Browsing by Subject "Flow cytometry"
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Item Open Access A novel, non-apoptotic role for Scythe/BAT3: a functional switch between the pro- and anti-proliferative roles of p21 during the cell cycle.(2012) Yong, Sheila T.Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis, a form of programmed cell death, has been extensively studied. However, since the developmental defects observed in Bat3‐null mouse embryos cannot be explained solely by defects in apoptosis, I investigated whether BAT3 is also involved in regulating cell‐cycle progression. Using a stable‐inducible Bat3‐knockdown cellular system, I demonstrated that reduced BAT3 protein level causes a delay in both the G1/S transition and G2/M progression. Concurrent with these changes in cell‐cycle progression, I observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21. p21 is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Additionally, I observed that the p21 turnover rate was also reduced in Bat3‐knockdown cells released from G2/M synchronization. My findings indicate that in Bat3‐knockdown cells, p21 continues to be synthesized during cell‐cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent cell‐cycle delay. Finally, I showed that BAT3 co‐localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. My study reveals a novel, non‐apoptoticrole for BAT3 in cell‐cycle regulation. By maintaining low p21 protein level during G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 into S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation, an event that promotes G2/M progression. BAT3 modulates these pro‐ and anti‐proliferative roles of p21 at least in part by regulating the translocation of p21 between the cytoplasm and nucleus of the cells to ensure proper functioning and regulation of p21 in the appropriate intracellular compartments during different cell‐cycle phases.Item Embargo Aptamers as Reversible Sorting Ligands in Dual FACS and MACS: Antisense and Nuclease-Mediated Approaches(2023) Requena, MartinFluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS) are two essential tools for cell separation in research and medicine. Antibodies, the gold standard in both of these methods, are effective ligands for cell-surface biomarkers, but their irreversible binding precludes a wide variety of downstream medical and experimental applications. Aptamers – nucleic acid ligands with a defined three-dimensional structure that enables them to bind a molecular target with a high degree of specificity – offer a viable alternative for this particular obstacle because their RNA- or DNA-based chemistry enables their removal from cellular targets. In these studies, we present examples of successful sorting of cells and removal of the targeting aptamers with MACS and FACS using both the previously-published antisense-based method of post-sorting aptamer removal and a more general approach using nuclease-based digestion of targeting aptamers on the cell surface after cell isolation. We believe this work can be used in a number of potential post-sorting applications where targeting ligands or attached magnetic or fluorescent moieties could interfere with experimental or clinical results.
Item Open Access Computational Methods for Comparative Analysis of Rare Cell Subsets in Flow Cytometry(2013) Frelinger, Jacob JeffreyAutomated analysis techniques for flow cytometry data can address many of the limitations of manual analysis by providing an objective approach for the identification of cellular subsets. While automated analysis has the potential to significantly improve automated analysis, challenges remain for automated methods in cross sample analysis for large scale studies. This thesis presents new methods for data normalization, sample enrichment for rare events of interest, and cell subset relabeling. These methods build upon and extend the use of Gaussian mixture models in automated flow cytometry analysis to enable practical large scale cell subset identification.
Item Open Access Multiple Testing Embedded in an Aggregation Tree With Applications to Omics Data(2020) Pura, JohnIn my dissertation, I have developed computational methods for high dimensional inference, motivated by the analysis of omics data. This dissertation is divided into two parts. The first part of this dissertation is motivated by flow cytometry data analysis, where a key goal is to identify sparse cell subpopulations that differ be- tween two groups. I have developed an algorithm called multiple Testing Embedded on an Aggregation tree Method (TEAM) to locate where distributions differ between two samples. Regions containing differences can be identified in layers along the tree: the first layer searches for regions containing short-range, strong distributional differences, and higher layers search for regions containing long-range, weak distributional differences. TEAM is able to pinpoint local differences and under mild assumptions, asymptotically control the layer-specific and overall false discovery rate (FDR). Simulations verify our theoretical results. When applied to real flow cytometry data, TEAM captures cell subtypes that are overexpressed in cytomegalovirus stimulation vs. control. In addition, I have extended the TEAM algorithm so that it can incorporate information from more than one cell attribute, allowing for more robust conclusions. The second part of this dissertation is motivated by rare variant association studies, where a key goal is to identify regions of rare variants, which are associated with disease. This problem is addressed via a flexible method called stochastic aggregation tree-embedded testing (SATET). SATET embeds testing of genomic regions onto an aggregation tree, which provides a way to test association at various resolutions. The rejection rule at each layer depends on the previous layer, and leads to a procedure that controls the layer-specific FDR. Compared to methods that search for rare-variant association over large regions, such as protein domains, SATET can pinpoint sub-genic regions associated with disease. Numerical experiments show FDR control for different genetic architectures and superior per- formance compared to domain-based analyses. When applied to a case-control study in amyotrophic lateral sclerosis (ALS), SATET identified sub-genic regions in known ALS-related genes, while implicating regions in new genes not previously captured by domain-based analyses.
Item Open Access The Immunology Quality Assessment Proficiency Testing Program for CD3⁺4⁺ and CD3⁺8⁺ lymphocyte subsets: a ten year review via longitudinal mixed effects modeling.(Journal of Immunological Methods, 2014-07) Bainbridge, J; Wilkening, CL; Rountree, W; Louzao, R; Wong, J; Perza, N; Garcia, A; Denny, TNSince 1999, the National Institute of Allergy and Infectious Diseases Division of AIDS (NIAID DAIDS) has funded the Immunology Quality Assessment (IQA) Program with the goal of assessing proficiency in basic lymphocyte subset immunophenotyping for each North American laboratory supporting the NIAID DAIDS HIV clinical trial networks. Further, the purpose of this program is to facilitate an increase in the consistency of interlaboratory T-cell subset measurement (CD3(+)4(+)/CD3(+)8(+) percentages and absolute counts) and likewise, a decrease in intralaboratory variability. IQA T-cell subset measurement proficiency testing was performed over a ten-year period (January 2003-July 2012), and the results were analyzed via longitudinal analysis using mixed effects models. The goal of this analysis was to describe how a typical laboratory (a statistical modeling construct) participating in the IQA Program performed over time. Specifically, these models were utilized to examine trends in interlaboratory agreement, as well as successful passing of proficiency testing. Intralaboratory variability (i.e., precision) was determined by the repeated measures variance, while fixed and random effects were taken into account for changes in interlaboratory agreement (i.e., accuracy) over time. A flow cytometer (single-platform technology, SPT) or a flow cytometer/hematology analyzer (dual-platform technology, DPT) was also examined as a factor for accuracy and precision. The principal finding of this analysis was a significant (p<0.001) increase in accuracy of T-cell subset measurements over time, regardless of technology type (SPT or DPT). Greater precision was found in SPT measurements of all T-cell subset measurements (p<0.001), as well as greater accuracy of SPT on CD3(+)4(+)% and CD3(+)8(+)% assessments (p<0.05 and p<0.001, respectively). However, the interlaboratory random effects variance in DPT results indicates that for some cases DPT can have increased accuracy compared to SPT. Overall, these findings demonstrate that proficiency in and among IQA laboratories have, in general, improved over time and that platform type differences in performance do exist.