Introdução: Das formas clínicas das leishmanioses, a forma clássica da leishmaniose visceral (LV) é a forma mais grave da doença, afetando órgãos como baço, fígado e linfonodos. Como a doença apresenta um comprometimento intersticial secundário à infecção pela Leishmania, existe o envolvimento de outros órgãos. No pulmão, o envolvimento se manifesta pela pneumonite intersticial. Essa alteração foi provada por estudo anatomopatológico em hamsteres, cães e homens. Embora as pesquisas pulmonares na doença avaliem as alterações ultraestruturais provocadas pela leishmaniose, não existem estudos que avaliem o impacto dessas sobre a função pulmonar. Objetivo: Caracterizar o distúrbio ventilatório em pacientes internado com LV por espirometria. Métodos: Foram avaliados transversalmente 20 pacientes com diagnóstico confirmado por Kalazar detect, mielograma e/ou sorologia. Os parâmetros medidos foram a capacidade vital forçada (CVF), volume expiratório forçado do primeiro segundo (VEF1), índice de Tiffeneau e fluxo expiratório forçado (25-75%). Posteriormente, foram utilizados, na análise estatística, o teste não paramétrico de Mann-Whitney, teste exato de Fisher, não paramétrico de Wilcoxon e o coeficiente de correlação de Speraman. Nível de significância com p < 0,05. Resultados: A espirometria mostrou-se alterada em 14 pacientes (70%). O padrão de distúrbio ventilatório apresentado foi somente restritivo. Em relação aos dados laboratoriais, os pacientes com hipoalbuminemia apresentaram espirometria alterada. Não foi achada correlação estatisticamente significativa entre tempo de medicação, consumo de tabaco, infecção, sintomas respiratórios, ocupação, tempo de sintomas. Conclusão: Os achados da espirometria evidenciaram volumes pulmonares reduzidos, com diminuição da CVF e, em 55% dos pacientes com VEF1, também diminuído. Neste estudo, demonstrou-se que a alteração da função pulmonar está, provavelmente, relacionada à fibrose pulmonar que ocorre na LV, como descrito / The clinical forms of leishmaniasis, the classic form of visceral leishmaniasis (VL) is the most severe form of the disease, affecting organs such as the spleen, liver and lymph nodes. As the disease presents an interstitial deterioration secondary to the infection by Leishmania, there is the involvement of other organs. In the lung, the involvement is manifested by the interstitial pneumonitis. This alteration has been proved by anatomopathological studies in hamsters, dogs and men. Although the researches concerning this lung disease assess the ultrastructural alterations caused by leishmaniasis, there are no studies evaluating the impact of these on lung function. Objective: To characterize the ventilatory disorder in patients hospitalized with VL by spirometry. Methods: 20 patients were transversely evaluated with diagnosis confirmed by Kalazar Detect, myelogram and / or serology. The measured parameters were the forced vital capacity (FVC), forced expiratory volume in one second (FEV1), Tiffeneau index and forced expiratory flow (25-75%). Thereafter, the non-parametric Mann-Whitney test, Fisher\'s exact test, non-parametric Wilcoxon and Spearman correlation coefficient were used in the statistical analysis. Significance level of p < 0.05. Results: The spirometry was altered in 14 patients (70%). The presented ventilatory disorder pattern was only restrictive. Regarding the laboratory data, patients with hypoalbuminemia presented altered spirometry findings. It was not found statistically significant correlation between time of medication, smoking, infections, respiratory symptoms, occupation, time of symptoms. Conclusion: The findings of spirometry showed reduced lung volumes, with decreased FVC and in 55% of patients with FEV1 also decreased. In this study it was shown that the change in lung function is probably related to pulmonary fibrosis that occurs in the VL as described in anatomicopathological studies conducted earlier

Yung, Kam Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 107-129). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / PUBLICATION --- p.ix / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xi / LIST OF ABBREVIATIONS --- p.xii / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- "The biology, diagnostics and management of lung cancer" --- p.2 / Chapter 1.1 --- "Basic biology, classification and diagnostics" --- p.2 / Chapter 1.1.1 --- Epidemiology and etiology of lung cancer --- p.2 / Chapter 1.1.2 --- Clinical Presentation and Diagnostics of Lung Cancer --- p.3 / Chapter 1.2 --- Treatment of lung cancer --- p.9 / Chapter 1.2.2 --- Radiotherapy --- p.10 / Chapter 1.2.3 --- Chemotherapy --- p.11 / Chapter CHAPTER 2: --- Epidermal Growth Factor Receptor Mutations in Lung Cancer --- p.13 / Chapter 2.1 --- The Epidermal Growth Factor Receptor --- p.13 / Chapter 2.2 --- Overexpression of EGFR in NSCLC --- p.14 / Chapter 2.3 --- The development of EGFR inhibitors --- p.15 / Chapter 2.3.1 --- Monoclonal Antibodies --- p.16 / Chapter 2.3.2 --- Small-molecule inhibitors --- p.17 / Chapter 2.3.2.1 --- Gefitinib --- p.17 / Chapter 2.3.2.2 --- Erlotinib --- p.19 / Chapter 2.3.2.3 --- Other small-molecule inhibitors --- p.20 / Chapter 2.4 --- Mutations of EGFR in NSCLC --- p.21 / Chapter 2.4.1 --- Activating Mutations conferring sensitivity to tyrosine kinase inhibitors --- p.21 / Chapter 2.4.2 --- Secondary mutations associated with resistance to tyrosine kinase inhibitors --- p.23 / Chapter 2.5 --- EGFR gene amplification --- p.24 / Chapter 2.6 --- Detection of EGFR mutations --- p.25 / Chapter 2.7 --- Aim of the thesis --- p.31 / Chapter SECTION II: --- DETECTION OF EGFR MUTATIONS IN TUMOR AND PLASMA SAMPLES BY MASS SPECTROMETRY AND DIGITAL PCR --- p.33 / Chapter CHAPTER 3: --- Detection of EGFR mutations by mass spectrometric methods --- p.34 / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.1.1 --- Principles of Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.34 / Chapter 3.1.2 --- The MassARRAY Homogenous MassEXTEND (hME) assay --- p.35 / Chapter 3.1.3 --- The Single-Allele Base Extension Reaction (SABER) and the Allele-Specific Base Extension Reaction (ASBER) --- p.36 / Chapter 3.2 --- Materials and Methods --- p.36 / Chapter 3.2.1 --- The protocol for the detection of EGFR exon 21 point mutation by Mass Spectrometric Methods --- p.37 / Chapter 3.3 --- Results --- p.42 / Chapter 3.4 --- Discussion --- p.49 / Chapter CHAPTER 4: --- Evaluation of the detection limit and sensitivity of the digital PCR assays --- p.51 / Chapter 4.1 --- Introduction --- p.51 / Chapter 4.1.1 --- The theoretical basis of digital PCR quantification and the relationship with the Poisson distribution --- p.51 / Chapter 4.1.2 --- Assessment of Assay Detection Limit --- p.54 / Chapter 4.1.3 --- Comparing Digital PCR with sequencing after conformation sensitive gel electrophoresis (CSGE) --- p.59 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Design of digital PCR assay for the detection of EGFR exon21 L858R point mutation --- p.59 / Chapter 4.2.2 --- Design of digital PCR assay for the detection of EGFR exon19 deletion --- p.60 / Chapter 4.2.3 --- The protocols of digital PCR assays for EGFR mutation detection --- p.64 / Chapter 4.2.4 --- Single molecule detection test --- p.65 / Chapter 4.2.5 --- Artificial mixtures of mutant and wild-type DNA --- p.66 / Chapter 4.2.6 --- Sequencing after CSGE --- p.66 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Results of the single molecule detection test and artificial mixture analysis --- p.67 / Chapter 4.3.2 --- Results of CSGE and sequencing compared with digital PCR --- p.73 / Chapter 4.4 --- Discussion --- p.75 / Chapter CHAPTER 5: --- Detection of EGFR mutations in prospectively collected tumor samples of NSCLC patients --- p.77 / Chapter 5.1 --- Introduction --- p.77 / Chapter 5.2 --- Materials and Methods --- p.78 / Chapter 5.2.1 --- Sample preparation and DNA extraction of tumor tissues --- p.78 / Chapter 5.3 --- Results --- p.79 / Chapter 5.4 --- Discussion --- p.82 / Chapter CHAPTER 6: --- Detection of EGFR mutations in prospectively collected plasma samples of NSCLC patients --- p.85 / Chapter 6.1 --- Introduction --- p.85 / Chapter 6.2 --- Materials and Methods --- p.87 / Chapter 6.2.1 --- Sample preparation and DNA extraction of plasma samples --- p.87 / Chapter 6.3 --- Results --- p.88 / Chapter 6.3.1 --- Digital PCR analysis of EGFR mutations in plasma samples of NSCLC patient --- p.88 / Chapter 6.3.2 --- Variations in plasma EGFR mutation concentration after TKI treatment --- p.93 / Chapter 6.4 --- Discussion --- p.96 / Chapter SECTION III: --- CONCLUDING REMARKS --- p.100 / Chapter CHAPTER 7: --- Conclusion and future perspectives --- p.101 / Chapter 7.1 --- Mass spectrometric analysis --- p.101 / Chapter 7.2 --- Microfluidics Digital PCR --- p.102 / Chapter 7.3 --- Future perspectives --- p.105 / References --- p.107

Em cães, a comprovação da real viabilidade da pneumonectomia direita, bem como, o estudo das complicações resultantes deste procedimento cirúrgico, tornam-se importantes diante da pequena quantidade de estudos na literatura específicos sobre pneumonectomia nesta espécie. O objetivo deste trabalho foi realizar um estudo experimental para avaliar a viabilidade da pneumonectomia direita em cães, através da avaliação paramétrica, hemogasométrica, ecocardiográfica, radiográfica, broncoscópica e da mecânica respiratória. Foram utilizados 10 cães, sadios, machos e fêmeas, adultos, sem raça definida, pesando entre 13 e 32 kg. Todos os cães foram submetidos à intubação seletiva e toracotomia direita no 5º espaço intercostal, onde foi realizada a pneumonectomia. Foi realizado estudo temporal aos sete, 30 e 60 dias de pós-operatório, onde foi feita avaliação ecocardiográfica, radiográfica e broncoscópica. A avaliação paramétrica e hemogasométrica foi realizada antes da indução anestésica, uma hora após extubação, 48 horas, sete, 30 e 60 dias após o procedimento cirúrgico. A avaliação da mecânica respiratória foi realizada antes da indução anestésica, durante a cirurgia, 48 horas, sete, 30 e 60 dias após o procedimento cirúrgico. Estas avaliações foram consideradas importantes para determinar as possíveis complicações relacionadas com a técnica anestésica, cirúrgica, assim como, as complicações resultantes deste procedimento cirúrgico. Os resultados encontrados foram analisados estatisticamente. Apesar das alterações dos índices paramétricos, hemogasométricos e da mecânica respiratória, todos os cães apresentaram compensação das trocas gasosas após retirada do pulmão direito. Em relação a avaliação da mecânica respiratória, os volumes pulmonares não se modificam de maneira acentuada. Ocorreu aumento das pressões de pico e resistência das vias aéreas devido ao emprego da sonda de duplo lúmen e ressecção pulmonar. Não houve diferença significativamente estatística nos níveis da pressão sistólica e média no tronco pulmonar. Entretanto, alguns cães apresentaram hipertensão pulmonar leve e transitória, sendo que o período de maior incidência foi aos 30 dias de pós-operatório. A fração de ejeção do ventrículo direito manteve-se normal durante o estudo. No entanto, nos mesmos cães que apresentaram hipertensão pulmonar, houve diminuição significativa da fração de ejeção do ventrículo direito aos 60 dias de pós-operatório. Na avaliação radiográfica, observamos que a expansão do pulmão remanescente causou deslocamento do coração e pulmão para hemitórax direito. Nas imagens broncoscópicas pós-operatórias não foram observadas quaisquer sinais de infecção, deiscência, fístula e estenose da sutura em coto brônquico. Concluiu-se que a realização da pneumonectomia direita é plenamente viável no cão, permitindo evolução paramétrica, hemogasométrica, ecocardiográfica, radiográfica, broncoscópica e da mecânica respiratória satisfatória em todos os cães. / In dogs, the evidence of the viability of right pneumonectomy, as well as the study of the complications resulting from surgical procedure, are very important against the small quantity of specific studies on pneumonectomy in this species. The objective of this project was carrying out an experimental study to evaluate the viability of right pneumonectomy in dogs, through parametric, hemogasometric, echocardiographic, radiographic, broncoscopic and mechanical respiratory evaluation. Ten adult mongrel, healthy dogs, both male and female were utilited weighing from 13 to 32 kg. The dogs were submitted to selective intubation and right thoracotomy at the 5th intercostal space, where the pneumonectomy was performed. A temporal study was made in 7, 30 and 60 days after the surgery, where echocardiographic, radiografic and bronchoscopic evaluation were done. The parametric and hemogasometric evaluations was made before anesthetic induction, one hour after extubation, in 48 hour, seven, 30 and 60 days after the surgical procedure. The mechanical respiratory evaluation was before anesthetic induction, during surgery, 48 hour, seven, 30 and 60 days after the surgical procedure. These evaluations were considered important to determine the probable complications connected to the anesthetic, surgical techniques, as well as the resulting complications from these procedures. The results were analyzed statistically. Besides the alterations of the parametric, hemogasometric and mechanical respiratory rates, all the dogs presented compensations on the gaseous exchanges after removing right lung. Regarding the mechanical respiratory evaluation, the pulmonary volume has not changed accentually. There was an increase of the peak pressures and resistance of the respiratory tract due to the use of a double lumen probe and lung ressection. There wasn\'t any significant statistically difference on the levels of systolic and average pressure on the pulmonary trunk. However, some dogs showed light and transitory pulmonary hypertension, and the highest occurrence period happened 30 days after surgery. The fraction of ejection of the right ventricle remained normal during the study. However, at the same dogs who presented pulmonary hypertension, there was a significant decrease of the fraction of the right ventricle 60 days after surgery. On the radiographic evaluation, we have observed that the expansion of the remaining lung caused heart and lung displacement to the right hemithorax. Any signs of infection, dehiscence, fistula or suture stenosis in the main right bronchial stump were observed on the post-operative bronchoscopic images. It follows that the right pneumonectomy is completely feasible the dogs, allowing a parametric, hemogasometric, echocardiographic, radiographic, bronchoscopic and mechanical respiratory satisfactory evaluation in all the dogs.

In this work, airflow and particle transport are studied using mathematical and image-based models of pulmonary acinus. Numerical results predict that airflow in the presence of wall motion in a three-dimensional honey-comb like geometry is characterized by the presence of a recirculation region within the alveolar cavity and a weak entraining flow between alveolar duct and cavity. Alveolar flow in distal generations is characterized by higher alveolar flow rates, larger entrainment of ductal flow and absence of recirculatory flow inside alveoli. The study of transport constitutes assessment of mixing visualized by the tracking of massless particles and the study of transport and deposition of aerosols. The phenomenon of steady streaming is found to hold the key to the origin of kinematic mixing in the alveolus, the alveolar mouth and the alveolated duct. This mechanism provides the explanation for observed folding of material lines and increases in material surface area, and has no bearing on whether the geometry is expanding or if flow separates within the cavity or not. Streaming results in non-zero drift of particles between the beginning and end of a breathing cycle. Based on flow conditions and resultant convective mixing measures, we conclude that significant convective mixing in the duct and within an alveolus could originate only in the first few generations of the acinar tree as a result of non-zero inertia, flow asymmetry and large KC number. Evidence of streaming and related Lagrangian drift is also observed in image-based acinar models. Finally, particle deposition calculations are performed on the models of pulmonary acinus considered in this study.

<p>Because of the difficulties in concluding whether a person has drowned or not, information that could be relevant for postmortal diagnosis of drowning was studied. With postmortal CT images lung volume, mean attenuation, anterior-posterior difference, lung density profile and amount of water within the lungs were investigated.</p><p>The report also evaluates three examples of software that calculates lung volume from postmortal CT images: Siemens’ Syngo Pulmo CT, Siemens‘ Volume Evaluation and GE Medical Systems’ Volume Viewer. The method used at autopsy was also studied. The repeatability and validity were tested and sources of errors identified.</p><p>Repeatability and validity for the three tested types of software were acceptable, while the method used at autopsy had to be improved. The study also showed that lung volume related to length, anterior-posterior difference and lung density profile seemed to vary between drowned and other deceased. These measures might conclude whether a person has drowned.</p>

Purpose. Low-dose computed tomography is an important imaging modality for screening and surveillance of lung cancer. The goal of this study was to determine the extent to which dose could be minimized while maintaining diagnostic accuracy through knowledgeable selection of reconstruction techniques. Methods. An anthropomorphic phantom was imaged on a 320-slice volumetric CT scanner. Detectability of small solid lung nodules was evaluated as a function of dose, patient size, reconstruction filter and slice thickness by means of 9-alternative forced-choice observer tests. Results. Nodule detectability decreased sharply below a threshold dose level due to increased image noise. For large body habitus, optimal (smooth) filter selection reduced dose by a factor of ~3. Nodule detectability decreased for slice thicknesses larger than the nodule diameter. Conclusions. Radiation dose can be reduced well below current clinical protocols. Smooth reconstruction filters and avoidance of large slice thickness permits lower-dose techniques without tradeoff in diagnostic performance.

Rationale: Ventilation inhomogeneity (VI) may be an early sign of obstructive airway disease. The lung clearance index (LCI) has been suggested as a sensitive marker of VI, although it has not been well characterized in young children in health and in those with CF and asthma. Objective: To determine if LCI can detect VI in asymptomatic infants and preschool-age subjects with CF or wheeze/asthma compared to healthy controls. Methods: Sulphur hexafluoride (SF6) multiple breath washout (MBW) testing was completed in all subjects. Results: LCI was found to be dependent on age in a large healthy cohort. Accounting for age, LCI was significantly elevated in disease groups compared to healthy controls in early childhood, illustrating early presence of VI in wheezy infants and the progression of disease in CF. Furthermore, the effects of breathing pattern and the variability of MBW parameters showed positive associations with age and VI.

Ischemia-reperfusion (IR) injury is a severe complication in lung transplantation characterized by inflammation, alveolar damage, and hypoxemia. Alpha 1-antitrypsin (A1AT), a protease inhibitor, is currently used clinically for the treatment of A1AT deficiency emphysema. A1AT has been shown to have the potential to reduce IR injury through its anti-inflammatory and anti-apoptotic effects. We hypothesized that A1AT will ameliorate IR injury through these effects. We tested A1AT in two models of IR: a cell culture model of simulated lung transplantation and a rat in situ pulmonary ligation model. In cell culture, we found that A1AT exerts its protective effects by inhibiting cell death and inflammatory cytokine release in a dose-dependent manner. In the rat pulmonary ischemia-reperfusion model, we found that A1AT improved lung function by inhibiting apoptosis and inflammation. There is potential for future application of A1AT in the treatment of IR injury in lung transplantation.

Rationale: Ventilation inhomogeneity (VI) may be an early sign of obstructive airway disease. The lung clearance index (LCI) has been suggested as a sensitive marker of VI, although it has not been well characterized in young children in health and in those with CF and asthma. Objective: To determine if LCI can detect VI in asymptomatic infants and preschool-age subjects with CF or wheeze/asthma compared to healthy controls. Methods: Sulphur hexafluoride (SF6) multiple breath washout (MBW) testing was completed in all subjects. Results: LCI was found to be dependent on age in a large healthy cohort. Accounting for age, LCI was significantly elevated in disease groups compared to healthy controls in early childhood, illustrating early presence of VI in wheezy infants and the progression of disease in CF. Furthermore, the effects of breathing pattern and the variability of MBW parameters showed positive associations with age and VI.

Ischemia-reperfusion (IR) injury is a severe complication in lung transplantation characterized by inflammation, alveolar damage, and hypoxemia. Alpha 1-antitrypsin (A1AT), a protease inhibitor, is currently used clinically for the treatment of A1AT deficiency emphysema. A1AT has been shown to have the potential to reduce IR injury through its anti-inflammatory and anti-apoptotic effects. We hypothesized that A1AT will ameliorate IR injury through these effects. We tested A1AT in two models of IR: a cell culture model of simulated lung transplantation and a rat in situ pulmonary ligation model. In cell culture, we found that A1AT exerts its protective effects by inhibiting cell death and inflammatory cytokine release in a dose-dependent manner. In the rat pulmonary ischemia-reperfusion model, we found that A1AT improved lung function by inhibiting apoptosis and inflammation. There is potential for future application of A1AT in the treatment of IR injury in lung transplantation.