2024-03-29T05:00:33Zhttp://digital.lib.washington.edu/dspace-oai/requestoai:digital.lib.washington.edu:1773/228752017-11-20T22:35:13Zcom_1773_4888col_1773_19658
Comparison of Lateral Flow Immunoassays to Current Stool Evaluation Methods for the Detection of Giardia and Cryptosporidium
Mullinax, Laurianne Tarver
Butler-Wu, Susan
Cryptosporidium; Giardia; Intestinal Parasite; Lateral Flow Immunoassay
Thesis (Master's)--University of Washington, 2013
NONE
2013-07-23T18:32:30Z
2013-07-23T18:32:30Z
2013-07-23
2013
Thesis
Mullinax_washington_0250O_11508.pdf
http://hdl.handle.net/1773/22875
en_US
Copyright is held by the individual authors.
oai:digital.lib.washington.edu:1773/233422017-11-20T22:35:50Zcom_1773_4888col_1773_19658
Evaluation of a CD4-aptamer for Flow Cytometry-based Enumeration of T-cell Subsets
Huang, Hsin-Hsuan
Wu, David
Aptamer; CD4 Counts; Flow Cytometry
Thesis (Master's)--University of Washington, 2013
Introduction: Aptamers are engineered oligonucleotides that can be selected to bind with high specificity and affinity. As aptamers are cost-effective (estimated to be ~1,000-fold cheaper than antibodies) and also temperature-stable, aptamers may be useful in resource-limited settings for T-cell subset quantification. Here, we evaluated the performance characteristics of a CD4 aptamer for T-cell subset quantification. Methods: A CD4-biotinylated-streptavidin-PE aptamer provided by SomaLogic, Boulder, CO was combined with CD3-PC5, CD8-ECD and CD45-FITC antibodies in the multicolor flow cytometric assay; a CD4-A594 antibody was also added as an internal control for the CD4 aptamer. Residual patient samples were evaluated using the aptamer versus antibody reagents. Both the proportion of CD4 T cell and absolute CD4 T cell count were analyzed to evaluate the clinical performance of the aptamer reagent. Results and Conclusion: The CD4 aptamer showed comparable results to the established single platform method, which uses CYTO-STAT reagent (CD45F/CD4RD1/CD8ECD/CD3PC5) and an FC-500 flow cytometry system, with r2 >0.92 of CD4 T-cell% measurement,. Further, both the percentage of CD4 T cells and absolute T-cell count showed excellent agreement over the range of T cell proportions (8.6% to 50%) and absolute T-cell counts (150 cells/ul to 1342 cells/ul) as assessed by the CD4 aptamer as compared to an internal CD4-A594 antibody control, with r2 >0.99 and 9 cells /ul in mean difference. Our results suggest that a CD4 aptamer can perform comparably to an established CD4 antibody for CD4 T-cell subset quantitation and supports further development of aptamers as potential cost-effective reagents in resource-limited regions.
2013-07-25T17:45:24Z
2013-07-25T17:45:24Z
2013-07-25
2013
Thesis
Huang_washington_0250O_11895.pdf
http://hdl.handle.net/1773/23342
en_US
Copyright is held by the individual authors.
oai:digital.lib.washington.edu:1773/240612017-11-20T22:35:13Zcom_1773_4888col_1773_19658
MicroRNA Biomarkers Released by Platelet Activation
Wang, Jie
Pritchard, Colin C
biomarkers; microvesicles; miRNAs; platelet; platelet activation; protein-associated miRNA
Thesis (Master's)--University of Washington, 2013
MicroRNAs are a group of small noncoding RNA molecules that have been implicated in a variety of human diseases. Because of their remarkable stability and abundant quantity in blood, circulating microRNAs are promising as noninvasive biomarkers for diagnosis or prognosis. Platelets are likely to be a substantial contributor to circulating miRNAs. Upon activation, platelets shed membrane-bound microparticles, microvesicles and protein complexes into circulation. Recent studies suggest that circulating miRNAs are protected by encapsulation in vesicles or binding with Argonaute2 (Ago2) protein complex from blood RNase activity. However, the mechanisms of how platelets release miRNAs into circulation, and the role of platelet activation in miRNA release remains unclear. Therefore, I characterized four highly expressed platelet-released microRNAs: miR-16; miR-142-3p; miR-223 and let-7a, using size-exclusion chromatography. My results show that miRNAs are released by platelet activation. Further, my data suggests that platelet-released miRNAs are primarily associated with vesicle-free protein complexes rather than microparticles and microvesicles.
2013-11-14T20:49:14Z
2013-11-14T20:49:14Z
2013-11-14
2013
Thesis
Wang_washington_0250O_12278.pdf
http://hdl.handle.net/1773/24061
en_US
Copyright is held by the individual authors.
oai:digital.lib.washington.edu:1773/251102017-11-20T22:35:52Zcom_1773_4888col_1773_19658
Measurement of Cystatin C in Dried Blood Spot Specimens
Vogl, Paul Timothy
Wener, Mark
Biomarker; Cystatin C; Dried Blood Spots
Thesis (Master's)--University of Washington, 2013
Dried blood spot (DBS) technologies can be used in a variety of clinical and research settings. Among other advantages, the use of DBS specimens facilitate the incorporation of biomarkers into demographic research, especially for large global studies conducted in remote areas with little or no laboratory infrastructure. Here, we report the results of an evaluation of a new method for measuring cystatin C (cysC) in DBS. We adapted a commercial ELISA kit designed to measure cysC in human plasma and were able to measure cysC concentrations across the physiologic range in DBS. Determinations from DBS were linearly correlated and near directly equivalent to cysC concentrations in DBS-matched plasma--intercept=-0.07 (95% confidence interval=-0.17to -0.01) and slope=0.98 (95% confidence interval=0.92 to 1.12), R=0.94. Bland Altman analysis of cysC concentrations in DBS vs. plasma revealed no bias and acceptable levels of agreement (Bias -0.02, 95% confidence interval=-0.21 to 0.17). The DBS-ELISA demonstrates utility as a screening tool; sensitivity and specificity for cysC concentrations outside the upper limit of the reference interval (0.51 mg/L to 1.02 mg/L) were 68% and 95% respectively; however, the DBS-ELISA had difficultly correctly assigning CKD stage from DBS. We further describe an intra-donor difference in cysC concentration between venous blood collected from venipuncture and capillary blood collected by fingerstick. The DBS-ELISA assay provides an opportunity to explore kidney function in large community-based studies.
2014-02-24T18:27:30Z
2014-02-24T18:27:30Z
2014-02-24
2013
Thesis
Vogl_washington_0250O_12654.pdf
http://hdl.handle.net/1773/25110
en_US
Copyright is held by the individual authors.
oai:digital.lib.washington.edu:1773/254292017-11-20T22:35:52Zcom_1773_4888col_1773_19658
Evaluation of Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry for Clinical Microbiology Applications
Bauerle, Elizabeth
Butler-Wu, Susan
bacterial identification; blood culture; MALDI-TOF-MS; Sepsityper
Thesis (Master's)--University of Washington, 2014
MALDI-TOF-MS has the ability to streamline identification of microorganisms in the microbiology lab. In this project, we focused on two main aims. First, the implementation of this technology for direct blood culture identification. Within this aim we determined the limit of detection for the protein purification method we planned on using in our laboratory, determined handling conditions for the samples, and evaluated the MALDI-TOF-MS for concordance with our current workflow and reports. Second, customization of the commercial library for Cryptococcus gattii was performed. Within this aim, we improved representation of the species in the MALDI-TOF-MS library, and also explored ways to determine the subspecies based on gathered spectra.
2014-04-30T16:22:16Z
2015-12-14T17:55:49Z
2014-04-30
2014
Thesis
Bauerle_washington_0250O_12730.pdf
http://hdl.handle.net/1773/25429
en_US
Copyright is held by the individual authors.
oai:digital.lib.washington.edu:1773/330702017-11-20T22:36:16Zcom_1773_4888col_1773_19658
A Comparison of Disc Diffusion and Microbroth Dilution Methods for the Detection of Antibiotic Resistant Subpopulations in Gram Negative Bacilli
Luc, Matthew
Butler-Wu, Susan
Thesis (Master's)--University of Washington, 2015
Two of the different types of antibiotic susceptibility tests available are the solid media based Kirby Bauer method and the liquid media based microbroth dilution method. The microbroth dilution method has become more widely used due to its ability to become automated, however, the Kirby Bauer method may be able to detect subpopulations of resistant bacteria that would be visualized as inner colonies in the zone of diffusion. Using Gram negative rod isolated collected from the UWMC clinical microbiology laboratory, the presence of inner colonies was screened and both methods were compared to determine if the microbroth dilution method was able to detect the additional resistance of the subpopulations. The data went to show that the microbroth dilution method was not detecting the additional resistance in about 68% of the isolates tested. It was unable to be determined if the results were due a limitation of the microbroth dilution method or whether it is an artifact of the testing methods, but it does open up the possibilities of further testing to determine the cause of the disparity in results.
2015-05-11T19:56:19Z
2015-05-11T19:56:19Z
2015
Thesis
Luc_washington_0250O_14147.pdf
http://hdl.handle.net/1773/33070
en_US
Copyright is held by the individual authors.
oai:digital.lib.washington.edu:1773/334792016-08-19T23:57:36Zcom_1773_4888col_1773_19658
Detection of MegaTAL-induced HIV pol Mutation By Droplet Digital PCR
Liang, Shu
Jerome, Keith R
ddPCR; HIV; MegaTAL; Mutation detection
Thesis (Master's)--University of Washington, 2015
Reliable detection of low-level mutations in HIV-1 provirus is still a challenge. Current methods regularly used in laboratories, Surveyor assay and bulk sequencing, only have a limit of detection about 10 ~ 20%. In the Jerome lab, a potential cure for HIV, megaTALs, is under development and a method to evaluate its outcome is in need. In this thesis, a droplet digital PCR assay that can detect low-level target mutations in HIV provirus induced by megaTALs was developed. This assay was validated with both plasmid and cell line samples. This assay had demonstrated high analytical linearity (r2 > 0.99) and repeatability (CVs < 7%). The Limit of Blank of this assay is 0.56%. The Limit of Detection of this assay is 1.06%. The Limit of Quantitation of this assay is 2.19%. This assay showed higher accuracy than Surveyor assay. The results of the same sample generated by this assay were comparable to those of clonal sequencing and Illumina sequencing. This assay has approved that ddPCR can be use as a reliable mutation detection method in HIV with high precision and accuracy.
2015-09-29T17:54:33Z
2015
Thesis
Liang_washington_0250O_14555.pdf
http://hdl.handle.net/1773/33479
en_US
Copyright is held by the individual authors.
oai:digital.lib.washington.edu:1773/334802017-11-20T22:35:13Zcom_1773_4888col_1773_19658
OPTIMIZATION OF FLOW CYTOMETRIC SORTING IN THE HEMATOPATHOLOGY LABORATORY
Thomas, Anju
Fromm, Jonathan
Thesis (Master's)--University of Washington, 2015
University of Washington Abstract OPTIMIZATION OF FLOW CYTOMETRIC SORTING IN THE HEMATOPATHOLOGY LABORATORY Anju Thomas Chair of Supervisory Committee: Dr. Jonathan Fromm MD, PhD Department of Laboratory Medicine A subset of neoplasms in Hematopathology show rare neoplastic cells in a background of abundant non-neoplastic reactive cells. Developing a method of enriching for neoplastic cells, in in combination with efficient DNA extraction for molecular applications, could be a valuable tool for evaluation of the rare cell neoplasm (RCN). The purpose of this study is to optimize the procedures for flow cytometric cell sorting, affording enrichment of neoplastic cells of RCN. This project also addressed validation of an effective and automated DNA extraction method using minimal numbers of sorted cells. The detection level limit for clonality based molecular assay was also established used abnormal and normal sorted cells. In this study, we considered different technical aspects of the flow sorting such as how nozzle sizes will affect recovery of the various sorted cells. For this experiment, we used a megakaryocytic lineage induced cell ine, a Hodgkin lymphoma cell line (L428), and normal T lymphocytes. Another technical consideration evaluated, compared different precision modes using various percentage of the spiked cell to calculate the efficiency of sorting. Finally, these studies used sorted cells for comparison of two DNA extraction technique to determine which method afforded a greater yield of DNA for subsequent molecular studies; the B cell clonality assay platform was used for sorting abnormal and normal cells to establish detection levels. The studies suggest that it is necessary to have larger nozzle sizes for rare cell neoplastic sorting if large neoplastic cells from RCN. The precision modes study demonstrated that the combination of yield mode and purity mode is the preferable method for rare cells sorting. Comparison of DNA extraction methods proved that the EZ viral kit methods are more effective and sensitive when using the sorted cells than other DNA extraction methodologies; the limit of detection with B cell clonality assay determined that the level of detection using sorted cells could be hundred fold better than with unsorted bulk specimen. These studies demonstrated that by combining a reliable enrichment method with an effectve DNA extraction (for downstream molecular techniques) would improve the diagnostic value of these tests.
2015-09-29T17:54:35Z
2015
Thesis
Thomas_washington_0250O_14639.pdf
http://hdl.handle.net/1773/33480
en_US
Copyright is held by the individual authors.
oai:digital.lib.washington.edu:1773/364192016-07-15T10:22:19Zcom_1773_4888col_1773_19658
Method Development and Validation of Vitamin D2 and Vitamin D3 using Mass Spectrometry
Riley, Devon
Hoofnagle, Andrew
Thesis (Master's)--University of Washington, 2016-06
Vitamin D has long been known to maintain bone health by regulating calcium and phosphorous homeostasis. In recent years, scientists have discovered additional physiological roles for vitamin D. The complex interaction between the active vitamin D hormone and its metabolic precursors continues to be a rich area of research. Fundamental to this research is the availability of accurate and precise assays. Few published assays for vitamins D2 and D3 have contained sufficient details on method validation or performance characteristics. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay developed for this thesis has undergone a rigorous validation and proven to yield a sensitive and specific method that exceeds the capabilities of all previously published methods. Developing and validating a novel assay is often complicated by the lack of established acceptability standards. This thesis explores this challenge, specifically for establishing meaningful interpretations and qualification standards of the lower limit of the measuring interval. Altogether, future research focused on vitamins D2, D3 and the Vitamin D pathway can benefit from this robust LC-MS/MS assay and the associated quality parameters outlined in this thesis.
2016-07-14T16:34:30Z
2016-07-14T16:34:30Z
2016-06
Thesis
Riley_washington_0250O_15723.pdf
http://hdl.handle.net/1773/36419
en_US
oai:digital.lib.washington.edu:1773/369802016-09-23T10:23:54Zcom_1773_4888col_1773_19658
Development of A Flow Cytometry Assay for Measurement of CD4+ T Cell Proliferation
He, Ying
Morishima, Chihiro
CD4+ T cell
Flow cytometry
in vitro stimulation
Ki67 protein
Proliferation
Thesis (Master's)--University of Washington, 2016-06
Measurement of CD4+ T cell function has been widely utilized in numerous studies. In clinical medicine, in vitro tests to measure CD4+ T cell functions such as activation and proliferative capacity have been established for decades. The Ki67 molecule is a nuclear protein present exclusively in proliferating cells. Ki67 protein expression is strictly correlated to cell proliferation and to the active phases of the cell cycle, although it’s functional role during cell proliferation is unclear. The purpose of this study was to define the optimal flow cytometric protocol for the detection of in vitro CD4+ T cell proliferation using Ki67. The ultimate goal is to develop a clinical-grade assay that can be used for the diagnosis and management of patients.
2016-09-22T15:40:53Z
2016-06
Thesis
He_washington_0250O_16274.pdf
http://hdl.handle.net/1773/36980
en_US
oai:digital.lib.washington.edu:1773/397932017-08-12T11:02:20Zcom_1773_4888col_1773_19658
Characterizing Aztreonam Resistance in Pseudomonas aeruginosa through Artificial Selection and Whole Genome Sequencing
McLean, Kathryn Ann
Salipante, Stephen
antibiotic resistance
aztreonam
Cystic fibrosis
Pseudomonas aerugnionsa
whole genome sequencing
Microbiology
Thesis (Master's)--University of Washington, 2017-06
While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole genome sequencing to investigate chromosomally-encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI. While individual mutations conferred modest resistance gains, high-level resistance (1024 µg/mL) was achieved through the accumulation of multiple variants. Despite being largely stable when passaged in the absence of antibiotics, aztreonam resistance was associated with slowed in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance, and raise the possibility that antibiotic treatment could inadvertently select for hyper-virulence phenotypes.
2017-08-11T22:45:20Z
2017-08-11T22:45:20Z
2017-06
Thesis
McLean_washington_0250O_16999.pdf
http://hdl.handle.net/1773/39793
en_US
Table S1-Summary of Bacterial Strains.docx; text; Table S1: Summary of Bacterial Strains.
Table S2-Growth Rates Before and After LB Passaging.xlsx; spreadsheet; Table S2: Growth Rates Before and After LB Passaging.
Table S3- No Selection MIC.xlsx; spreadsheet; Table S3: No Selection MIC.
Table S4- No Selection variants.xlsx; spreadsheet; Table S4: No Selection Variants.
Figure S1.pdf; pdf; Figure S1.
Figure S2- phage assay.pdf; pdf; Figure S2.
none
oai:digital.lib.washington.edu:1773/397942017-08-12T11:02:31Zcom_1773_4888col_1773_19658
Establishing Prehospital Transfusion with Airlift Northwest and Harborview Medical Center
Louzon, Max J
Hess, John R
Aeromedical Evacuation
Blood bank
Blood storage
Transfusion
Medicine
Thesis (Master's)--University of Washington, 2017-06
Harborview Medical Center (HMC) routinely received patients transferred from outside facilities. These patients would be supported with blood products sent by the transferring facility, and on arrival these products would be discarded due to unvalidated shipping methods. This process put a strain on local blood suppliers and small transfusion services. The local aero-medical evacuation provider, Airlift Northwest (ALNW), also had no blood products to support trauma patients being transferred from the scene of the injury. These patients only began receiving transfusion support upon arrival at HMC. HMC partnered with ALNW to begin supplying blood products to support the transfer of patients. This process would entail validating a system to ensure the blood products would be kept at a storage temperature between 1-6℃. Since ALNW had six air bases throughout Washington and Alaska, the Credo Series 4 EMT cooler was validated to store the blood products for up to seven days to minimize transport between HMC and ALNW. To ensure an appropriate temperature was maintained in the cooler, a temperature recorder was placed in the cooler along with the blood products. HMC also used new blood products, low titer plasma and liquid plasma, to support prehospital transfusion. Low titer plasma (LTP) was defined as blood type A plasma with a titer of anti-B as ≤1:200. LTP was occasionally used in place of the traditional universal type AB plasma due to inventory control purposes. Typically HMC stocks thawed plasma that expires five days after thawing. To support ALNW holding blood products for seven days, liquid plasma was used as it expires 26 days after donation. All staff at HMC and ALNW were trained in handling the coolers as well as the applicable usage procedures. HMC began supplying blood products to ALNW in June 2015 and since then 24 patients have been transfused. Originally Boeing Field was the only site supported by HMC allowing for quick resolution of the problems encountered with the coolers, temperature recorders, and blood products. Since June 2015, the program has expanded to Olympia with plans to support the other air bases. The program for prehospital transfusion has also gained the interest of the organ transplant team and a separate validation will be taking place.
2017-08-11T22:45:21Z
2017-08-11T22:45:21Z
2017-06
Thesis
Louzon_washington_0250O_17038.pdf
http://hdl.handle.net/1773/39794
en_US
CC BY
oai:digital.lib.washington.edu:1773/408022018-01-20T11:34:03Zcom_1773_4888col_1773_19658
Comparison of Clinical Features Between Inpatient and Outpatient Cases of Clostridium difficile Infection
Belger, Marcus Jerrod
Fang, Ferric C
CDI
Clostridium diffcile
FilmArray
GeneXpert
Health sciences
Microbiology
Thesis (Master's)--University of Washington, 2017
The Microbiology Laboratories at Harborview Medical Center and the University of Washington Medical Center evaluated the Biofire FilmArray Gastrointestinal Panel, a multiplex PCR assay to conventional stool culture. The FilmArray can detect both toxin A (tcdA) and toxin B genes (tcdB) in Clostridium difficile. C. difficile is not detected by conventional stool culture. Instead, both laboratories use the Cepheid GeneXpert C. diffcile assay to rapidly detect the toxin B gene (tcdB). These two different test methods and the testing requirements provided an opportunity to compare clinical features of patients whom CDI was detected by targeted testing to those whom CDI was an unexpected finding detected by the multiplex PCR assay. A retrospective observational cohort study was performed on one-hundred forty cases of diagnosed CDI. A comparison of risk factors, clinical presentation, and responses to CDI-specific therapy was done between inpatients and outpatient cases. Analysis of the results showed that inpatients and outpatients were considerably similar in all those categories. There is a significant proportion of the CDI burden, with potential of cases overlooked, in the outpatient setting.
2018-01-20T00:57:15Z
2018-01-20T00:57:15Z
2017
Thesis
Belger_washington_0250O_18091.pdf
http://hdl.handle.net/1773/40802
en_US
none
oai:digital.lib.washington.edu:1773/420382018-08-01T10:13:07Zcom_1773_4888col_1773_19658
Regulation of Phenotypic Heterogeneity in Salmonella: Bifunctional Regulators, RcsB and YifA, Shape Flagellar Bistability
Chisholm, Claire Rozell
Cookson, Brad T
Flagella
Phenotypic Heterogeneity
RcsB
Regulation
Salmonella
YifA
Microbiology
Molecular biology
Health sciences
Thesis (Master's)--University of Washington, 2018
Heterogeneous expression of flagellar and invasion genes is crucial for Salmonella virulence. Such a division of labor enables an invasive subpopulation to trigger inflammation in the infected host and alter the gut environment to favor growth of Salmonella. Meanwhile, nonflagellated cells are better able to evade host defenses systemically and persist. This bistable expression requires the master regulator of the flagellar cascade, FlhD4C2, to be kept in low concentration by post-translational regulators YdiV, ClpXP, and STM14_2047. In this study, we demonstrated novel roles for two previously identified regulators of flagellar expression. RcsB and YifA are independently able to repress the transcription of both flhDC and STM14_2047 in several conditions, but their control of flagellar expression is environmentally responsive. We have identified conditions where RcsB and YifA act as either repressors or promoters of flagellar expression and hypothesize that this conditional regulation increases fitness in varied environments.
2018-07-31T21:06:16Z
2018
Thesis
Chisholm_washington_0250O_18774.pdf
http://hdl.handle.net/1773/42038
en_US
none
oai:digital.lib.washington.edu:1773/420392018-08-01T10:13:30Zcom_1773_4888col_1773_19658
Validation of an ELISA Method for Epstein-Barr Virus Antibodies in Dried Blood Spot Specimens and Correlation of EBV Serology with DNA Detection and Cytomegalovirus Serology
Wang, Chen-Yu
Wener, Mark H
CMV
Cytomegalovirus
DBS
Dried blood spot
EBV
Epstein-Barr virus
Immunology
Virology
Thesis (Master's)--University of Washington, 2018
Dried blood spot (DBS) technology offers several advantages over conventional liquid sample types in terms of cost, storage and stability especially in large population-based or field research. The Epstein-Barr virus (EBV) antibody level is widely accepted as a biomarker for psychosocial stress, based on the notion that stress triggers downregulation of cellular immune function and causes the reactivation of latent EBV and in turn, the increase of EBV IgG level as a response. Our project has two goals: First, we validated an ELISA method for measuring EBV antibody in DBS. We found a strong correlation between EBV IgG in plasma and DBS sample types (R2 = 0.90). Second, we examined the correlations between EBV serology and EBV PCR results and cytomegalovirus (CMV) serology in DBS and serum in hopes of exploring a more direct or alternate means of measuring herpesvirus reactivation. We found that EBV IgG level poorly predicted viremia in serum (R2 = 0.08) and viral load in DBS [F (1, 85) = 1.93, P = 0.17] and that EBV and CMV serology correlated weakly (r = 0.43).
2018-07-31T21:06:16Z
2018-07-31T21:06:16Z
2018
Thesis
Wang_washington_0250O_18392.pdf
http://hdl.handle.net/1773/42039
en_US
none
oai:digital.lib.washington.edu:1773/428802018-11-28T11:33:34Zcom_1773_4888col_1773_19658
Antibody Characterization for use in Clinical Mass Spectrometry
Moore, Andrea
Hoofnagle, Andrew
Baird, Geoffrey
antibody
immunoaffinity enrichment
KLOTHO
mass spectrometry
procollagen
screen
Chemistry
Biochemistry
Biology
Thesis (Master's)--University of Washington, 2018
Many biomarkers in human serum are in low abundance. Immunoaffinity enrichment via anti-peptide antibody and analysis via liquid chromatography tandem mass spectrometry (LC-MS/MS) is an attractive workflow that depends on the production of new antibody reagents. Although there are different methods available to characterize antibodies such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and LC-MS/MS, using the same platform for screening and assay development is likely beneficial. Antibody apparent dissociation constant (KD) and on rate and off rate (kon and koff) may be equally as critical as recovery efficiency for screening. A high throughput, manual, in-house antibody LC-MS/MS screen was developed to screen 846 supernatants with antibodies to specific peptides of KLOTHO and Procollagen type III C-terminal Pro-peptide (P3CP) to find the best 15 antibodies and characterize them for future assay development.
2018-11-28T03:13:13Z
2018-11-28T03:13:13Z
2018
Thesis
Moore_washington_0250O_19269.pdf
http://hdl.handle.net/1773/42880
en_US
none
oai:digital.lib.washington.edu:1773/438702019-08-15T10:42:34Zcom_1773_4888col_1773_19658
Contributions and Evolutionary Potential of ampC Mutations to confer Aztreonam Resistance in Pseudomonas aeruginosa
Lee, Duan-kun
Salipante, Stephen J
Molecular biology
Microbiology
Health sciences
Thesis (Master's)--University of Washington, 2019
Aztreonam is a synthetic monobactam antibiotic that is increasingly used for chronic suppressive therapy in Cystic Fibrosis (CF) patients with Pseudomonas aeruginosa airway infection. P. aeruginosa can accumulate mutations in the chromosomal ampC β-lactamase which promote aztreonam resistance, but the extent to which spontaneous mutations can increase ampC activity against aztreonam remains incompletely explored. To address this question, here we both examined ampC mutations that occur in P. aeruginosa clinical isolates derived from CF patients treated with aztreonam and additionally performed in vitro evolution of ampC for enhanced aztreonam hydrolysis. First, to catalog spontaneous ampC mutations that confer increased aztreonam resistance in P. aeruginosa, we sequenced a collection of 60 pre- and post-treatment clonal isolate pairs showing elevated resistance levels and identified 14 isolates with treatment-associated mutations in that gene. The 12 alleles, containing 13 unique mutations, had a variable effect on the ability of ampC to confer elevated aztreonam resistance relative to the wild-type allele (range 8-fold decrease to 64-fold increase) when expressed either in Escherichia coli or a P. aeruginosa laboratory strain deficient for native ampC. The alleles conferred variable resistance to ampicillin, ceftazidime, and cefpirome, but meropenem resistance was consistently unaffected. To more fully evaluate the potential of point mutations to increase ampC activity against aztreonam, we subsequently performed artificial evolution and aztreonam selection of plasmid-borne ampC expressed in E. coli. We recovered mutants showing up to 4,096-fold increases in aztreonam resistance (128 ug/mL) relative to wild-type ampC, with mainained or enhanced resistance provided to other β-lactam drugs. Collectively, these findings indicate that mutations in ampC which result in elevated aztreonam resistance are relatively frequent in P. aeruginosa isolates from CF patient lungs, and that the maximum potential for ampC to evolve activity against this synthetic antibiotic are greater than previously appreciated.
2019-08-14T22:25:27Z
2019-08-14T22:25:27Z
2019
Thesis
Lee_washington_0250O_19915.pdf
http://hdl.handle.net/1773/43870
en_US
CC BY-NC-ND
oai:digital.lib.washington.edu:1773/438712019-08-15T10:42:35Zcom_1773_4888col_1773_19658
A Tale of Two Validations: Molecular Diagnostic Assays for the Detection of Clinically Significant Alterations
McGregor, Ayako J
Lockwood, Christina M
Laboratory Developed Test
Molecular Assay Validation
Molecular Diagnostics
Next Generation Sequencing
Real-time PCR
Validation
Genetics
Thesis (Master's)--University of Washington, 2019
Laboratory developed tests (LDTs) have become an integral part of laboratory medicine with applications to a variety of complex genetic testing and are at the frontlines of clinical medicine. Proper validation and implementation of LDTs is essential to ensure accurate and high quality results to aid in not only the diagnostic work-up but also for prognostic information and identification of therapeutic options for patients. With numerous techniques and technologies applied to LDTs, it is important to understand which ones are the best methods to apply to a given clinical problem. To best learn this, two validations were performed for two different genetic assays: first a real-time PCR SNP genotyping assay for Apolipoprotein L1 (ApoL1) variant alleles (G1 and G2 alleles) to assess the risk of developing chronic kidney disease (CKD) and secondly a comprehensive next generation sequencing (NGS) assay, OncoPlex version 6 (OncoPlex v6), to detect somatic mutations through an expanded panel of 340 genes. Both validations proved excellent performance characteristics. The accuracy of the real-time PCR genotyping assay had an average of 99.4% and a perfect inter-run and intra-run reproducibility of 100%. Using a simple real-time PCR methodology could potentially determine the patient’s risk for CKD before they progress to the advance stages of the disease. OncoPlex v6 demonstrated exceptional performance characteristics with approximately a 2.5-fold increase in average sequencing coverage and a 3.7-fold increase in percent on-target sequencing. The accuracy and precision of OncoPlex v6 across all classes of alterations was above 97% and 92% respectively, with the majority of variants not identified either present at low variant allele fractions or demonstrating low-level copy gains or losses. In addition to excellent performance characteristics, the modular capture chemistry of OncoPlex v6 demonstrates improvements over prior versions by reduced capture cost, as well as superior sequencing quality and faster turnaround time. Both assays are capable of providing critical information that can impact prognosis, diagnosis, and treatment options.
2019-08-14T22:25:27Z
2019-08-14T22:25:27Z
2019
Thesis
McGregor_washington_0250O_20053.pdf
http://hdl.handle.net/1773/43871
en_US
none
oai:digital.lib.washington.edu:1773/438722019-08-15T10:42:36Zcom_1773_4888col_1773_19658
Inherited chromosomally integrated HHV-6 demonstrates tissue-specific RNA expression in vivo
Peddu, Vikas
Greninger, Alexander
HHV-6
Human Herpesvirus-6
iciHHV-6
Inherited chromosomally integrated HHV-6
Virology
Bioinformatics
Microbiology
Thesis (Master's)--University of Washington, 2019
Human herpesvirus-6A and 6B (HHV-6A, HHV-6B) are highly prevalent viruses unique in that they, besides Epstein-Barr virus, are the only human herpesviruses capable of chromosomal integration. When a chromosomal integration event happens in a germ cell the resulting progeny will have one copy of the virus in all of their cells, referred to as inherited chromosomally integrated human herpesvirus-6 (iciHHV-6). Active HHV-6 infections in those who are iciHHV-6 positive confound current DNA based PCR assays, highlighting a necessity for mRNA-based assays. As the transcriptional state of iciHHV-6 has not been defined in vivo, it is unclear which HHV-6 genes best determine actively infected iciHHV-6 individuals. Here, we screened DNA- Seq and RNA-Seq data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A and 4 iciHHV-6B positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, the HHV-6 genes U90 and U100 were expressed in the brain, testis, breast, adrenal gland, lungs, salivary gland, esophagus, skeletal muscle, colon, tibial nerve and artery, adipose tissue, heart, skin, and thyroid. Additionally, high levels of HHV-6 gene expression were detected in the brain, testis, esophagus, and adrenal gland. We also analyzed data from the Synapse Mount Sinai Brain Bank and found similar brain tissue expression in the 4 iciHHV-6 positive samples. We found no expression of U38, a viral polymerase subunit found to be expressed in HHV-6 active infections, in GTEx whole blood RNAseq samples. However, using an RT-qPCR assay specific to the HHV-6A and -6B U38 genes, we saw that U38 was expressed in some iciHHV-6 positive PBMC samples submitted for iciHHV-6 testing.
2019-08-14T22:25:28Z
2019-08-14T22:25:28Z
2019
Thesis
Peddu_washington_0250O_20364.pdf
http://hdl.handle.net/1773/43872
en_US
CC BY-NC
oai:digital.lib.washington.edu:1773/450612020-02-05T11:34:30Zcom_1773_4888col_1773_19658
Design and Development of Hemogram, a Web Application for a Remote Examination of Peripheral Blood Smears from Resource-Limited Clinics in Nepal
Bhandari, Sukra Raj
Hoffman, Noah G
Diagnostic Medicine
Peripheral Blood Smears
Tele-hematology
Information technology
Health sciences
Thesis (Master's)--University of Washington, 2019
Examination of peripheral blood smears is a simple medical laboratory test that can provide signicant diagnostic information. Nevertheless, this test is manual, time-consuming, and demands highly experienced medical technologists and/or pathologists. The gravity of this test is far more signicant in resource-limited clinics in developing countries where advanced laboratory testing is beyond their capacity. Hemogram, a web application, is developed as a prototype project that aims at providing a user-friendly yet robust platform to remotely examine peripheral blood smears by experienced laboratory professionals. Implementing Hemogram to resource-limited clinics can provide added laboratory support in diagnosing hematologic disorders and alleviate the problem of shortage of laboratory professionals. This thesis presents various facets of Hemogram, including the design and development of its database schema, user interface, and application logic.
2020-02-04T19:22:29Z
2020-02-04T19:22:29Z
2019
Thesis
Bhandari_washington_0250O_20989.pdf
http://hdl.handle.net/1773/45061
en_US
none
oai:digital.lib.washington.edu:1773/471982021-08-28T10:44:55Zcom_1773_4888col_1773_19658
Accurate quantification of placental (fetal) fraction by tissue specific cell-free DNA analysis.
Adil, Mohamed
Pritchard, Colin
Ha, Gavin
Cell-free DNA
Fetal fraction
Sequencing
Tissue of origin
Molecular biology
Bioinformatics
Pathology
Thesis (Master's)--University of Washington, 2021
Background:Noninvasive liquid biopsy analytes such as cell-free DNA (cfDNA) have advanced the field of precision medicine. It is most widely used in noninvasive prenatal testing (NIPT) to screen for untreatable aneuploidies from maternal plasma. An important parameter for the success of the test is being able to accurately calculate the fraction of fetal cfDNA (FF). Recent advances in analysis of cfDNA’s fragmentation patterns have shown to reveal the tissue of origin which can be used to quantify the tissue’s fraction. Here we apply this concept to estimate the FF from shallow whole genome sequencing (sWGS).
Method:
In this study, we present an approach to quantify the placental fraction which is a surrogate for fetal fraction (FF). This is because the majority of fetal DNA in maternal plasma originates from placental tissue. We perform cfDNA fragmentation analysis using cfDNA tissue specific maps (cfDTM) to identify features that correlate with fetal fraction. We then apply machine learning strategies to accurately quantify FF from sWGS. The method is further validated using additional sample sets.
Results:
Fragmentation analysis of cfDNA using (cfDTMs) identified features that correlate highly with FF as calculated by a gold standard method – quantification of chromosome-Y derived fragments in mothers carrying male fetuses. We further identified Transcription factors associated with placental and hematopoietic origin. Finally, by training a model we were able to estimate the FF with very high accuracy. Furthermore, we show that the method is sex independent and is not affected by chromosomal aneuploidy.
Conclusion:
Using cfDNA samples we developed and validate a method of estimating the fraction of a tissue of interest using clinically routine sWGS techniques. Using placenta as our target tissue we were able to accurately quantify placental (fetal) fraction. This has immediate clinical application for NIPT to improve the accuracy of FF estimation reducing the number of false negatives. We anticipate this approach is generalizable which suggests that tissue-of-origin analysis of circulating cfDNA can be further applied to other clinically relevant use cases such as transplant rejection, tissue injury and in oncology as a prognostic and predictive biomarker.
2021-08-26T18:03:05Z
2021-08-26T18:03:05Z
2021
Thesis
Adil_washington_0250O_23117.pdf
http://hdl.handle.net/1773/47198
en_US
CC BY
oai:digital.lib.washington.edu:1773/478632021-10-30T10:51:52Zcom_1773_4888col_1773_19658
The spectrum of mosaic mutations in megalencephaly and other growth disorders by ultra-deep targeted next-generation sequencing (NGS)
Madkhali, Nawal
Shirts, Brian
Genetics
Thesis (Master's)--University of Washington, 2021
This is a retrospective study aimed at analyzing genetic variants and levels of mosaicism identified in a cohort of patients clinically tested for brain and body overgrowth phenotypes between 2014 and 2019 through the Megaplex multi-gene panel offered at the University of Washington. We analyzed the megaplex data to further characterize the molecular basis of overgrowth phenotypes and to optimize future interpretation and analysis of this panel.
In this study, we examined samples from 180 clinical patients diagnosed with brain and body overgrowth disorders. An additional 33 samples were collected from parents to determine the inheritance of compelling variants. The panel consisted of 37 genes known to be associated with brain and body overgrowth disorders. DNA was extracted from peripheral blood in 169 (53.8%) of the samples, 69 (22.8%) in skin fibroblast, 68 (21.8%) in tissue, 3 (0.9%) in saliva, 2 (0.6%) in cell-free DNA (cfDNA), and 2 (0.6%) in unknown samples. Capture-based Next-Generation sequencing (NGS) was performed using custom-designed SureSelect probe libraries and analyzed using short read sequencing on Illumina HiSeq 2000 or MiSeq sequencers. Identified mutations were confirmed and analyzed using a custom in-house bioinformatics pipeline.
Of the 213 individuals tested, 128 (41.0%) had pathogenic and likely pathogenic mutations. Most of these variants were in PIK3CA (N =49, 38%) and PTEN 17 (13.3%). There were no pathogenic or likely pathogenic mutations reported in 161 cases (51.6%). In this study, variants of uncertain significance were reported in 23 (7.4%) of cases.
Ultra-deep NGS can efficiently identity mosaic mutations in megalencephaly and overgrowth disorders including detecting low levels of mosaicism, compared to Sanger sequencing and standard-depth NGS testing. Detecting mosaic mutations using deep NGS testing improves the clinical yield and provides a better understating of the spectrum of mosaic mutations underlying these phenotypes.
2021-10-29T16:16:03Z
2021-10-29T16:16:03Z
2021
Thesis
Madkhali_washington_0250O_23503.pdf
http://hdl.handle.net/1773/47863
en_US
none
oai:digital.lib.washington.edu:1773/481532022-01-27T11:44:57Zcom_1773_4888col_1773_19658
Hematologic parameters and bone marrow flow cytometry features in patients with GATA2 and RUNX1 germline mutations ; Evaluation of frequency of 11q aberrations in patients with Burkitt-like lymphoma and other aggressive B cell lymphomas: correlation with histopathology and clinical characteristics
HO, CHIA-CHEN
Eckel, Ashley
Burkitt-like lymphoma
GATA2
Germ-line
Hematologic maliganacies
RUNX1
Medicine
Thesis (Master's)--University of Washington, 2021
For patients presenting with cytopenias, the clinical presentation and histopathological distinction between an inherited (germline) bone marrow failure syndrome and an acquired bone marrow failure/myelodysplastic syndrome (MDS) can be challenging. Prompt and precise diagnosis of is critical to inform appropriate management before a leukemic transformation. GATA2 and RUNX1 are known germline pre-disposition mutations affecting master transcription factors important in lineage development. Here we aimed to develop a screening assay to help assess patients found to have germline GATA2 mutation and assist in germline variant curation. We compared the hematologic parameters and early bone marrow flow cytometric features in 8 patients with germline GATA2 mutations to 15 patients with aplastic anemia at our institution. To further consider the specificity of the findings in GATA2 deficiency patients, we compared their findings to 9 patients with germline RUNX1 mutations. Consistent with prior studies, our patients with GATA2 mutations showed less pronounced anemia and thrombocytopenia than those with aplastic anemia. Findings in the marrow, including the absence of hematogones, and reduction in NK cells, also support prior studies. Furthermore, compared to patients with RUNX1 mutations, patients with GATA2 mutations showed significantly lower B cells (of lymphocytes) and NK cells (of lymphocytes), and higher T cells (of lymphocytes). Interestingly, we also identified patients with RUNX1 mutations to have significantly more PDCs than patients with GATA2 mutations. Differences in our findings from prior studies, including similar peripheral blood absolute white counts, absolute neutrophils, absolute monocytes, absolute lymphocyte counts, and B cells or monocytes in the marrows of our patients with germline GATA2 mutations, are due to the small size of patient cohorts given the rarity of these germline variants as well as to variable disease status at presentation for evaluation/inclusion in these studies (i.e., pre-MDS, MDS). The combined findings contributed to the understanding of how these mutations establish variable pre-leukemic states predisposing to myeloid malignancies and supported the development of effective screening strategies. Burkitt-like lymphoma with 11q aberration (BLL-l1q) is one of the newly described provisional entities with morphologic features, immunophenotype, and gene expression profile similar to Burkitt lymphoma (BL) but lacks MYC rearrangement. Whether this entity is a distinct category or a variant of BL, diffuse large B cell lymphoma (DLBCL), or high-grade B cell lymphoma is still controversial. Few cases have been reported, leading to poor understanding of clinical presentations, treatment regimens, or full understanding of the landscape of mutations/copy-number alterations in BLL-11q. Here we aimed to evaluate the frequency of 11q aberration in our patients and their histopathologic characteristics, clinical presentations, and responses to therapy received. Thus, we performed genomic array on 10 BL patients in all ages, 5 DLBCL or HGBCL patients with CD10 or BCL6 positive and BCL2 negative under 60 years old (“Burkitt-like” group), and 4 DLBCL or HGBCL patients with CD10 or BCL6 positive and BCL2 positive under 60 years old. No typical 11q gain/loss pattern were found in our patients according to WHO classification at this time point. One patient in the BL group was identified with 11q terminal deletion, which is within the regions identified by prior BLL-11q studies. In addition to 11q aberrations, other findings in the BL group and the DLBCL or HGBCL with BCL2+ group further reflect previous studies on patients with BL and DLBCL. We concluded that the groups of patients are currently too limited for meaningful statistical assessment of the frequency of 11q aberrations, but suggest that 11q aberrations are not highly frequent in our “Burkitt-like” cases. We also observed the limitations on performing genomic array on older FFPE samples. Our future directions include requesting and running more recent samples, using stored RNA for transcriptome, and considering other significant findings in Burkitt-like patients without 11q aberrations.
2022-01-26T23:20:01Z
2021
Thesis
HO_washington_0250O_23701.pdf
http://hdl.handle.net/1773/48153
en_US
CC BY-NC-ND
oai:digital.lib.washington.edu:1773/483962022-04-20T10:47:59Zcom_1773_4888col_1773_19658
DNA Cleavage During Pyroptosis
Almalki, Khawlah
Fink, Susan
Immunology
Thesis (Master's)--University of Washington, 2022
Programmed cell death is a normal process in living organisms that removes damaged or infected cells. DNA fragmentation is an important aspect of some forms of programmed cell death, and is a well-recognized feature of apoptosis. DNA damage is also present during pyroptosis, a form of pro-inflammatory programmed cell death mediated by caspase-1. This enzyme cleaves and activates the pore-forming protein, gasdermin D, leading to cell swelling and plasma membrane rupture. The goal of this thesis was to better understand the mechanism and consequences of DNA damage during pyroptosis. We used Salmonella and lethal toxin as pyroptosis inducers and confirmed the presence of DNA damage. We found that release of the DNA-associated nuclear protein HMGB1 also occurs during pyroptosis. Extracellular calcium, which enters pyroptotic cells through gasdermin D pores, is required for both DNA damage and HMGB1 release from the nucleus. However, gasdermin D pores alone are insufficient to stimulate DNA damage. Lastly, we found that DNA from cells undergoing pyroptosis does not appear to resemble apoptotic DNA fragmentation using agarose gel electrophoresis. Together, these results provide further insight into the process of DNA damage during pyroptosis.
2022-04-19T23:41:07Z
2022-04-19T23:41:07Z
2022
Thesis
Almalki_washington_0250O_23843.pdf
http://hdl.handle.net/1773/48396
en_US
none
oai:digital.lib.washington.edu:1773/484132024-02-21T19:41:55Zcom_1773_4888col_1773_19658
Hedgehog signaling in Joubert syndrome
Gomez, Arianna Ericka
Doherty, Daniel
Molecular biology
Thesis (Ph.D.)--University of Washington, 2022
Joubert syndrome (JS) is a mostly recessive, neurodevelopmental condition diagnosed by the molar tooth sign (MTS) on axial brain imaging. Currently, >40 genes are associated with JS, all of which encode proteins that function in and around the primary cilium, a microtubule-based signaling organelle. The association of >40 cilium-related JS genes with the JS-specific brain malformation suggests a unifying cilium-related disease mechanism across genetic causes; however, the mechanistic details remain unknown. Aberrant Hedgehog (Hh) signaling has been implicated in model systems representing more than half of the JS genes, but specific alterations in the Hh signaling cascade have not been characterized across genetic causes of JS in a single model system. To determine whether a consistent disruption in Hh signaling underlies JS, we evaluated Hh signaling using standard assays for two key steps in a single model system. Using the CRISPR-Cas9 system in hTERT RPE-1 cells, I engineered biallelic frameshift variants in 9 representative JS-associated genes encoding ciliary proteins that localize to each of the major sub-compartments of the cilium: tip, cilium-proper, transition zone, and basal body. Transition zone mutants displayed fewer cilia, and multiple mutants exhibited abnormal cilium length; however, these defects were not uniformly present across mutants. Similarly, relocalization of the key Hh pathway component SMO in response to pathway stimulation was blunted in several mutants across compartments, most severely in the one transition zone mutant that made enough cilia to be tested. Finally, induction of the Hh target gene GLI1 was blunted in cilium-proper and transition zone mutants, but not in tip and basal body mutants. My work indicates that blunted Hh signaling is a key mechanistic feature associated with JS gene dysfunction, potentially identifying a target for pharmacologic treatments. Future work will focus on determining whether JS gene dysfunction impacts the dynamic range, timing, and other aspects of Hh signal transduction, or if we need to look beyond Hh to identify a unifying mechanism underlying JS.
2022-04-19T23:41:32Z
2022
Thesis
Gomez_washington_0250E_23977.pdf
http://hdl.handle.net/1773/48413
en_US
CC BY
oai:digital.lib.washington.edu:1773/486662022-07-16T11:01:26Zcom_1773_4888col_1773_19658
Understanding Ovarian Cancer Risk: Pathogenic TP53 mutation burden in peritoneal fluid is associated with BRCA germline mutation status
Tee, Xin Ray
Risques, Rosana
aging
cancer risk prediction
duplex sequencing
early ovarian cancer detection
peritoneal fluid
somatic mutation
Molecular biology
Genetics
Oncology
Thesis (Master's)--University of Washington, 2022
Individuals with germline BRCA1 and BRCA2 mutations (BRCA carriers) have an increased risk of developing high-grade serous ovarian cancer (HGSOC), the most common and deadliest subtype of ovarian cancer. While HGSOC is initiated by TP53 mutations, these mutations are also now recognized as a common feature of normal human aging. We hypothesize that BRCA carriers harbor increased TP53 mutation burden, underlying their predisposition to HGSOC. We used high-fidelity, high-depth duplex sequencing to analyze mutations in TP53 in Paps and peritoneal fluid of 25 BRCA1 carriers, 21 BRCA2 carriers, and 11 BRCA non-carriers. Mutations were annotated and compared across groups, sample types, and with the TP53 UMD HGSOC database. TP53 coding and pathogenic mutation burden were associated with age in Paps and peritoneal fluid of all individuals. BRCA1 carriers older than 45 years old had a significantly higher pathogenic mutation burden than BRCA2 carriers and controls of the same age in peritoneal fluid, but not in Paps, Peritoneal fluid from BRCA1 carriers older than 45 years old had the most significant enrichment of hotspot TP53 mutations with 36.2% of all mutations identified in hotspot codons compared to 7.7% expected by chance (p = 5x10-8). 2.6% (6 out of 230) of peritoneal fluid TP53 variants were also found in Paps but not in blood. BRCA1 carriers harbor an excess of TP53 pathogenic mutations in peritoneal fluid after age 45, coinciding with the age of increased HGSOC risk. Paps have no resolution to detect differences in TP53 mutation burden due to germline status.
2022-07-14T22:01:57Z
2022
Thesis
Tee_washington_0250O_24366.pdf
http://hdl.handle.net/1773/48666
en_US
Supplementary Figures.pdf; pdf; Supplementary Figures.
Supplementary Tables.xlsx; spreadsheet; Supplementary Tables.
CC BY-NC
oai:digital.lib.washington.edu:1773/486672022-07-16T11:01:27Zcom_1773_4888col_1773_19658
Retrospective Flow Cytometric Data Review of Classic Hodgkin Lymphoma Cases to Explore Disease Outcomes
Tang, Claire
Fromm, Jonathan R
biomarker
classic Hodgkin Lymphoma
flow cytometry
immunophenotype
tumor microenvironment
Oncology
Medicine
Thesis (Master's)--University of Washington, 2022
Flow cytometry is an emerging method in detecting and diagnosing classic Hodgkin Lymphoma (cHL). There is increasing evidence that the tumor microenvironment (TME) plays a significant role in the prognosis and clinical outcome of the disease. In this study, ten different parameters were assessed and correlated to outcome. A retrospective cross-sectional study was performed by analyzing flow cytometric data collected from 62 patients at presentation who were diagnosed with cHL. Correlation was made from TME cellular composition to clinical outcome. The composition of the TME varied between patients who achieved complete remission compared to those who relapsed or whose disease had progressed. The T-cell rosetted fraction of Hodgkin-Reed Sternberg (HRS) cells showed a significant correlation to outcome. However, the %CD20 B-cells, %CD5 T-cells, %HRS cells, percentage of activated B-cells and T-cells characterized by CD71 expression, ratio of CD4+/CD8+ T-cells, %eosinophils, %neutrophils, and eosinophil/neutrophil ratio did not show significant correlation to outcome. This study shows that the fraction of T-cell rosetted Hodgkin-Reed Sternberg cells at presentation may be a biomarker in predicting outcome in cHL.
2022-07-14T22:01:58Z
2022-07-14T22:01:58Z
2022
Thesis
Tang_washington_0250O_24531.pdf
http://hdl.handle.net/1773/48667
en_US
none
oai:digital.lib.washington.edu:1773/495582023-01-21T11:25:55Zcom_1773_4888col_1773_19658
Characterization of Thiomuscimol: A Novel Small Molecule Pyroptosis Inhibitor
Anderson, Marisa
Fink, Susan L
cell death
innate immunology
macrophage
pyroptosis
salmonella
thiomuscimol
Immunology
Biology
Microbiology
Thesis (Master's)--University of Washington, 2022
Pyroptosis, a form of pro-inflammatory programmed cell death mediated by caspase- 1 and dependent on gasdermin D, is a crucial element of the innate immune response. Pyroptosis arises from activation of sensor proteins which respond to threats to host health, leading to the formation of multiprotein complexes known as inflammasomes that induce production of pro-inflammatory cytokines. The downstream effects of pyroptosis such as cell rupture and the release of inflammatory signals are implicated in the progression of a number of diseases, indicating that having the ability to control pyroptosis has great therapeutic potential. GABAA receptor agonist muscimol inhibits pyroptotic lysis. The goal of this thesis was to better understand the mechanism of the muscimol analog thiomuscimol, which inhibits pyroptosis in a unique manner. We used Salmonella typhimurium and anthrax lethal toxin as pyroptosis inducers, and we identified the target concentrations of thiomuscimol and that the effects of thiomuscimol were not due to cytotoxicity nor were boosted by a pretreatment. Then we determined that thiomuscimol binds in a reversible manner, and that it inhibits caspase-3 in addition to caspase-1.Finally, we determined that thiomuscimol is capable of both diffuse active caspase-1 inhibition as well as foci-bound caspase-1 inhibition. Together, these results provide insight into thiomuscimol’s mechanism, contributing to the understanding of small molecule pyroptosis inhibition.
2023-01-21T05:00:32Z
2022
Thesis
Anderson_washington_0250O_25049.pdf
http://hdl.handle.net/1773/49558
en_US
none
oai:digital.lib.washington.edu:1773/500742023-08-16T11:47:44Zcom_1773_4888col_1773_19658
Highly Saturated Transposon Sequencing identifies genes impacting Staphylococcus aureus pathogenesis in macrophages
Lo, HsinYu
Salipante, Stephen J.
cystic fibrosis
facultatively intracellular pathogens
genomics
Staphylococcus aureus
Tn-Seq
transposons
Microbiology
Molecular biology
Genetics
Thesis (Master's)--University of Washington, 2023
Staphylococcus aureus is a facultative intracellular pathogen in many host cell types,facilitating its persistence in chronic infections. The genes contributing to intracellular
pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus
invasion and survival within human macrophages using two laboratory strains (ATCC2913 and
JE2). We developed an in vitro transposition method to produce saturated transposon mutant
libraries in S. aureus, and performed Tn-Seq to identify candidate genes with significantly altered
abundance following macrophage invasion. While some significant genes were strain-specific,
107 were identified in common across both S. aureus strains, with most (n=105) being required
for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally
validate phenotypic contributions for a select subset of genes. Of the 20 genes passing validation,
7 had a previously identified role in S. aureus virulence, and 13 were newly implicated. Validated
genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of de novo mutations occurring in groups (n=237)
of clonally-related S. aureus isolates from the airways of chronically infected individuals with
cystic fibrosis (CF) revealed significantly greater rates of in vivo selection in candidate genes than
factors not associated with macrophage invasion. This study implicates a core set of genes
necessary to support macrophage invasion by S. aureus, highlights strain-specific differences in
phenotypic effects of effector genes, and provides evidence for selection of candidate genes
identified by Tn-Seq analyses during chronic airway infection in CF patients in vivo.
2023-08-14T17:00:33Z
2023
Thesis
Lo_washington_0250O_25306.pdf
http://hdl.handle.net/1773/50074
en_US
Supp Table 1 primer_gblockb.xlsx; spreadsheet; .
Supp Table 2 time0_test_ATCC_SS.xlsx; spreadsheet; .
Supp Table 3 time0_test_JE2 copy.xlsx; spreadsheet; .
Supp Table 4 atcc_je2_overlap 105 genes.xlsx; spreadsheet; .
Supp Table 5 control_test_ATCCb.xlsx; spreadsheet; .
Supp Table 6 control_test_JE2.xlsx; spreadsheet; .
Supp Table 7 Crisrpri.xlsx; spreadsheet; .
Supp Table 8 ATCC in vivo.xlsx; spreadsheet; .
Supp Table 9 JE2 in vivo.xlsx; spreadsheet; .
none
oai:digital.lib.washington.edu:1773/506292023-09-28T10:39:35Zcom_1773_4888col_1773_19658
A simple rapid flow cytometry assay to assess malaria vaccine responses in mice
Shankar Raman, Shruthi
Murphy, Sean Dr.
Shears, Melanie Dr.
Microbiology
Thesis (Master's)--University of Washington, 2023
Malaria is a global threat affecting nearly half of the world's population. Efforts to control the disease include vector control, accurate testing, and post-infection treatment. Despite substantial progress in these control measures, they do not provide comprehensive protection against malaria. This leads to the need for a more effective control measure, and a malaria vaccine would indeed be helpful as a public health tool. Thus, in the Murphy Laboratory, we are on the hunt for a more effective second-generation pre-erythrocytic malaria vaccine that induces a strong CD8 T cell response and offers long term protection against subsequent malarial infections. In this thesis, we discuss in detail, a surrogate marker assay: the upregulation of CD11a / downregulation of CD8a on CD8 T cells post antigenic experience. This assay serves as a valuable tool to measure vaccine responses in mice, enabling us to evaluate the efficacy of novel vaccines developed in our laboratory. By examining the specific changes in the expression of CD11a and CD8a on CD8 T cells following exposure to antigens, we gain insights into the immune response stimulated by the vaccines.
2023-09-27T17:16:47Z
2023
Thesis
ShankarRaman_washington_0250O_25959.pdf
http://hdl.handle.net/1773/50629
en_US
none
oai:digital.lib.washington.edu:1773/510522024-02-13T11:21:11Zcom_1773_4888col_1773_19658
HIV evolution during ART failures revealed by using long-read sequencing and bioinformatics tools
WANG, SHIYI
Torbett, Bruce E
Antiviral Drug Resistance
Combination Antiretroviral Therapy
Computational Biology
HIV
Nanopore Sequencing
Virology
Bioinformatics
Thesis (Ph.D.)--University of Washington, 2023
HIV resistance often leads to antiretroviral therapy (ART) failures, involving two crucial mutation categories: drug-resistance mutations (DRMs) and compensatory mutations. DRMs reside in HIV enzyme active sites (protease, reverse transcriptase, and integrase), hindering drug binding, while compensatory mutations restore enzyme stability and function, compensating for DRMs. With the increase of drug potency, more compensatory mutations are involved in compensating for one DRM, forming complex mutational patterns. However, the interplay between DRMs and compensatory mutations remains elusive. In this thesis work, I combined a long-read sequencing approach and bioinformatic tools to unveil the complex mutational patterns driving HIV resistance development. Long-read sequencing yielded 4.5kb gag-pol sequences from individual HIV genomes within clinical serum samples, preserving co-varying mutations critical for pattern identification. Mutational patterns were inferred based on pairwise correlations detected in the sequencing data and quantified using a custom bioinformatic tool. I utilized Hamming-distance-based phylogenetic analysis (HDBPA) and paired post-ART HIVs with their pre-ART most recent common ancestors (MRCAs) based on sequence similarity. In this way, I divided mutations in mutational patterns into different categories (mutations inherited from pre-ART MRCA, and mutations acquired during ART) and revealed the order of mutation development. I demonstrated the utility of this approach by studying the HIV evolution in two PWHs facing ART failures. The findings revealed different mutational patterns selected and enriched during ART and inferred evolutionary pathways taken by HIVs during resistance development.
Alongside substitution mutation involved in HIV evolution, I participated in a collaborative study, aiming to measure linkage disequilibrium between recombination events and SNVs. The findings revealed novel correlations between p6Gag insertions and Gag cleavage site mutations in drug-resistant HIV genomes.
Taken together, my work deciphered mutational patterns and recombination events driving HIV evolution during ART using long-read sequencing and custom bioinformatics tools. The findings of this study indicated interactions both within HIV proteins and among proteins, which could guide anti-viral drug design. The methods introduced could be used for identifying complex mutational patterns required for resistance development and revealing the order of mutation development in HIV as well as other fast-evolving viruses and bacteria.
2024-02-12T23:37:57Z
2023
Thesis
WANG_washington_0250E_26304.pdf
http://hdl.handle.net/1773/51052
en_US
CC BY-ND
oai:digital.lib.washington.edu:1773/510532024-02-13T11:21:11Zcom_1773_4888col_1773_19658
The Relationship Between Neurosteroid Structures and Inhibition of Pyroptotic Lysis
Huston, Hanna Clare
Fink, Susan L.
Inflammation
Macrophage
Neurosteroid
Pyroptosis
Immunology
Medicine
Biochemistry
Thesis (Master's)--University of Washington, 2023
Pyroptosis is a form of programmed cell death with inflammatory biologicalconsequences. In moderation, pyroptosis helps to regulate and maintain a healthy immune
response in the presence of invading pathogens. However, excessive, or inappropriate
pyroptosis causes harmful consequences including inflammation and damage to tissues and
organs. There are several steps within the pyroptotic cascade that result in lysis and
release of cellular components implicated in harm to healthy cells and tissues. Previously
identified small molecule inhibitors, like the amino acid glycine, have been shown to inhibit
pyroptosis-mediated cell lysis, in vitro. Glycine is an ion channel modulator and
examination of other ion channel modulators led to the discovery that pregnenolone sulfate
also inhibits cell lysis in cells undergoing pyroptosis. Pregnenolone sulfate is a more potent
inhibitor than glycine, but the mechanism of action for the protection is unknown. To begin
to fill the gap in knowledge of how pregnenolone sulfate, and other small molecules, may be
interacting with pyroptotic cells to prevent membrane lysis, we tested a rationally selected
library of neurosteroids with similar structures to pregnenolone sulfate for inhibitive
activity. We found some neurosteroids retain the same level of activity in comparison to
pregnenolone sulfate, but others exhibit a partial or total loss in activity. No single aspect of
the structure was responsible for inhibitive activity; different parts of the molecule
contribute to inhibition of pyroptosis. Together, the results of this thesis lay the foundation
for a deeper knowledge of what mechanisms may provide protection and how we can design
future inhibitors with these mechanisms in mind.
2024-02-12T23:37:57Z
2024-02-12T23:37:57Z
2023
Thesis
Huston_washington_0250O_26396.pdf
http://hdl.handle.net/1773/51053
en_US
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