Matching Items (43)
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 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
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 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
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: (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
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 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
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 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
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 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
Description
Sea ice algae dominated by diatoms inhabit the brine channels of the Arctic sea ice and serve as the base of the Arctic marine food web in the spring. I studied sea ice diatoms in the bottom 10 cm of first year land-fast sea ice off the coast of Barrow, AK, in spring of 2011, 2012, and 2013. I investigated the variability in the biomass and the community composition of these sea-ice diatoms between bloom phases, as a function of overlying snow depth and over time. The dominant genera were the pennate diatoms Nitzschia, Navicula, Thalassiothrix, and Fragilariopsis with only a minor contribution by centric diatoms. While diatom biomass as estimated by organic carbon changed significantly between early, peak, and declining bloom phases (average of 1.6 mg C L-1, 5.7 mg C L-1, and 1.0 mg C L-1, respectively), the relative ratio of the dominant diatom groups did not change. However, after export, when the diatoms melt out of the ice into the underlying water, diatom biomass dropped by ~73% and the diatom community shifted to one dominated by centric diatoms. I also found that diatom biomass was ~77% lower under high snow cover (>20 cm) compared to low snow cover (<8 cm); however, the ratio of the diatom categories relative to particulate organic carbon (POC) was again unchanged. The diatom biomass was significantly different between the three sampling years (average of 2.4 mg C L-1 in 2011, 1.1 mg C L-1 in 2012, and 5.4 mg C L-1 in 2013, respectively) as was the contribution of all of the dominant genera to POC. I hypothesize the latter to be due to differences in the history of ice sheet formation each year. The temporal variability of these algal communities will influence their availability for pelagic or benthic consumers. Furthermore, in an Arctic that is changing rapidly with earlier sea ice and snowmelt, this time series study will constitute an important baseline for further studies on how the changing Arctic influences the algal community immured in sea ice.
ContributorsKinzler, Kyle (Author) / Neuer, Susanne (Thesis advisor) / Juhl, Andrew (Committee member) / Hartnett, Hilairy (Committee member) / Arizona State University (Publisher)
Created2014
Description
In 2010, a monthly sampling regimen was established to examine ecological differences in Saguaro Lake and Lake Pleasant, two Central Arizona reservoirs. Lake Pleasant is relatively deep and clear, while Saguaro Lake is relatively shallow and turbid. Preliminary results indicated that phytoplankton biomass was greater by an order of magnitude in Saguaro Lake, and that community structure differed. The purpose of this investigation was to determine why the reservoirs are different, and focused on physical characteristics of the water column, nutrient concentration, community structure of phytoplankton and zooplankton, and trophic cascades induced by fish populations. I formulated the following hypotheses: 1) Top-down control varies between the two reservoirs. The presence of piscivore fish in Lake Pleasant results in high grazer and low primary producer biomass through trophic cascades. Conversely, Saguaro Lake is controlled from the bottom-up. This hypothesis was tested through monthly analysis of zooplankton and phytoplankton communities in each reservoir. Analyses of the nutritional value of phytoplankton and DNA based molecular prey preference of zooplankton provided insight on trophic interactions between phytoplankton and zooplankton. Data from the Arizona Game and Fish Department (AZGFD) provided information on the fish communities of the two reservoirs. 2) Nutrient loads differ for each reservoir. Greater nutrient concentrations yield greater primary producer biomass; I hypothesize that Saguaro Lake is more eutrophic, while Lake Pleasant is more oligotrophic. Lake Pleasant had a larger zooplankton abundance and biomass, a larger piscivore fish community, and smaller phytoplankton abundance compared to Saguaro Lake. Thus, I conclude that Lake Pleasant was controlled top-down by the large piscivore fish population and Saguaro Lake was controlled from the bottom-up by the nutrient load in the reservoir. Hypothesis 2 stated that Saguaro Lake contains more nutrients than Lake Pleasant. However, Lake Pleasant had higher concentrations of dissolved nitrogen and phosphorus than Saguaro Lake. Additionally, an extended period of low dissolved N:P ratios in Saguaro Lake indicated N limitation, favoring dominance of N-fixing filamentous cyanobacteria in the phytoplankton community in that reservoir.
ContributorsSawyer, Tyler R (Author) / Neuer, Susanne (Thesis advisor) / Childers, Daniel L. (Committee member) / Sommerfeld, Milton (Committee member) / Arizona State University (Publisher)
Created2011
Description
The oceans play an essential role in global biogeochemical cycles and in regulating climate. The biological carbon pump, the photosynthetic fixation of carbon dioxide by phytoplankton and subsequent sequestration of organic carbon into deep water, combined with the physical carbon pump, make the oceans the only long-term net sink for anthropogenic carbon dioxide. A full understanding of the workings of the biological carbon pump requires a knowledge of the role of different taxonomic groups of phytoplankton (protists and cyanobacteria) to organic carbon export. However, this has been difficult due to the degraded nature of particles sinking into particle traps, the main tools employed by oceanographers to collect sinking particulate matter in the ocean. In this study DNA-based molecular methods, including denaturing gradient gel electrophoresis, cloning and sequencing, and taxon-specific quantitative PCR, allowed for the first time for the identification of which protists and cyanobacteria contributed to the material collected by the traps in relation to their presence in the euphotic zone. I conducted this study at two time-series stations in the subtropical North Atlantic Ocean, one north of the Canary Islands, and one located south of Bermuda. The Bermuda study allowed me to investigate seasonal and interannual changes in the contribution of the plankton community to particle flux. I could also show that small unarmored taxa, including representatives of prasinophytes and cyanobacteria, constituted a significant fraction of sequences recovered from sediment trap material. Prasinophyte sequences alone could account for up to 13% of the clone library sequences of trap material during bloom periods. These observations contradict a long-standing paradigm in biological oceanography that only large taxa with mineral shells are capable of sinking while smaller, unarmored cells are recycled in the euphotic zone through the microbial loop. Climate change and a subsequent warming of the surface ocean may lead to a shift in the protist community toward smaller cell size in the future, but in light of these findings these changes may not necessarily lead to a reduction in the strength of the biological carbon pump.
ContributorsAmacher, Jessica (Author) / Neuer, Susanne (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Lomas, Michael (Committee member) / Wojciechowski, Martin (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
Many studies over the past two decades examined the link between climate patterns and discharge, but few have attempted to study the effects of the El Niño Southern Oscillation (ENSO) on localized and watershed specific processes such as nutrient loading in the Southwestern United States. The Multivariate ENSO Index (MEI) is used to describe the state of the ENSO, with positive (negative) values referring to an El Niño condition (La Niña condition). This study examined the connection between the MEI and precipitation, discharge, and total nitrogen (TN) and total phosphorus (TP) concentrations in the Upper Salt River Watershed in Arizona. Unrestricted regression models (UMs) and restricted regression models (RMs) were used to investigate the relationship between the discharges in Tonto Creek and the Salt River as functions of the magnitude of the MEI, precipitation, and season (winter/summer). The results suggest that in addition to precipitation, the MEI/season relationship is an important factor for predicting discharge. Additionally, high discharge events were associated with high magnitude ENSO events, both El Niño and La Niña. An UM including discharge and season, and a RM (restricting the seasonal factor to zero), were applied to TN and TP concentrations in the Salt River. Discharge and seasonality were significant factors describing the variability in TN in the Salt River while discharge alone was the significant factor describing TP. TN and TP in Roosevelt Lake were evaluated as functions of both discharge and MEI. Some significant correlations were found but internal nutrient cycling as well as seasonal stratification of the water column of the lake likely masks the true relationships. Based on these results, the MEI is a useful predictor of discharge, as well as nutrient loading in the Salt River Watershed through the Salt River and Tonto Creek. A predictive model investigating the effect of ENSO on nutrient loading through discharge can illustrate the effects of large scale climate patterns on smaller systems.
ContributorsSversvold, Darren (Author) / Neuer, Susanne (Thesis advisor) / Elser, James (Committee member) / Fenichel, Eli (Committee member) / Arizona State University (Publisher)
Created2012