During wintertime temperature inversion episodes the concentrations of particulate air pollution, also defined as particulate matter (PM), in Utah’s Cache Valley have often been highest in the nation, with concentrations surpassing more populated and industrial areas. This has attracted much local and national attention to the area and its pollution. The Cache Valley has recently been declared to be in non-attainment of provisions of Federal law bringing to bear Federal regulatory attention as well. While there is epidemiological evidence indicating that PM is detrimental to public health, there is much less information indicating by which biological and molecular mechanisms PM can exert harm. This study was undertaken to better understand the mechanisms by which ambient PM collected in the Cache Valley can be harmful to human lung cells. Cache Valley PM was found to be mildly cytotoxic only at concentrations that were much greater than physiologically achievable, and such concentrations were difficult to obtain with the limited amounts of captured ambient PM. The limited cytotoxicity was despite apparent PM-induced pro-apoptotic signaling such as caspase-3 upregulation, and activation of caspase-12 and calpain. Cache Valley PM was found to be stressful to cells, triggering endoplasmic reticulum stress and the unfolded protein response. Cache Valley PM was also found to be inflammogenic leading to activation of pro-inflammatory transcription factors, increases in the release of pro-inflammatory cytokines and chemokines, as well as the upregulation of the activating receptors of these cytokines. The proinflammatory effects and absence of apoptosis, despite pro-apoptotic signaling of the Cache Valley PM on human lung cells appeared to stem from increased activation of the central pro-growth protein Akt with subsequent inactivation of the tumor suppressor P-TEN. These findings have indicated novel mechanisms of PM-related cellular stress and inflammation contributing needed information on what may be underlying mechanisms of PM associcated illnesses.

Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality as well as delayed clearance of the virus. Efforts to improve influenza infection outcomes in neonates typically center on prevention, although current vaccines fall short of complete protection and can only be administered in humans after 6 months of life. We propose that as the neonatal immune system responds differently than the adult immune system, interventions that are efficacious or tolerable in adults cannot be guaranteed to perform the same in neonates. T cell vaccines that target conserved influenza virus epitopes have been proposed for conferring protection to multiple influenza virus strains, but if T cell vaccines will be used in infants and adults, neonates must be able to respond to the same T cell antigens as adults. Mouse pups responded to influenza virus peptide PA224-233 but not NP366-374 during influenza virus infection in contrast to the codominant adult response. Mice infected as pups also generated diminished T cell memory compared to mice infected as adults and displayed skewed immunodominance during secondary infection. Adult bone marrow derived dendritic cells (BMDCs) improved viral clearance when loaded with influenza virus and promoted NP366-374-specific CD8+ T cell responses in infected pups. BMDC peptide vaccination could stimulate PA224-233-specific but not NP366-374-specific CD8+ T cell responses in pups, but, PA224-233 vaccination offered no protection to pups during lethal infection. These data suggest that altered immunodominance must be considered when stimulating CD8+ T cell responses in adults and neonates. Immaturity and active suppression of immune responses are both factors in neonatal vulnerability to disease. Specifically, active suppression of neonatal immunity by regulatory T cells (Tregs) has been proposed as a driving factor in diminished neonatal immunity, but removing these cells can compromise viral defense or increase deleterious inflammation. Mice that lacked Tregs displayed compromised anti-influenza antibody responses and decreased lymph node responses during influenza virus infection. A high proportion of pup Tregs also expressed Gata3. Transgenic pups with a Treg specific Gata3 knockout displayed an increase in Tbet expression in both conventional and regulatory T cells and an increase in IFNγ producing CD4+ T cells in the lungs during infection. These data suggest that Tregs are required for effective humoral responses to influenza virus and that Gata3 expression influences Treg suppressive function in neonates.

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.