Acute respiratory distress syndrome (ARDS) and infant respiratory distress syndrome (IRDS) are pulmonary diseases with a mortality rate of ∼40% and 75,000 deaths annually in the United States. Mechanical ventilation restores airway patency and gas transport but leads to ventilator-induced lung injury. Surfactant replacement therapy alleviates these effects in IRDS, but is ineffective due to surfactant delivery difficulties and deactivation by vascular proteins leaking into the airspace in ARDS. Here, we demonstrated that surfactant function can be substantially improved (up to 50%) in situ in an in vitro pulmonary airway model using unconventional flows that incorporate a short-term retraction of the air-liquid interface, leading to a net decrease in cellular damage. This research may provide a starting point for developing novel ventilation waveforms to improve surfactant function in edematous airways. / acase@tulane.edu

Lung cancer is the leading cause of cancer-related death for both men and women in the United States, despite being the second-most frequent cancer diagnosis for both sexes. This high mortality rate is due to the majority of cases being diagnosed after the primary lung cancer has metastasized. In an effort to reduce mortality associated with lung cancer by diagnosing lung cancer at an earlier stage, screening of high-risk populations has been employed. One screening tool, computed tomography (CT), has been shown to reduce mortality by 20%, compared to screening for lung cancer by chest x-ray. This was achieved by earlier stage diagnosis of lung cancer in participants screened with CT. The use of chest CT in lung cancer screening has also led to increased numbers of false-positives - benign lung nodules that are marked as suspicious for lung cancer. These false-positives result in unnecessary invasive follow-up procedures and costs while incurring additional emotional stress on the patient. In an effort to reduce the number of false-positives, a computer-aided diagnostic (CAD) tool can be designed to determine the probability of malignancy of a lung nodule based on objective measurements. While current CAD models characterize the pulmonary nodule's shape, density, and border, analyzing the parenchyma surrounding the nodule is an area that has been minimally explored. By quantifying characteristics, or features, of the surrounding tissue, this project explores the hypothesis that textural differences in both the nodule and surrounding parenchyma exist between malignant and benign cases. By incorporating these features, performance in the measures of sensitivity, specificity and accuracy can be improved over CAD tools that rely on nodule characteristics alone. A CAD program was developed for the computation of features from a pulmonary nodule. A region of interest containing a nodule and surrounding parenchyma was extracted from a CT scan. Several novel feature extraction techniques were developed, including a three-dimensional application of Laws' Texture Energy Measures to quantify the textures of the parenchyma surrounding the nodule and the nodule itself. In addition, the densities of the nodule and surrounding parenchyma were summarized through metrics such as mean, variance, and entropy of the intensities within each region. Finally, the margins of the nodule were characterized by analyzing mean and variance of border irregularity. A total of 299 features were extracted. To illustrate proof of concept, the CAD program was applied to 27 regions of interest - 10 benign and 17 malignant. Through feature selection, 36 significant features were recognized (p-values < 0.05), including many textural and parenchymal features. These features were further reduced by forward feature selection to two features that summarized the dataset. A neural network was used to classify the cases in a leave-one-out method. Preliminary results yielded 92.6% accuracy in classification of test cases, with two benign nodules incorrectly classified as malignant. The significance of texture and parenchymal features supports the hypothesis that features extracted from the parenchyma have the potential to improve classification of nodules, aiding in the reduction of false-positives identified through CT screening. As more cases are incorporated into the database, these textural features will play a larger role.

BACKGROUND: Finite element models of lung motion can aid in understanding mechanically driven lung deformation. Current finite element models consider each lung half as a continuum, lacking the ability to capture the displacement discontinuity at fissures caused by lobe sliding. OBJECTIVE: The objective of this work was to develop and evaluate finite element models for simulating lung motion that incorporate the role of sliding at the lobe boundaries. METHODS: Finite element models were developed from 4DCT of tidal breathing from five cancer subjects. To allow sliding, the lobes were modeled as independent bodies within a pleural cavity shell. Pleural cavity deformation was obtained from deformable image registration of the lung segmentations. Contact between the pleural cavity and lobes prevented penetration and allowed sliding at all interfaces. Lung parenchyma was modeled as a homogeneous, 2-parameter, Neo-Hookean finite elastic model. The parameters of the Neo-Hookean model, C1 and D1, were optimized by perturbation within realistic reported ranges; defined by the equivalent infinitesimal elasticity parameters: Young's modulus (from 0.7 kPa to 70 kPa) and ν (from 0.2 to 0.49). The frictional coefficient at fissures was perturbed between 0 (free sliding) and 1.5 (no sliding). 1,960 finite element analyses were performed across the five subjects. The optimal parameter ranges were evaluated by average landmark error and percentage of converged solutions. The developed finite element method, using optimized material and friction parameters, was further evaluated in a data set of six healthy subjects with image pairs spanning functional residual capacity (FRC) to total lung capacity (TLC). The finite element predicted displacement field for lobe sliding finite element models and continuum-based finite element models were compared using average landmark error and correlation with the lobe-by-lobe deformable image registration results. RESULTS AND DISCUSSION: The optimal parameters for Young's modulus were 49 kPa to 70 kPa and Poisson's ratio were 0.2 to 0.4. Variation of inter-lobar frictional coefficients did change displacement field accuracy assessed by landmark error or correlation to lobe-by-lobe deformable image registration. Characteristics of sliding predicted by the lobe sliding finite element models were consistent with characteristics in sliding observed in deformable image registration results. Also, variations in regional ventilation, quantified at the lobe level, were predicted by the finite element models and were shown to be influenced by the amount of lobe sliding allowed by the models.

Chronic obstructive pulmonary disease (COPD) is a growing health concern associated with high morbidity and mortality, and is currently the third-ranked cause of death in the United States. COPD is characterized by airflow limitation that is not fully reversible and includes chronic bronchitis, functional small airway disease, and emphysema. The interrelationship between emphysema and airway disease in COPD makes it a highly complex and heterogeneous disorder. Appropriate diagnosis of COPD is vital to administer targeted therapy strategies that can improve patient’s quality of life and reduce the frequency of COPD associated exacerbations. Although spirometry or pulmonary function tests are currently the gold standard for the diagnosis and staging of the disease, their lack of reproducibility and minimal information on regional characterization of the lung tissue destruction makes it hard to rely on to phenotype COPD population and predict disease progression. Quantification of COPD, as done by computed tomography (CT) methods has seen significant advancements, helping us understand the complex pathophysiology of this disease. The prospective and established techniques that are derived from CT imaging such as densitometry, texture, airway, and pulmonary vasculature-based analyses have been successful in regional characterization of emphysema related lung tissue destruction and airway disease related morphological changes in COPD patients. Although, these measures enriched our diagnostic and treating capability of COPD, they lack information on patient specific alterations in lung mechanics and regional parenchymal stresses. This valuable information can be achieved through the use of image registration protocols. Our main goal of this research work is to examine and evaluate the role of lung mechanical measures derived from CT image registration techniques in COPD diagnosis, phenotyping, and progression.

Les laminopathies regroupent des pathologies liées aux lamines. La Progeria est due à une mutation du gène LMNA entrainant la synthèse d’une protéine anormale : la progérine. Elle s’accumule dans le noyau et entraîne des dommages cellulaires aboutissant à une sénescence prématurée, à l’origine d’un vieillissement prématuré et accéléré des patients dont le décès survient vers l’âge de 14 ans. Les microARNs (miRs) sont des petits ARNs non-codants régulant l’expression des gènes. Dans le projet principal de ma Thèse, nous avons identifié par un miRNome en RT-qPCR, 14 miRs différentiellement exprimés dans les fibroblastes HGPS. Certains d’entre eux appartenant à la région 14q32.2-14q32-3, sont surexprimés à cause de modifications chromatiniennes. Nous avons ensuite étudié l’impact de la surexpression des miR-376a-3p et miR-376b-3p sur la régulation de l’autophagie. L’inhibition de ces miRNAs entraine une augmentation du niveau d’autophagie, associée à une diminution de la progérine. Une 2ème étude miRNome réalisée en NGS, a permis d’identifier d’autres miRNAs potentiellement impliqués dans la Progeria. Dans un second projet, nous avons analysé l’expression des lamines de type A dans des cellules tumorales métastatiques issues d’épanchements pleuraux de patients atteints d’adénocarcinome bronchique. Nous avons démontré que la diminution d’expression de la lamine A chez un groupe de patient est corrélée à un mauvais pronostic. Cette diminution pourrait être due à miR-9 qui cible directement l’ARNm de la prélamine A. Ces travaux de Thèse illustrent le rôle fondamental des lamines et suggère une place importante des miRNAs dans la physiopathologie de ces 2 types de laminopathies / Laminopathies are diseases linked to lamins. Progeria (HGPS) is a genetic disease caused by a mutation in LMNA gene leading to an abnormal protein called progerin. It accumulates in nucleus and causes cell damages leading to a premature senescence. Patient die around 14 years old. miRNAs are small non coding RNA regulating gene expression. In my main project, I identified with a miRNome approach by RT-qPCR, 14 differentially expressed miRNAs in dermal HGPS fibroblasts. We demonstrated that the overexpression of the miRNAs that belong to the 14q32 region was caused by chromatin modulation. Next, we studied the role of miR-376-3p and miR-376b-3p on autophagy and demonstrated that the inhibition of their overexpression increases autophagy and decreases progerin. A second miRNome by NGS identified other miRNAs potentially linked to HGPS pathophysiology. In my second project, I studied lamins expression in metastatic cells from pleural effusion of lung adenocarcinoma patients. We showed that the decreased expression of lamin A in a group of patients was correlated with poor prognosis, which could be linked to miR-9 expression. This thesis illustrates the fundamental role of lamins and suggest the role of miRNAs in the pathophysiology of this to types of laminopathies.

Computed tomography (CT) imagery is an important weapon in the fight against lung cancer; various forms of lung cancer are routinely diagnosed from CT imagery. The growth of the suspect nodule is known to be a prognostic factor in the diagnosis of pulmonary cancer, but the change in other aspects of the nodule, such as its aspect ratio, density, spiculation, or other features usable for machine learning, may also provide prognostic information. We hypothesized that adding combined feature information from multiple CT image sets separated in time could provide a more accurate determination of nodule malignancy. To this end, we combined data from multiple CT images for individual patients taken from the National Lung Screening Trial. The resulting dataset was compared to equivalent datasets featuring single CT images for each patient. Feature reduction and normalization was performed as is standard. The highest accuracy achieved was 83.71% on a subset of features chosen by a combination of manual feature stability testing and the Correlation-based Feature Selection algorithm and classified by the Random Forests algorithm. The highest accuracy achieved with individual CT images was 81.00%, on a feature set consisting solely of the volume of the nodule in cubic centimeters.

The effect of lateral electron disequilibrium on patient dose has been investigated. This has been achieved by dosimetry in lung and air cavity phantoms at megavoltage x-ray energies. The scatter function photon beam models for tissue inhomogeneity, such as the ETAR correction algorithm, currently implemented in commercial treatment planning systems do not predict the dose distribution accurately in many situations where lateral electron equilibrium does not exist. The lung phantom is made up of solid water slabs and lung analogue slabs. Using a thimble ionization chamber, a Markus ionization chamber and TLDs the problems of central axis dose reduction and penumbral flaring in lung for x-rays have been investigated. It is found that the ETAR correction predicts the dose at mid lung with varying degrees of accuracy depending on the field size. It was found that internal body cavities, depending on their size, experience underdose or overdose in the distal surfaces of the cavities when compared with the results predicted by an ETAR correction algorithm. Therefore, this energy is not recommended for use in situations where cavities arise / Master of Science (Hons)

Lung cancer (LC) is often diagnosed at advanced stage and as a result, survival rates are low. Recent studies describe exhaled breath and exhaled breath condensate (EBC) as a potential non-invasive method of sampling the airways for assessing inflammation of the respiratory system, and possibly for the early detection of LC. It was hypothesised that higher concentrations of markers and protein will be present in the EBC of LC patients compared to those of normal controls and healthy smokers, and may aid in assessing lung status. Methods: The gaseous phase of breath was investigated for volatile organic compound (VOC) patterns using an electronic nose (eNose) system, in addition to off-line measurements of carbon monoxide (CO) and nitric oxide (NO) levels. The aqueous phase, EBC, was collected during tidal breathing through a glass collection device cooled to 4??C by ice. Nitrite/nitrate (NOx) and pH levels were determined by a fluorescent modification of the Griess method, and silicon chip sensor pH meter, respectively. Protein levels in EBC were examined with a bicinchoninic acid (BCA) assay, silver staining and PAGE techniques, while the levels of tumour markers, CYFRA 21-1 and CEA, were quantified by enzyme-linked immunosorbent assays (ELISA). Results: The eNose machine was not able to produce characteristic VOC profiles from exhaled breath unique to each study group, while no significant difference was observed for mean NOx concentrations in the LC group when compared to other subjects (p=0.8824). Higher protein levels were found in the EBC of LC patient compared to normal controls (p=0.0204), with subsequent measurements of elevated CEA levels observed in the LC group when compared to non-smokers and smokers (p=0.023). Conclusion: This study showed that protein can be detected in the exhaled breath condensate of patients, with a significantly elevated amount in the samples from newly diagnosed LC patients. The mechanism for these differences remains to be determined but may be related to inflammatory changes within the airway, such as vascular protein leakage and release of mediators. Future work may aim to identify the upregulated proteins, and focus on proteomics and tissue microarrays to explore candidate proteins.

The fetal lung undergoes extensive physiological and biochemical maturation prior to birth in preparation for its postnatal function as an organ for gas exchange. Pulmonary surfactant, a substance that reduces surface tension and prevents alveolar collapse, is produced by type II pneumocytes within the lung. Reduced ability to produce surfactant leads to neonatal respiratory distress syndrome. Synthesis of the phospholipid component of surfactant, phosphatidylcholine (PC), is stimulated by fibroblast-pneumocyte factor (FPF), a protein expressed by fibroblast cells within the fetal lung. Although its function is well known, the identity of this important protein has remained a mystery. Recent research has suggested that FPF may be neuregulin-1, a growth factor found in many tissues during development. Enhanced synthesis of PC (and therefore detection of FPF) is measured using a tissue culture-based method. Primary cultures of lung fibroblasts and type II pneumocytes are prepared, and fibroblast-conditioned medium (FCM) is exposed to the type II cells. Resultant PC synthesis is measured using radioisotope-labeled PC-precursor and a chloroform-based lipid extraction method. Initial results using this method were very inconsistent, so a study was undertaken to determine which parts of the method could be contributing to this inconsistency. Cell density of type II cultures (measured in μg DNA.plate-1) was shown to have a significant effect on results. Treatment of fibroblasts with 100 nM dexamethasone and exposure of type II cultures to the resultant FCM caused a mean 9.17% increase in PC synthesis, but when only type II cultures with a cell density below 25 μg DNA.plate-1 were analyzed, this value increased to 17.56%. Type II cultures with cell density above this threshold value showed a mean increase in synthesis of only 3.39%. The consistent application of [3H]-choline chloride also had a significant effect on results. Experiments utilizing phorbol 12-myristate 13-acetate to stimulate fibroblasts were very inconsistent. The mean activity of the initial [3H]-choline chloride solution prepared for these experiments was found to be 2.04 μCi.mL-1, compared to a mean of 4.79 μCi.mL-1 for all other experiments. Observations from this section of the study led to considerable revision of the method used to measure PC synthesis. Quadrupolar ion trap mass spectrometry (MS) was used to analyze FCM and determine if neuregulin-1 (NRG1) could be FPF. A mass spectrum was obtained for recombinant NRG1, with predominant ions of 1068, 1142 and 1246 m/z. All three of these ions were also detected in both control and dexamethasone-treated FCM. Partial fragmentation of 1068 m/z of NRG1 was achieved using MS2, and generated a base peak of 1047 m/z. This fragmentation was also observed in 1068 m/z from FCM. LC/MS was utilized to quantify NRG1 in FCM, using a standard curve generated using recombinant NRG1. Control FCM had a NRG1 concentration of 19.85 μg.mL-1, while the concentration in dexamethasone treated FCM was 41.59 μg.mL-1. FCM which had given no positive response to dexamethasone when tested using the indirect cultured cell system had a control NRG1 concentration of 20.85 μg.mL-1, and a dexamethasone treated concentration of 22.84 μg.mL-1. These values were not significantly different from the control value for FCM in those fibroblast cultures that had generated a positive response to dexamethasone. Results of this section of the study have provided strong evidence that NRG1 is a major component of FPF, and a review of the NRG1 signaling pathway further supports this conclusion. Insulin-like growth factors (IGFs) are functionally related to neuregulins and are known to be important in fetal development. The effect of IGF-II on synthesis of surfactant PC and its subsequent secretion from type II pneumocytes was studied. In terms of PC synthesis, IGF-II was tested at concentrations of 0.4, 0.6 and 0.8 μM. The mean increase in synthesis was found to be 6.00, 6.15 and 6.91%, respectively. These values were not significantly different from control values. Secretion of PC was tested over the concentration range of 0.1 to 1.6 μM, with no significant effect observed. Possible inhibition by IGF-II was also studied, using the known stimulants of secretion, neuromedin C and isoproterenol. No significant effect on the enhanced level of secretion was observed when IGF-II was added with either secretagogue. Lack of an appropriate receptor and/or the possibility that cultured cells may not exactly mimic the situation in vivo are probably the reasons IGF-II has no effect on either synthesis or secretion.

Doctor of Philosophy (PhD) / The H69CIS200 cisplatin-resistant and H69OX400 oxaliplatin-resistant cell lines developed as part of this study, are novel models of low-level platinum resistance. These resistant cell lines do not have common mechanisms of platinum resistance such as increased expression of glutathione or decreased platinum accumulation. Rather, these cell lines have alterations in their cell cycle allowing them to proliferate rapidly post drug treatment in a process known as ‘regrowth resistance’. This alteration in cell cycle control has come at the expense of DNA repair capacity. The resistant cell lines show a decrease in nucleotide excision repair and homologous recombination repair, the reverse of what is normally associated with platinum resistance. The alterations in these DNA repair pathways help signal the G1/S checkpoint to allow the cell cycle to progress despite the presence of DNA damage. The decrease in DNA repair capacity has also contributed to the development of chromosomal alterations in the resistant cell lines. Similarities in chromosomal change between the two platinum resistant cell lines have been attributed to inherent vulnerabilities in the parental H69 cells rather than part of the mechanism of resistance. The H69CIS200 and H69OX400 resistant cells are cross-resistant to both cisplatin and oxaliplatin. This demonstrates that oxaliplatin does not have increased activity in low-level cisplatin-resistant cancer. Oxaliplatin resistance also developed more rapidly than cisplatin resistance suggesting that oxaliplatin may be less effective than cisplatin in the treatment of SCLC. The resistant cell lines have also become hypersensitive to taxol but show no alterations in the expression, polymerisation or morphology of tubulin. Rather, the PI3K/Akt/mTOR pathway is involved in both platinum resistance and taxol sensitivity as both are reversed with rapamycin treatment. mTOR is also phosphorylated in the resistant cell lines indicating that platinum resistance is associated with an increase in activity of this pathway. The mechanism of regrowth resistance in the platinum-resistant H69CIS200 and H69OX400 cells is a combination of activation of PI3K/Akt/mTOR signalling and alterations in control of the G1/S cell cycle checkpoint. However, more work remains to determine which factors in these pathways are governing this novel mechanism of platinum resistance.