Chronic obstructive pulmonary disease (COPD), a progressive and nonreversible disease, is a leading cause of mortality and morbidity throughout the world. Detecting COPD early in the disease process will help in decreasing later stage COPD severity. Because airway inflammation is a hallmark of COPD, it has been proposed that measuring exhaled nitric oxide, a marker of inflammation, in exhaled breath condensate could prove to be an inexpensive and efficient method to detect COPD in outpatient settings. Using the hypothetico-deductive theory as a guideline, this study used secondary data from the National Health and Nutrition Examination Survey 2007 to 2010 to test the association between exhaled nitric oxide (eNO), COPD, and COPD severity. In addition, this study explored whether occupation modifies the association between eNO and COPD. Descriptive statistics, chi-square analyses, and regression analyses were used to analyze data from a sample size of 10,214 individuals. The prevalence of COPD was 7.2%, based on self-reported physician diagnoses and 11.4% based on prebronchodilator spirometry analysis, strengthening the argument that COPD is often under- or misdiagnosed in clinical settings. This study found no statistically significant association between eNO, COPD, and COPD severity, and occupational status did not appear to modify the association between eNO and COPD. The findings of this study highlight the importance of using objective measures such as spirometry in clinical settings for early diagnosis and management of COPD. Early diagnosis helps to slow the progression of the disease, resulting in fewer related comorbidities and complications.

Background: Asthma is a heterogeneous disease. Diagnosis relies on symptom evaluation and lung function tests using spirometry. Symptoms can be vague. Spirometry is effort-dependent and does not reliably evaluate small airways. Allergic asthma in preschool children is not easily separated from episodic wheeze. Exhaled NO (FeNO) is a marker of allergic Th2-cytokine-driven airway inflammation. However, FeNO is not feasible in preschoolers with current devices and algorithms. Alveolar NO is an estimate of small airway involvement. Forced oscillometry (FOT) is an effort-independent lung function test assessing both large and small airways. Aims: To study clinical and methodological aspects of FeNO, alveolar NO and lung function indices by FOT. Methods: Asthmatic children and young adults and healthy controls, were included in the studies. FeNO at 50 mL/s was performed in all studies (in study III with an adapted single-breath method with age-adjusted exhalation times). FeNO at multiple exhalation flow rates were performed in studies I, II and IV to calculate alveolar NO, as was spirometry. FOT indices were assessed in study IV. Results: The exhalation time needed to reach steady-state NO was < 4 s in subjects aged 3-4 years, and was related to subject height. FeNO was higher in ICS-naïve asthmatic children than in controls. ICS-naïve asthmatic preschool children had FeNO < 20 ppb. The oral contribution to FeNO was similar in asthmatic and healthy youths. Multiple flow rates and modelling of alveolar NO were feasible in children aged 10-18 years. Alveolar NO correlated to asthma characteristics, though not when axial diffusion correction was applied. FOT resistance measures were associated with asthma diagnosis, and small airway FOT measures were associated with asthma control, in adolescents. Conclusion: An adapted FeNO method is feasible from 4 years, and exhalation time is related to child height. Our findings emphasise the need to refine clinical cut-offs for FeNO in younger children. FOT variables discriminate between asthmatics and controls, much like spirometry. The information provided by FOT is additive to that from spirometry. Further studies of exhaled NO dynamics and FOT indices of small airways are warranted to evaluate new treatment options and possibly improve asthma control.

Pacientes submetidos à cirurgia cardiotorácica com circulação extracorpórea (CEC) apresentam reações inflamatórias, desencadeadas por este procedimento e pela isquemiareperfusão, que acarretam disfunção pulmonar e lesão do endotélio vascular no pósoperatório. Estes processos relativos à cirurgia cardíaca podem afetar a produção e o consumo do óxido nítrico (NO) no pulmão e no endotélio vascular, principalmente na sua participação no processo inflamatório. No pulmão, o NO se difunde com facilidade sendo detectado na via aérea na fase gasosa ou no fluído pulmonar como nitrito e o nitrato. A presença do NO na via aérea pode em algumas situações refletir a sua produção pelo epitélio da via aérea e pelo endotélio microvascular pulmonar. No entanto, os processos que envolvem a produção e o consumo do NO na via aérea ainda não estão totalmente esclarecidos. O condensado do exalado pulmonar (CEP) é um fluido obtido através do resfriamento do ar exalado através de um método totalmente não invasivo e que pode ser utilizado para a investigação do NO nas vias aéreas distais. No desenvolvimento deste estudo foi utilizado um aparato de coleta artesanal e de baixo custo para obtenção do CEP. Assim, foi coletado o CEP de pacientes coronariopatas e valvopatas submetidos à cirurgia cardíaca com CEC nos períodos pré- operatório e pós-operatório (4 horas após a CEC e 12, 24, 48 e 72 horas após a extubação). As concentrações plasmáticas de nitrito/nitrato destes pacientes também foram avaliadas através de amostras de sangue colhidas imediatamente após cada coleta do CEP. O CEP e o plasma foram analisados pela técnica de quimioluminescência para obter as concentrações de nitrito/nitrato. O aparato utilizado obteve utilização reprodutível na rotina clínica da cirurgia cardíaca nesta pesquisa em pacientes sob ventilação espontânea e ventilação mecânica. Não foram encontradas diferenças significativas (p>0,05) nas concentrações de nitrito/nitrato no CEP e plasma entre os pacientes coronariopatas e valvopatas no período pré-operatório. Concentrações significativamente maiores de nitrito/nitrato (p=0,017) foram encontradas no CEP, mas não no plasma, de pacientes que utilizaram nitrato por via oral (dinitrato de isossorbida) no período pré-operatório. Em pacientes que não utilizaram medicação contendo doador de NO (nitroglicerina ou nitroprussiato de sódio) por via endovenosa no pósoperatório ocorreu uma tendência à elevação das concentrações de nitrito/nitrato no CEP que foi numericamente significativa no período de 48 horas do pós-operatório (p=0,008). Em pacientes que receberam ou não doador de NO no pós-operatório as concentrações de nitrito/nitrato foram significativamente maiores nos períodos de 48 horas (p=0,005) e 72 horas (p=0,037) do pós-operatório. A utilização de nitroglicerina (NTG) no período pósoperatório elevou significativamente as concentrações de nitrito/nitrato no CEP nos períodos de 12 horas (p=0,022), 48 horas (p=0,015) e 72 horas (0,048) e no plasma nos períodos de 12 horas (p=0,045). As concentrações de nitrito/nitrato plasmáticos foram significativamente reduzidas (p=0,045) no pós-operatório imediato 4 horas após a CEC em pacientes que receberam ou não doador de NO. A partir destes resultados pode se observar que não houve diferenças significativas nas concentrações de nitrito/nitrato no CEP dos pacientes coronariopatas e valvopatas no período pré-operatório, exceto elevação significativa encontrada em pacientes que utilizaram dinitrato de isossorbida por via oral. As concentrações de nitrito/nitrato no CEP tendem a elevar-se no período pós-operatório principalmente com a utilização de NTG. As concentrações de nitrito/nitrato no plasma foram significativamente reduzidas após 4 horas da CEC no pós-operatório imediato. No entanto foi observado elevação nas concentrações de nitrito/nitrato no plasma de pacientes que usaram NTG. Os achados deste estudo poderão contribuir em futuras pesquisas fornecendo dados comparativos a respeito da participação do NO na via aérea e no plasma nos processos que envolvem a cirurgia cardíaca nos períodos pré e pós-operatório. / Patients undergoing cardiothoracic surgery with cardiopulmonary bypass (CPB) have inflammatory reactions triggered by this procedure and by ischemia-reperfusion, which cause lung dysfunction and injury of vascular endothelium in the postoperative period. These processes related to cardiac surgery may affect the production and consumption of nitric oxide (NO) in the lung and vascular endothelium, especially in their participation in the inflammatory process. In the lung, NO diffuses easily and can be detected in the airway in the gas phase or in the lung fluid as nitrite and nitrate. The presence of NO in exhaled air may reflect the airway epithelium and lung microvascular endothelium in some situations. However, processes involving the production and consumption of NO in the airway are not totally understood. The exhaled breath condensate (EBC) is a fluid obtained by cooling exhaled air wich can be used for the investigation of NO in the distal airways. For this study we used an apparatus homemade and with low cost for collecting and obtain the EBC. The EBC was collected from patients with coronary artery disease and heart valve disease undergoing cardiac surgery with CPB in the preoperative and postoperative period (4 hours after CPB and 12, 24, 48 and 72 hours after extubation). Plasma concentrations of nitrite/nitrate in these patients were also evaluated using blood samples taken immediately after each collection of the EBC. The EBC and plasma were analyzed by chemiluminescence method to obtain the concentrations of nitrite/nitrate. The apparatus used was reproducible in clinical routine of cardiac surgery in patients under spontaneous ventilation or mechanical ventilation. There were no significant differences (p> 0.05) in concentrations of nitrite/nitrate in EBC and plasma in patients with coronary artery disease and heart valve disease in the preoperative period. Significantly higher concentrations of nitrite/nitrate (p=0.017) were found in EBC, but not in plasma of patients who used oral nitrate (isosorbide dinitrate) in the preoperative period. In patients who did not use medication containing NO donor (nitroglycerin or sodium nitroprusside) intravenously in the postoperative period there was a tendency to high concentrations of nitrite/nitrate in EBC that was numerically significant in the period of 48 hours postoperative (p=0.008). In patients who received or not NO donor in postoperative period the concentrations of nitrite/nitrate were significantly higher in periods of 48 hours (p=0.005) and 72 hours (p=0.037) after surgery. The use of nitroglycerin (GTN) in the postoperative period significantly elevated concentrations of nitrite/nitrate in the EBC in periods of 12 hours (p=0.022), 48 hours (p=0.015) and 72 hours (0.048) and plasma in period of 12 hours (p=0.045). The concentrations of nitrite/nitrate were significantly reduced (p=0.045) in the immediate postoperative period 4 hours after CPB in patients who received or not NO donor. From these results we could observe that there were no significant differences in concentrations of nitrite/nitrate in the EBC of patients with heart valve disease and coronary artery disease in the preoperative period, but we found a significant increase in these concentrations in patients who used isosorbide dinitrate orally. The concentrations of nitrite/nitrate in EBC presented a tendency to increase in postoperative period especially in patients who used GTN. The concentrations of nitrite/nitrate in plasma were significantly reduced after 4 hours of CPB in the immediate postoperative period. However, was observed that GTN tended to increase the concentration of nitrite/nitrate in plasma. These findings could help in future research providing comparative data about the role of NO in the airway and plasma in processes involving cardiac surgery in the pre and postoperative periods.

This thesis examines the overarching theme of inhaled corticosteroid (ICS) dose response effects on a variety of asthma outcome measures; with further importance placed on the application of these findings to personalising ICS dosing for the individual asthmatic. The introduction provides a detailed summary of the current recommendations for the treatment of adult asthma, with particular reference to the mechanism of action and clinical utility of ICS for the treatment of asthma. Current methods of assessing ICS dose response are presented, as well as the common influences that affect these responses. Novel therapeutic theories and the identification of specific asthmatic phenotypes are also introduced, in order to demonstrate the shift towards personalising treatment for asthma. The first two studies examine the dose response of ICS on two specific factors that influence asthma. The third study presents an examination of pharmacological manipulation of the ICS dose response using an additional agent. The following two studies address: how asthma outcomes relate to each other in patients receiving ICS; in addition to an overall assessment of the ICS dose response across a broad range of both ICS moieties and outcome measures. The final study examines for any detrimental effect of an ICS dose ramp on bone metabolism, an important potential long-term adverse effect of higher ICS dosing. The discussion draws together all the results obtained in relation to ICS dose response in asthma, and how these apply to current clinical practice for the individual patient. Furthermore, hypotheses are generated for areas of future study based on the findings from this work.

Pacientes submetidos à cirurgia cardiotorácica com circulação extracorpórea (CEC) apresentam reações inflamatórias, desencadeadas por este procedimento e pela isquemiareperfusão, que acarretam disfunção pulmonar e lesão do endotélio vascular no pósoperatório. Estes processos relativos à cirurgia cardíaca podem afetar a produção e o consumo do óxido nítrico (NO) no pulmão e no endotélio vascular, principalmente na sua participação no processo inflamatório. No pulmão, o NO se difunde com facilidade sendo detectado na via aérea na fase gasosa ou no fluído pulmonar como nitrito e o nitrato. A presença do NO na via aérea pode em algumas situações refletir a sua produção pelo epitélio da via aérea e pelo endotélio microvascular pulmonar. No entanto, os processos que envolvem a produção e o consumo do NO na via aérea ainda não estão totalmente esclarecidos. O condensado do exalado pulmonar (CEP) é um fluido obtido através do resfriamento do ar exalado através de um método totalmente não invasivo e que pode ser utilizado para a investigação do NO nas vias aéreas distais. No desenvolvimento deste estudo foi utilizado um aparato de coleta artesanal e de baixo custo para obtenção do CEP. Assim, foi coletado o CEP de pacientes coronariopatas e valvopatas submetidos à cirurgia cardíaca com CEC nos períodos pré- operatório e pós-operatório (4 horas após a CEC e 12, 24, 48 e 72 horas após a extubação). As concentrações plasmáticas de nitrito/nitrato destes pacientes também foram avaliadas através de amostras de sangue colhidas imediatamente após cada coleta do CEP. O CEP e o plasma foram analisados pela técnica de quimioluminescência para obter as concentrações de nitrito/nitrato. O aparato utilizado obteve utilização reprodutível na rotina clínica da cirurgia cardíaca nesta pesquisa em pacientes sob ventilação espontânea e ventilação mecânica. Não foram encontradas diferenças significativas (p>0,05) nas concentrações de nitrito/nitrato no CEP e plasma entre os pacientes coronariopatas e valvopatas no período pré-operatório. Concentrações significativamente maiores de nitrito/nitrato (p=0,017) foram encontradas no CEP, mas não no plasma, de pacientes que utilizaram nitrato por via oral (dinitrato de isossorbida) no período pré-operatório. Em pacientes que não utilizaram medicação contendo doador de NO (nitroglicerina ou nitroprussiato de sódio) por via endovenosa no pósoperatório ocorreu uma tendência à elevação das concentrações de nitrito/nitrato no CEP que foi numericamente significativa no período de 48 horas do pós-operatório (p=0,008). Em pacientes que receberam ou não doador de NO no pós-operatório as concentrações de nitrito/nitrato foram significativamente maiores nos períodos de 48 horas (p=0,005) e 72 horas (p=0,037) do pós-operatório. A utilização de nitroglicerina (NTG) no período pósoperatório elevou significativamente as concentrações de nitrito/nitrato no CEP nos períodos de 12 horas (p=0,022), 48 horas (p=0,015) e 72 horas (0,048) e no plasma nos períodos de 12 horas (p=0,045). As concentrações de nitrito/nitrato plasmáticos foram significativamente reduzidas (p=0,045) no pós-operatório imediato 4 horas após a CEC em pacientes que receberam ou não doador de NO. A partir destes resultados pode se observar que não houve diferenças significativas nas concentrações de nitrito/nitrato no CEP dos pacientes coronariopatas e valvopatas no período pré-operatório, exceto elevação significativa encontrada em pacientes que utilizaram dinitrato de isossorbida por via oral. As concentrações de nitrito/nitrato no CEP tendem a elevar-se no período pós-operatório principalmente com a utilização de NTG. As concentrações de nitrito/nitrato no plasma foram significativamente reduzidas após 4 horas da CEC no pós-operatório imediato. No entanto foi observado elevação nas concentrações de nitrito/nitrato no plasma de pacientes que usaram NTG. Os achados deste estudo poderão contribuir em futuras pesquisas fornecendo dados comparativos a respeito da participação do NO na via aérea e no plasma nos processos que envolvem a cirurgia cardíaca nos períodos pré e pós-operatório. / Patients undergoing cardiothoracic surgery with cardiopulmonary bypass (CPB) have inflammatory reactions triggered by this procedure and by ischemia-reperfusion, which cause lung dysfunction and injury of vascular endothelium in the postoperative period. These processes related to cardiac surgery may affect the production and consumption of nitric oxide (NO) in the lung and vascular endothelium, especially in their participation in the inflammatory process. In the lung, NO diffuses easily and can be detected in the airway in the gas phase or in the lung fluid as nitrite and nitrate. The presence of NO in exhaled air may reflect the airway epithelium and lung microvascular endothelium in some situations. However, processes involving the production and consumption of NO in the airway are not totally understood. The exhaled breath condensate (EBC) is a fluid obtained by cooling exhaled air wich can be used for the investigation of NO in the distal airways. For this study we used an apparatus homemade and with low cost for collecting and obtain the EBC. The EBC was collected from patients with coronary artery disease and heart valve disease undergoing cardiac surgery with CPB in the preoperative and postoperative period (4 hours after CPB and 12, 24, 48 and 72 hours after extubation). Plasma concentrations of nitrite/nitrate in these patients were also evaluated using blood samples taken immediately after each collection of the EBC. The EBC and plasma were analyzed by chemiluminescence method to obtain the concentrations of nitrite/nitrate. The apparatus used was reproducible in clinical routine of cardiac surgery in patients under spontaneous ventilation or mechanical ventilation. There were no significant differences (p> 0.05) in concentrations of nitrite/nitrate in EBC and plasma in patients with coronary artery disease and heart valve disease in the preoperative period. Significantly higher concentrations of nitrite/nitrate (p=0.017) were found in EBC, but not in plasma of patients who used oral nitrate (isosorbide dinitrate) in the preoperative period. In patients who did not use medication containing NO donor (nitroglycerin or sodium nitroprusside) intravenously in the postoperative period there was a tendency to high concentrations of nitrite/nitrate in EBC that was numerically significant in the period of 48 hours postoperative (p=0.008). In patients who received or not NO donor in postoperative period the concentrations of nitrite/nitrate were significantly higher in periods of 48 hours (p=0.005) and 72 hours (p=0.037) after surgery. The use of nitroglycerin (GTN) in the postoperative period significantly elevated concentrations of nitrite/nitrate in the EBC in periods of 12 hours (p=0.022), 48 hours (p=0.015) and 72 hours (0.048) and plasma in period of 12 hours (p=0.045). The concentrations of nitrite/nitrate were significantly reduced (p=0.045) in the immediate postoperative period 4 hours after CPB in patients who received or not NO donor. From these results we could observe that there were no significant differences in concentrations of nitrite/nitrate in the EBC of patients with heart valve disease and coronary artery disease in the preoperative period, but we found a significant increase in these concentrations in patients who used isosorbide dinitrate orally. The concentrations of nitrite/nitrate in EBC presented a tendency to increase in postoperative period especially in patients who used GTN. The concentrations of nitrite/nitrate in plasma were significantly reduced after 4 hours of CPB in the immediate postoperative period. However, was observed that GTN tended to increase the concentration of nitrite/nitrate in plasma. These findings could help in future research providing comparative data about the role of NO in the airway and plasma in processes involving cardiac surgery in the pre and postoperative periods.

acase@tulane.edu

Master of Science in Medicine / Rationale: Asthma management aims to achieve optimal control on the minimal effective dose of medication. We assessed the effectiveness of two algorithms to guide ICS dose in well-controlled patients on ICS+LABA in a double-blind study, comparing dose adjustment guided by exhaled nitric oxide (eNO) to clinical care algorithm(CCA) based on symptoms and lung function. Methods: We randomised non-smoking adult asthmatics on minimum FP dose 100μgs daily +LABA to ICS adjustment using eNO or CCA, assessed over 5 visits during 8 months treatment. Primary endpoints were asthma-free days and asthma related quality of life (QOL). Analysis was by mixed model regression and generalised estimating equations with log link. Results: 69 subjects were randomised (eNO:34, CCA:35) and 58 completed the study. At baseline mean FEV1 was 94% pred., mean eNO (200ml/sec) 7.1 ppb, median ACQ6 score 0.33. Median ICS dose was 500 μg (IQR 100-500) at baseline and 100 μg on both eNO (IQR 100-200) and CCA arms (IQR 100–100) at end of study. There were no significant differences between eNO and CCA groups in asthma-free days (RR=0.92, 95% CI 0.8–1.01), AQL (RRAQL<median = 0.95, 95% CI 0.8–1.1) or exacerbation-free days (HR = 1.03, 95%CI 0.6–1.7). Neither clinic FEV1 (overall mean difference FEV1 % pred. -0.24%, 95% CI -2.2–1.7) nor a.m. PEF (mean difference 1.94 L/min (95% CI -2.9–6.8) were significantly different. Similar proportions of subjects were treated for ≥1 exacerbation (eNO: 50%, 95% CI 32.1–67.9; CCA: 60%, 95% CI 43.9–76.2). Conclusion: Substantial reductions in ICS doses were achieved in well controlled asthmatics on ICS+LABA, with no significant differences in outcomes between eNO or clinically based algorithms.

Screening complex biological specimens such as exhaled air, tissue, blood and urine to identify biomarkers in different forms of cancer has become increasingly popular over the last decade, mainly due to new instruments and improved bioinformatics. However, despite some progress, the identification of biomarkers has shown to be a difficult task with few new biomarkers (excluding recent genetic markers) being considered for introduction to clinical analysis. This review describes recent advances in gas chromatographic methods for the identification of biomarkers in the detection, diagnosis and treatment of cancer. It presents a general overview of cancer metabolism, the current biomarkers used for cancer diagnosis and treatment, a background to metabolic changes in tumors, an overview of current GC methods, and collectively presents the scope and outlook of GC methods in oncology.

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.