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dc.contributor.advisorSoper, Prof. Steven A
dc.contributor.authorWijerathne, Harshani Nayomi
dc.date.accessioned2020-03-29T18:02:42Z
dc.date.available2020-03-29T18:02:42Z
dc.date.issued2019-08-31
dc.date.submitted2019
dc.identifier.otherhttp://dissertations.umi.com/ku:16763
dc.identifier.urihttp://hdl.handle.net/1808/30228
dc.description.abstractA major drawback of currently available stroke diagnosis methods, such as computed tomography (CT) and magnetic resonance (MRI), is that they cannot provide timely diagnosis within the narrow therapeutic time window of 4.5 h from stroke onset afforded by recombi-nant tissue plasminogen activator treatment. Upon initiation of a stroke event, CD15+ neu-trophils and CD8+ T cells are recruited and activated in response to the inflammatory stroke event and can release into blood extracellular vesicles (EVs) containing mRNA markers with altered expression profiles indicative of tissue damage. Our previous studies demonstrated that certain leukocyte subpopulations and gene expression profiling of these isolated sub-populations could be used to diagnose acute ischemic stroke (AIS) within 3 h. Here, our re-search goal was to develop a novel approach for the measurement of mRNA transcripts in EVs rather than cells as a possible diagnostic for AIS. To facilitate the development of the AIS diagnostic based on EVs, we developed a microfluidic device with a high-density array of antibody-modified micropillars for the affinity selection of CD8+ or CD15+ EVs with an analysis time less than the 4.5 h recombinant tissue plasminogen activator effective therapeu-tic time window. We successfully developed a microfluidic device with a high-density array of anti-body-modified micropillars for the affinity selection of CD8+ EVs, which could process 200 µL of plasma in 90%. Initial validation of these devices was per-formed using a model cell line Molt-3, which contained CD8+ T-cells. With the aid of fluo-rescence microscopy, we demonstrated that EVs can be affinity selected using the microflu-idic device with higher specificity compared to other EV isolation techniques, such as ul-trancentrifugation or PEG-precipitation that can improve the quality of the mRNA expression data. Transmission Electron Microscopy (TEM) and Nano Particle Tracking Analysis (NTA) revealed that the microfluidic device was capable of capturing and releasing enriched EVs with a short analysis time (<25 min). Gene expression analysis performed via droplet digital PCR revealed that for AIS, the genes we selected (PLBD1, MMP9, VCAN, FOS, CA4) pro-duce similar expression between the CD8+ T cells and EVs originating from these cells. The analysis of clinical samples, which used a 7-bed microfluidic device with 10 µm pillars and an interpillar spacing of 10 µm provided a higher dynamic range compared to a 3-bed device that used larger pillars (~90 µm) as well as significantly reduced processing time. In a blinded study performed for healthy and AIS patient samples, we were able to correctly identify 4/5 stroke patient samples and 4/5 healthy control samples. Although results reported here are very encouraging, more extensive studies are needed with a larger cohort of patient samples and healthy controls to clearly determine receiver operating characteristics for the use of EVs as a source of mRNA for AIS diagnosis. The research work I conducted on identification of mutations stabilizing Bacterioferritin associated ferredoxin is included in Appendix.
dc.format.extent246 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsCopyright held by the author.
dc.subjectAnalytical chemistry
dc.subjectBiochemistry
dc.subjectDroplet digital PCR
dc.subjectExtracellular vesicles
dc.subjectIschemic stroke
dc.subjectMicrofluidics
dc.subjectNanoparticle tracking analysis
dc.subjectTransmission Electron Microscopy
dc.titleAnalyzing Extracellular Vesicles as Potential Biomarkers of Stroke Using Polymer Microfluidic Devices
dc.typeDissertation
dc.contributor.cmtememberLunte, Prof. Susan
dc.contributor.cmtememberDunn, Prof. Robert
dc.contributor.cmtememberBenson, Prof. David
dc.contributor.cmtememberFriss, Prof. Liza
dc.thesis.degreeDisciplineChemistry
dc.thesis.degreeLevelPh.D.
dc.identifier.orcidhttps://orcid.org/0000-0002-9921-0731
dc.rights.accessrightsopenAccess


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