A 1kb HinfI DNA fragment, containing a repeat DNA sequence element was isolated from a Bordetella pertussis BP348 cosmid gene bank. This repeat DNA sequence has subsequently been named IS148. The insertion sequence was demonstrated by have a high copy number only in B.pertissus and to be absent in Bordetella parapertussis and Bordetella bronchiseptica. Using a restriction fragment of IS148 labelled with radioactivity as a probe in DNA or whole cell dot blots between 10-100 cells could be detected or from 100pb-1ng DNA. Furthermore, a pair of oligonucleotide primers was synthesised corresponding to a central region of IS148 that could generate a 242bp fragment upon PCR amplification. The use of PCR amplification with specific primers allows the detection of 0.5pg of B.pertussis chromosomal DNA and as little as one B.pertussis cell. The prn genes encoding the outer membrane P.70 and P.68 pertactin from B.parapertussis and B.bronchiseptica have been cloned, sequenced and expressed in Escherichia coli. Analysis of the DNA sequences reveal that the genes have open reading frames capable of encoding proteins with molecular weights of 95,177 (P.95, B.parapertussis) and 93,996 (P.94, B.bronchiseptica). These precursors molecular are processed to form the P.70 and P.68 antigens on the surface of B.parapertussis and B.bronchiseptica respectively. Heterologous expression of the full-length gene encoding P.95 and P.94 in E.coli result in similar processing, with the P.70 and P.68 antigens targetted to the bacterial outer membrane. Comparison of P.95, P.94 and P.93, encoding homologous proteins from B.parapertussis, B.bronchiseptica and B.pertussis, shows a high degree (> 90%) of homology. The major differences between all three proteins occur in the number of repeat of the two families (Gly-Gly-Xaa-Xaa-Pro)n and (Pro-Gln-Pro)n of reiterated sequence motifs.

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The studies contained in this dissertation were driven by a desire to improve the methods for sensory testing of cough for clinical research and practice. Two scientific gaps in the cough evaluation literature were identified and investigated using two specific studies on healthy young adult participants. The first study focused on validating an appropriate descriptor for cough sensations (Chapter 2) and the second study (Chapter 3) focused on evaluating magnitude estimation tools to measure cough sensations. The findings of this dissertation make several unique contributions to the cough literature. The first study systematically compared two descriptive responses to cough stimuli (i.e., capsaicin) within subjects in terms of both cough sensory and cough motor outcomes. Findings revealed two types of descriptive responses for capsaicin stimuli, warm/burn and urge-to-cough (UTC). The UTC descriptor was, however, more sensitive and a valid predictor of cough response. The second study systematically compared two magnitude estimation tools, the Modified Borg Scale (MBS) and the generalized Labeled Magnitude Scale (gLMS) to measure the UTC sensations. Findings revealed that both tools were reliable and valid in detecting UTC sensations and predicting cough response. However, a differential effect to detection of UTC sensations across neighboring stimuli concentrations were demonstrated by the two tools. This dissertation provides the first set of normative reference values for UTC responses across a wide range of sensory continua using the conventional metric, the MBS, and an additional metric, the gLMS. Limitations are acknowledged and future work is suggested.

Rationale: Methacholine and mannitol challenges are used clinically to assess airway hyperresponsiveness (AHR). Cough during (a) high-dose methacholine challenge in individuals with methacholine-induced cough and normal airway sensitivity and (b) mannitol challenge in some individuals with asthma both occur in the absence of significant declines in forced expiratory volume in one second (FEV1). We hypothesized mechanical responses to these challenges would reflect a continuum amongst subjects with: (i) asthma; (ii) cough variant asthma (CVA) and (iii) methacholine-induced cough and normal airway sensitivity due to varying degrees of impairment/preservation of the beneficial effects of deep inspirations. Purpose: To compare cough and airway responses to mannitol and high-dose methacholine challenges between these groups. Methods: Individuals with asthma or suspected CVA were invited to participate. Subjects were challenged with mannitol and high-dose methacholine in random order 2-14 days apart. Cough frequency, spirometry and esophageal-pressure were recorded at baseline and after each dose of mannitol and methacholine to a maximal decline in FEV1 of 15% and 50% respectively. Plethysmography was used to measure lung volumes at baseline, the dose nearest to a 15% decline in FEV1 during mannitol challenge (PD15) and 20% decline in FEV1 during methacholine challenge (PC20), and at the highest dose of methacholine. Measurements were compared: (a) between groups at PD15, PC20 and the highest dose of methacholine; and (b) within groups at PD15 and PC20, and the highest equivalent level of bronchoconstriction. Results: 22 subjects (17 female; 48.0±12.7 (mean±SD years)) who completed both challenges were included. All subjects coughed during both challenges. Mechanical responses to mannitol and high-dose methacholine challenges reflected a continuum amongst groups. Six of 8 subjects with asthma were mannitol postitive (PD15=115.2±100.0 mg) and were significantly more sensitive to mannitol compared to 3 of 5 mannitol positive subjects with CVA (PD15=533.6±88.3 mg; p=0.020) and 3 of 9 mannitol positive subjects with methacholine-induced cough and normal airway sensitivity (PD15=472.8±203.0 mg; p=0.037). At the highest equivalent level of bronchoconstriction, methacholine induced significant declines in FEF50% and FEF25-75% in all subjects groups while mannitol did not. Conclusion: Mechanical responses to mannitol and high-dose methacholine challenges reflected a continuum amongst groups. / Thesis (Master, Physiology) -- Queen's University, 2012-07-27 08:58:05.298

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