CLINICAL APPLICATIONS OF CHEMILUMINESCENCE DETECTION

This dissertation evaluates the potential of chemiluminescence analysis for two types of clinical applications: (1) rapid analysis with a flow injection system which consumes small reagent volumes (25 (mu)L) and (2) a new approach to chemiluminescence immunoassay. The first application studied uses the firefly reaction with a flow system to analyze for adenosine triphosphate (ATP). Two flow configurations called the valve within a valve and merging zones configurations were evaluated with respect to effect of parameters such as flow and sample volume on precision, sensitivity and sample throughput. With the appropriate choice of parameters, both systems achieve precisions of 1-2% relative standard deviation and throughputs of 5-10 measurements per minute. The merging zones configuration consumes smaller volumes of sample. The second project demonstrates a new approach to immunoassay based on chemical excitation of fluorophor labelled immunochemicals using the peroxyoxalate reaction. The effects of fluorophors, esters, and solvents, etc. on the chemiluminescence measurement were evaluated. The conditions for achieving the best precision (1-3% R.S.D.) with a high water component in the measurement were rhodamine B in Tris buffer adjusted to pH 8.00, 10(\u27-3) M bis(2,4,6-trichlorophenyl) oxalate (TCPO) in ethyl acetate and 10(\u27-3) M hydrogen peroxide in acetonitrile in the ratio 5:2:3. Under these conditions, the detection limit for rhodamine B is 10(\u27-9) M. The detection limit is established by variations in the background chemiluminescence. The possibility of both homogeneous and heterogeneous immunoassays was evaluated. It was shown that the chemiluminescence excitation efficiency decreases when fluorophors were bound to albumin but increases when the fluorophor was coupled to folic acid. The feasibility of a homogeneous immunoassay was demonstrated by binding albumin to rhodamine labelled antiserum. It was shown that the chemiluminescence intensity of the fluorophor labelled antibody decreases with increasing concentrations of bound antigen. The heterogeneous chemiluminescence immunoassay was evaluated by comparison with a commercially available fluorescence immunoassay kit by Bio Rad. Both immunoassays are limited by imprecision of mixing of the solvent systems for the chemiluminescence measurement

5’-UTR STRUCTURE AND IMPLICATIONS IN GENE REGULATION

The SERPINA1 gene encodes the protease inhibitor α-1-antitrypsin. Translation of SERPINA1 mRNAs is regulated, in part, through structural features in the 5'-untranslated region (5'-UTR). Compact hairpins in the 5'-UTR of human mRNAs are known to regulate translation; however, few studies have explored the influences of larger, complex structures on gene expression. The work presented in this thesis comprehensively examines the role of large-scale RNA structure in the 5'-UTR of the most highly transcribed isoform of SERPINA1, NM_000295. The native 5'-UTR forms a highly-structured, complex, three-helix domain, called Stem 1, with limited base pairing to the coding region. I systematically and comprehensively destabilized structure at nearly every nucleotide of the 5'-UTR of NM_000295 using a series of 42 six-nucleotide, spatially distinct mutants and measured the RNA structure and translation of each. A majority of mutants recapitulated native structure, however, mutations that disrupted Stem 1 created three non-native structure groups. Two non-native groups refolded to introduce a stable helix near the translation initiation site and decreased translation. Counterintuitively, stable, complex internal folding in Stem 1 sequesters RNA sequences to minimize structure at the translation start site and promote ribosome accessibility for optimized translation. Next, I characterized how Stem 1 impacts translation by introducing a series of point mutations in the three-helix junction of Stem 1. I discovered that translation effects could be predicted based on simple considerations of stability of the Stem 1 structure. Collectively, the data presented in this thesis indicate the highly nuanced role that complex, large-scale RNA structure plays a critical function in translation regulation in NM_000295 and that these motifs are finely tuned to optimize translation.Doctor of Philosoph

The effects of rare SERPINA1 variants on lung function and emphysema in SPIROMICS

Rationale: The role of PI (protease inhibitor) type Z heterozygotes and additional rare variant genotypes in the gene encoding alpha-1 antitrypsin, SERPINA1 (serpin peptidase inhibitor, clade A,member 1), in determining chronic obstructive pulmonary disease risk and severity is controversial. Objectives: To comprehensively evaluate the effects of rare SERPINA1 variants on lung function and emphysema phenotypes in subjects with significant tobacco smoke exposure using deep gene resequencing and alpha-1 antitrypsin concentrations. Methods: DNA samples from 1,693 non-Hispanic white individuals, 385 African Americans, and 90 Hispanics with >20 pack-years smoking were resequenced for the identification of rare variants (allele frequency,0.05) in 16.9 kB of SERPINA1. Measurements and Main Results: White PI Z heterozygotes confirmed by sequencing (MZ; n = 74) had lower postbronchodilator FEV1 (P = 0.007), FEV1/FVC (P = 0.003), and greater computed tomography-based emphysema (P = 0.02) compared with 1,411 white individuals without PI Z, S, or additional rare variants denoted as VR. PI Z-containing compound heterozygotes (ZS/ZVR; n = 7) had lower FEV1/FVC (P = 0.02) and forced expiratory flow, midexpiratory phase (P = 0.009). Nineteen white heterozygotes for five non-S/Z coding variants associated with lower alpha-1 antitrypsin had greater computed tomography-based emphysema compared with those without rare variants. In African Americans, a 59 untranslated region insertion (rs568223361) was associated with lower alpha-1 antitrypsin and functional small airway disease (P = 0.007). Conclusions: In this integrative deep sequencing study of SERPINA1 with alpha-1 antitrypsin concentrations in a heavy smoker and chronic obstructive pulmonary disease cohort, we confirmed the effects of PI Z heterozygote and compound heterozygote genotypes. We demonstrate the cumulative effects of multiple SERPINA1 variants on alpha-1 antitrypsin deficiency, lung function, and emphysema, thus significantly increasing the frequency of SERPINA1 variation associated with respiratory disease in at-risk smokers

Peroxyoxalate chemiluminescence detection for the highly sensitive determination of fluorescence-labeled chlorpheniramine with Suzuki coupling reaction.

A sensitive and selective high performance liquid chromatography-peroxyoxalate chemiluminescence (PO-CL) method has been developed for the simultaneous determination of chlorpheniramine (CPA) and monodesmethyl chlorpheniramine (MDCPA) in human serum. The method combines fluorescent labeling with 4-(4,5-diphenyl-1H-imidazole-2-yl)phenyl boronic acid using Suzuki coupling reaction with PO-CL detection. CPA and MDCPA were extracted from human serum by liquid-liquid extraction with n-hexane. Excess labeling reagent, which interfered with trace level determination of analytes, was removed by solid-phase extraction using a C18 cartridge. Separation of derivatives of both analytes was achieved isocratically on a silica column with a mixture of acetonitrile and 60 mM imidazole-HNO(3) buffer (pH 7.2; 85:15, v/v) containing 0.015% triethylamine. The proposed method exhibited a good linearity with a correlation coefficient of 0.999 for CPA and MDCPA within the concentration range of 0.5-100 ng/mL. The limits of detection (S/N = 3) were 0.14 and 0.16 ng/mL for CPA and MDCPA, respectively. Using the proposed method, CPA could be selectively determined in human serum after oral administration

The relationship between the "Great Awakening" and the transition from psalmody to hymnody in the New England colonies

This study examines the relationship between the first major religious revival in the New England colonies and the change from psalmody to hymnody in the mid-eighteenth century through an approach which integrates the two fields of theology and church music. The termination date is 1770, and the focus is Protestant congregational song in the three groups most influenced by Puritan thought: the Congregationalists, the Presbyterians, and the Baptists.While much has been written separately about the change in eighteenth-century sacred song and the Great Awakening itself, there has been little research that attempts to place the psalmody/hymnody issue within the larger context of the changing theological milieu. This study first examines the theological and ecclesiastical structures which provided the context for Reformed worship, and then explores how fundamental changes in those structures and thought systems impacted congregational song. In order to comprehend the major changes which occurred in the mid-eighteenth century in colonial America, chapters on the Reformed Church and the beginning and spread of psalmody, the New England colonies to 1700, and the beginning of English hymnody are included.Conclusions1. The primary conclusion of this study is that the Great Awakening is the single most important factor in the change from psalmody to hymnody in the New England colonies. It is not a peripheral factor as indicated in much of the research. Rather, it provides both the rationale and the means for the transition in church song. The Great Awakening represented a basic theological change from a theocentric to an anthropocentric viewpoint that subsequently required alterations in sacred song. The revival movement, through its evangelistic spirit, also provided the vehicle by which this change in psalmody was effected.2. The agitation of the 1720s as evidenced in the tracts and treatises did not affect the transition directly. However, it is indicative of the increasing discontent with traditional Calvinist theology.3. The Psalms and Hymns of Isaac Watts were not a primary reason for the change, but met the needs of the new anthropocentric theology of the Great Awakening that required a new language of praise.Thesis (D.A.)School of Musi