liquid loading, a common problem in microfluidic devices. An algorithm was developed to reject the effect of bubbles generated during the reaction process, to enable more accurate nucleic acid analysis. This genomic sensor has been validated at gene and gene expression levels using Synechocystis sp. PCC 6803 genomic DNA and total RNA. Results suggest that the detection limits reached 10 copies/μL and 100 fg/μL, respectively. This approach was highly quantitative, with linear standard curves down to 103 copies/μL and 1 pg/μL, respectively. In addition to environmental microbe characterization, this genomic sensor has been employed for viral RNA quantification during an infectious disease outbreak. As the Zika fever was spreading in America, a quantitative detection of Zika virus has been performed. The results show that the genomic sensor is highly quantitative from 10 copies/μL to 105 copies/μL. This suggests that the novel nucleic acid quantification technology is sensitive, quantitative, and robust. It is a promising candidate for rapid microbe detection and quantification in routine laboratories.

In the future, this genomic sensor will be implemented in in situ platforms as a core analytical module with minor modifications, and could be easily accessible by oceanographers. Deployment of this microbial genomic sensor in the field will enable new scientific advances in oceanography and provide a possible solution for infectious disease detection.
ContributorsCi, Shufang (Author) / Meldrum, Deirdre R (Thesis advisor) / Chao, Shih-hui (Committee member) / Neuer, Susanne (Committee member) / Arizona State University (Publisher)
Created2017