acase@tulane.edu

Asthma, current detection methods and metabolites proposed as asthma markers are described. The limitation of the disease diagnosis is outlined and metabolomics is introduced as the approach carried out within this research with the potential to measure the group metabolites that characterise the metabolic responses of a biological system to a specific disease. Chemistry underlying breathing, current breath collection and analytical techniques are described as well as detection and data processing technology associated within our research. A work-flow for the collection, analysis and processing of exhaled breath samples in respiratory diseases is described. The non-invasive sampling method allows collection of exhaled breath samples on children and adults without experiencing any discomfort. The analysis of exhaled breath samples using thermal desorption gas chromatography mass spectrometry outlines the use of retention index for the alignment of VOCs retention time shifting over time. This methodology enables the creation of a breath matrix for multivariate analysis data processing where each VOC is defined by retention index and most intense fragments of the mass spectrum. This methodology is tested in two cohorts of participants: paediatric asthma and severe asthmatic participants whose breath profiles are compared against healthy controls and within the two asthmatic phenotypes to prospect the markers that differentiate between the different groups. Eight candidate markers are identified to discriminate between asthmatic children and healthy children and seven markers between asthmatics undergoing therapy and healthy controls. The database from severe and paediatric asthma is compared, establishing seven non-age related markers between the two groups. A new interface is developed for the faster analysis of exhaled breath samples using thermal desorption ion mobility mass spectrometry. The interface front end has been modified and optimised to achieve the best sensitivity and resolution of VOCs in exhaled breath. A preliminary study carried out in a small cohort of volunteers shows the feasibility of the technique for the differentiation of asthmatic and healthy adults.

Angesichts der nach wie vor hohen Mortalität und Morbidität des Bronchialkarzinoms ist die Entwicklung geeigneter Methoden zur früheren Diagnostik eine wichtige Notwendigkeit, um die geringe durchschnittliche 5-Jahres-Überlebensrate von 15% – 18% zu steigern. Unter diesem Gesichtspunkt wurde in der vorliegenden Arbeit das Atemkondensat von Patienten mit Bronchialkarzinom als nicht-invasiv und kostengünstig zu gewinnendes Medium auf das Vorliegen eines potentiellen Screeningmarkers – dem methylierten Tumorsuppressor-Gen p16 – untersucht. Dazu wurde ein Versuchsablauf entwickelt, bei dem trotz des geringen DNA-Gehaltes im Atemkondensat p16-Methylierungen nachgewiesen werden konnten. Die letztendlich etablierte Methode war eine methylierungsspezifische nested-PCR mit anschließendem Restriktionsverdau durch das Restriktionsenzym BstUI. Des Weiteren erfolgte die Untersuchung von in Paraffin eingebetteten Tumorpräparaten der Patienten. In der anschließenden statistischen Auswertung wurde der Einfluss von verschiedenen Faktoren wie COPD-Grad, Tumorlage, Tumorart, Nikotinabusus und stattgehabte Chemo- oder Strahlentherapie auf den Methylierungsstatus des p16-Gens analysiert.

Exhaled breath condensate (EBC) is a non-invasive method of investigating airway inflammation associated with nitric oxide (NO) and the metabolites nitrite/nitrates (NOx) in diseases such as chronic obstructive pulmonary disease (COPD), but some of the variables affecting the results are unknown. It was hypothesised that 1) EBC would be influenced by lung volumes and the type of EBC collection device; 2) fractional exhaled NO (FENO) and EBC NOx in COPD patients would be altered by smoking and glucocorticosteroids (GCS); 3) cigarette smoke could contribute to the EBC NOx concentration while it may also decrease FENO indirectly by converting airway NO to NOx. It was found that EBC volume was significantly correlated with both tidal volume and minute volume. Comparing four EBC collection devices demonstrated greater efficiency with the ECoScreen?? than siliconised glass tubes or RTube?? but it gave factitiously high NOx levels. Total EBC protein levels over a 10-minute collection were significantly higher using the ECoScreen?? than either glass or RTube?? devices. A cross-sectional study of 96 COPD patients and 80 age-matched control subjects demonstrated that FENO levels in COPD patients were significantly higher than normal subjects when comparing either the combined groups or appropriate two subgroups: ex-smokers and smokers. GCS treatment demonstrated no significant effect on either FENO levels or EBC NOx, but EBC NOx was elevated in smokers. In vitro, cigarette smoke extract (CSE) induced significantly higher NOx and asymmetric dimethylarginine (ADMA) levels in A549 cells when compared with control media. The anti-oxidant, NAC pre-treatment partially reversed the elevated NOx levels but not the ADMA levels. This thesis is the first to report FENO and EBC NOx in COPD patients in an appropriate sample size to be able to evaluate each subgroup, and the increased EBC NOx levels found in smokers in vivo was consistent with the elevated NOx level in response to CSE observed in vitro. These data indicate that smoking-related airway inflammation and activation of the NO pathway are complex with both an increase in ADMA, NO, NOx and may be regulated by oxidative stress rather than the nitric oxide synthase (NOS) pathway.

<p>Two thirds of patients with diabetes avoid regularly monitoring their blood glucose levels because of the painful and invasive nature of current blood glucose detection. As an alternative to blood sample collection, exhaled breath condensate (EBC) has emerged as a promising non-invasive sample from which to monitor glucose levels. However, the inconsistency in the methods used to collect EBC significantly impacts the reliability of reported analyte concentrations in EBC. Furthermore, this dilute sample matrix requires a highly sensitive glucose biosensor to enable robust and accurate glucose detection at the point-of-care. Together, a reliable collection method and sensitive detection system can enable accurate modeling of glucose transport from blood to breath that is reflective of airway glucose homeostasis.</p> <p> I address this research gap by simultaneously designing a standardized EBC collection method that allows for separation of dead space and alveolar air and developing a competitive fluorescent biosensor that can resolve micromolar glucose concentrations changes. First, I develop a low-cost, automated condenser that selectively collects exhaled breath that has been exchanged with lung fluid based on the detection of higher breath temperatures that are characteristic of the lower respiratory regions. Using this device, I investigate the relationship between blood and EBC glucose in diabetic and normoglycemic human subjects. Next, I engineer the exquisitely sensitive <i>E. coli</i> glucose binding protein (GBP) with a chemo-enzymatic tag to selectively conjugate it to highly photostable quantum dots (QDs). Finally, I take advantage of the competitive binding of glucose (K_D=0.35 µM) and galactose (K_D=1.4 µM) to GBP to develop a fluorescent glucose biosensor using the GBP-QD conjugate.</p>

The aim of this study was to evaluate the relationship of oxidative stress (OS) marker s in exhaled breath condensate (EBC) in adult patients with cystic fibrosis (CF) to the severity of lung disease, nutritional status and systemic antioxidants and inflammatory markers, as well as to short - and medium - term development of pulmonary function and nutritional status, and finally to assess the response to treatment with inhaled corticosteroids (ICS). Methods: CF patients were examined in a stable phase of the disease during routine outpatient controls. EBC was collected using E C oScreen device (J aeger) in CF patients and in control group members. Nitrites and nitrates and 8 - isoprostane were examined using liquid chromatography and competitive enzyme immunoanalysis, respectively, in EBC as OS markers. Demographic data including the dominant pathogen of airway colonization and ICS treatment were recorded in CF patients. Lung function tests, chest X-ray s, nutritional statuses and systemic antioxidants and inflammatory markers were also examined using standard methods. The values of OS markers in EBC in patients with CF were compared with the control group and correlated to clinical parameters. Lung function tests and nutritional status es in CF patients were examined in one, three and five years intervals...

The ultimate goal of this research is to develop an efficient, reproducible and low cost method for analysis of VOCs in complex mixtures such as those in exhaled breath and in headspace of fungi cultures. In Chapter I; analytical methods for volatile biomarkers identification is reviewed In Chapter II, active SPME GCMS was employed to analyze VOCs in the breath of a single healthy male and a single female. The goal was to determine the extent of intra-individual variations in the VOC profiles. In Chapter III, a preliminary study was carried out in a greenhouse to determine the pathogenicity of different isolates of M. phaseolina on soybeans. This will allow, in future studies, the matching of VOC profiles of different isolates of M. phaseolina with their relative pathogenicity. This is a key step towards the development of an early warning system for the detection of pathogenic M. phaseolina fungus contaminations.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13579号 / 論医博第2301号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 古川 壽亮, 教授 大鶴 繁, 教授 平井 豊博 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

My thesis installation emerged from an interest in visualizing breath. The resulting work came to exist at the intersection between art, biology, and performance. The unicorn tapestries were used as a generative point of departure to explore the preservation and transformation of images through time, by time, and with time. Reproductions of the six tapestries were each etched into paper and then submerged into solutions of Phenol Red dye, Ferric Ferrocyanide (also known as Prussian Blue), and various forms of sodium chloride. Exhaled breath was used to encrust these images of the tapestries into physical objects which gradually crystallized and changed colors in response to viewers within the surrounding space. An invasive plant species native to Richmond, Virginia was utilized as a catalyst to re-absorb Carbonic acid from exhaled breath and convert the objects back to their original (or not so original) states. The final piece became the active remnants from an inquiry which quickly snowballed into so much more than I had initially planned. The following text is written as a supplemental reading to trace some of my influences at the periphery of the piece while mapping my thought processes.

Exercise has become a vital component of the therapy regimen prescribed to cystic fibrosis (CF) patients due to its systemic benefits, such as increased sputum expectoration, attenuation of the expected 2-3% annual decline in pulmonary function, and extended life expectancy. However, exercise still is not viewed as being as beneficial as pharmacological treatments by many CF patients and can be intimidating. My aims in this study were two-fold; first, to determine the ideal exercise intensity for individuals with CF; and second, to determine if exercise at this ideal intensity could provide improvements in ion regulation in the lungs, which was measured using exhaled breath condensate (EBC) collection and nasal potential difference (NPD), that were comparable to one of their standard pharmacological therapies, albuterol. I hypothesized that with moderate intensity exercise, Na⁺ absorption would decrease from baseline due to Na⁺ channel inhibition, rather than increase or remain unchanged, as was expected with albuterol, and cause an even greater increase Cl- secretion compared to albuterol due to activation of both CF-dependent and independent Cl- efflux with exercise. CF (n=14) and healthy (n=16) subjects completed three visits, a baseline screening and two treatment visits. I collected EBC at baseline, 30- and 60-minutes post-albuterol administration on one visit, and at baseline and during three separate 15 min exercise bouts at low, moderate and high intensity on the other visit. Following the EBC collection, NPD was performed at 30- and 80-minutes post albuterol or following moderate and high intensity exercise. We also measured spirometry and diffusing capacity of the lungs for nitric oxide (DLNO) during each visit at the various time points. In CF subjects, moderate intensity exercise resulted in greater improvements in DLNO (39 ± 29vs.15 ± 22% change from baseline, exercise vs. albuterol respectively), similar levels of bronchodilation compared to 60-minutes post-albuterol administration, no change in Na⁺ absorption, and a four-fold increase in Cl- secretion. Our results suggest that moderate intensity exercise is the best dose for CF patients, and can provide comparable changes as its pharmacological counterpart albuterol, when compared over a short term duration.