Browsing by Subject "Activation"
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Item Characterization of Natural Killer Cell Activation and Functionality for Cell Therapy Applications(2020-12) One, JenniferNatural killer (NK) cells are a promising emerging allogeneic cell therapy due to their cytotoxic effector and cytokine producing functions and lack of induction of Graft-vs-Host Disease. For allogeneic clinical applications, NK cells isolated from a single donor must be expanded through an efficient large-scale biomanufacturing process to treat many patients and produce an economical off-the-shelf therapy. Critically, the cultured NK cells must maintain functionality post-expansion to be an effective cellular therapy. Through multiple rounds of activation with K562 artificial antigen-presenting cells (aAPCs), the quantity of NK cells can expand by several orders of magnitude. However, the changes that chronic stimulation might induce in cell cycle status, metabolism, and ex vivo functionality of NK cells are not well understood. Even less is known regarding how changes in these cell characteristics may influence their in vivo functionality. In this work, we conducted a systematic evaluation of the activation and expansion process of NK cells through transcriptome analysis, dynamics of chromatin accessibility, metabolic characterization, and phenotypic analysis of exhaustion and senescence over time. By understanding the transcriptional and epigenetic signature of the K562-activated NK cells, we have identified potential genes and transcription factors that may regulate K562 activation in order to develop a bioprocess that phases out these feeder cells from the culture process. Furthermore, rigorous characterization of NK cell growth and receptor expression during expansion revealed that prolonged stimulation results in an immature, exhausted, cytokine-producing phenotype over time. Changes in NK growth kinetics corresponds to shifts in NK cytotoxicity in select target cancer cell lines, indicating proliferative potential may be an indicator of a good donor for clinical use. Collectively, understanding the effects of activation and consequent proliferation of NK cells would remove a major roadblock in the biomanufacturing of NK cells, thus laying the groundwork for their potential use as an off-the-shelf allogeneic cellular therapy. Insights into mechanisms underlying activation and expansion provide a path to develop strategies to eliminate K562 aAPCs altogether, which would be desirable from a regulatory standpoint and further ease the transition from benchtop to biomanufacturing as well as improve efficacy in the clinic.Item Quantitation of the metabolic activation of phenanthrene in smokers:potential use in the assessment of lung cancer susceptibility(2012-06) Wang, JingPolycyclic aromatic hydrocarbons (PAHs) in cigarette smoke are among the most likely causes of lung cancer. PAHs require metabolic activation to initiate the carcinogenic process. Phenanthrene (Phe), a non-carcinogenic PAH, was used as a surrogate of benzo[α]pyrene (BaP) and related PAHs to study the metabolic activation of PAHs in smokers. A dose of 10 μg deuterated Phe ([D10]Phe) was administered to 25 healthy smokers in a crossover design, either as an oral solution or by smoking cigarettes containing [D10]Phe. Intensive blood and urine sampling was performed to quantitate the formation of deuterated r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene ([D10]PheT), a biomarker of the diol epoxide metabolic activation pathway. In both the oral and smoking arms an average of approximately 6% of the dose was metabolically converted to diol epoxides, with a large inter-subject variability in the formation of [D10]PheT observed. Two diagnostic plots were developed to identify subjects with large systemic exposure and significant lung contribution to metabolic activation, respectively. The combination of the two plots led to the identification of subjects with substantial local exposure. These subjects produced, in one single pass of [D10]Phe through the lung, a [D10]PheT exposure equivalent to the systemic exposure of a typical subject, which may be an indicator of lung cancer susceptibility. Polymorphisms in PAH metabolizing genes of the 25 subjects were also investigated. The integration of phenotyping and genotyping results indicated that GSTM1 null subjects produced approximately 2-fold more [D10]PheT than did GSTM1 positive subjects. A population pharmacokinetic analysis was also tried as an alternative approach to identify subjects with extensive activation, but the analysis was not completely successful. The preliminary statistical analysis indicated a correlation between renal function and an individual’s capacity to activate PAHs, which may deserve further investigation. A simulation project based on the pharmacokinetic data of 25 subjects was carried out to simulate the metabolic activation of [D10]Phe in 350 subjects after oral dosing and smoking. The simulation suggested that the collection of a 6-hr urine sample in both treatment arms should be conducted in the future large-scale trials in order to efficiently assess the hypothesized correlation between extensive activation and high lung cancer risk.Item Reactions of copper complexes with dioxygen and oxo transfer reagents: toward elusive copper-oxyl species.(2010-06) Hong, SungjunThe binding and activation of dioxygen by Cu ions is central to the function of numerous biological systems. Among the enzymes activate dioxygen for the functionalization of organic substrates, those catalyzed by the mononuclear copper enzymes dopamine β-monooxygenase (DβM) and peptidylglycine α-hydroxylating monooxygenase (PHM) are less understood. Despite extensive research on these enzymes, the exact nature of the active species responsible for substrate functionalization is not resolved, with two provocative proposals involving either a CuII-superoxo or a mononuclear CuII-oxyl species having been put forth. The goal of this research is to understand the reaction catalyzed by the PHM and DβM enzymes on a fundamental chemical level via a small molecule synthetic model approach, with particular emphasis on generating and/or characterizing a Cu-oxygen species that is capable of performing similar reactions to those seen in the DβM and PHM enzymes. Chapter 1 contains a general overview of dioxygen activation in biological systems and gives a review of the structure and proposed catalytic mechanisms of DβM and PHM, followed by a summary of recent synthetic efforts toward mononuclear Cu/O2 adducts and Cu-oxyl species. Chapter 2 describes the synthesis and characterization of the copper(I) complexes of the electron-deficient β-diketiminate and analogous 4- nitroformazan supporting ligands, and their O2-reactivity studies, portions of which have been previously reported.1 Chapter 3 describes a bio-inspired synthetic route toward a mononuclear Cu-oxyl species that involves decarboxylation of copper(I)-α- ketocarboxylate complexes by dioxygen; portions of the work have been communicated previously.2 Chapter 4 then describes results obtained from reactions of copper(I) complexes of bidentate N-donor ligands with pyridine- and trimethylamine N-oxides or PhIO. Portions of this work were previously reported.3