Dissertations / Theses on the topic 'Alveolar mechanics'

Estudo clínico prospectivo, em 11 pacientes com SARA ou LPA, avaliando o comportamento regional da densidade do tecido pulmonar e do colapso alveolar ao longo dos três eixos do espaço. Foram realizadas tomografias seriadas, após manobra de recrutamento inicial e após níveis de PEEP progressivamente decrescentes. Regressão linear múltipla (R2=0.83) mostrou importante gradiente no eixo gravitacional (p<0.001) e não no sentido céfalo-caudal (p<0.001), nem da direita para a esquerda (p<0.05). Isto corrobora o conceito do pulmão líquido, em que a resultante das pressões exercidas pelo diafragma, estruturas mediastinais e derrames seria transmitida uniformemente pelo tecido pulmonar. Cada um destes níveis isogravitacionais tem uma pressão crítica de fechamento (Pclosing), que é maior do que a pressão superimposta calculada. PEEP tem um efeito homogeneizador sobre o parênquima pulmonar. Dentre os parâmetros clínicos estudados, Pflex mostrou a pior correlação com colapso pulmonar documentado enquanto PO2 e a complacência máxima se mostraram equivalentes.
A prospective clinical study performed on 11 patients with ARDS or ALI with the intention of studying the regional behavior of lung tissue density and alveolar collapse along the three spatial axes. An initial recruitment maneuver was followed by multiple semi-complete CT scans at descending levels of PEEP. Multiple linear regression (R2=0.83) showed a gravitational gradient of densities and collapse (p<0.001) and no cephalo-caudal (p<0.001) or right-toleft increase (p<0.05), corroborating the liquid-like behavior of the lung. Pressure exerted by mediastinal structures, chest wall and effusions is transmitted uniformly throughout the lung. PEEP has a homogenizing effect on lung parenchyma. Among commonly used clinical surrogates, Pflex showed the worst correlation with actual lung collapse, while arterial PO2 and compliance were equivalent.

In this thesis, we describe several imaging techniques specifically designed and developed for the assessment of pulmonary structure, function and pathology. We then describe the application of this technology within appropriate biological systems, including the identification, tracking and assessment of lung tumors in a mouse model of lung cancer. The design and development of a Large Image Microscope Array (LIMA), an integrated whole organ serial sectioning and imaging system, is described with emphasis on whole lung tissue. This system provides a means for acquiring 3D pathology of fixed whole lung specimens with no infiltrative embedment medium using a purpose-built vibratome and imaging system. This system enables spatial correspondence between histology and non-invasive imaging modalities such as Computed Tomography (CT), Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET), providing precise correlation of the underlying 'ground truth' pathology back to the in vivo imaging data. The LIMA system is evaluated using fixed lung specimens from sheep and mice, resulting in large, high-quality pathology datasets that are accurately registered to their respective CT and H&E histology. The implementation of an in vivo micro-CT imaging system in the context of pulmonary imaging is described. Several techniques are initially developed to reduce artifacts commonly associated with commercial micro-CT systems, including geometric gantry calibration, ring artifact reduction and beam hardening correction. A computer controlled Intermittent Iso-pressure Breath Hold (IIBH) ventilation system is then developed for reduction of respiratory motion artifacts in live, breathing mice. A study validating the repeatability of extracting valuable pulmonary metrics using this technique against standard respiratory gating techniques is then presented. The development of an ex vivo laser scanning confocal microscopy (LSCM) and an in vivo catheter based confocal microscopy (CBCM) pulmonary imaging technique is described. Direct high-resolution imaging of sub-pleural alveoli is presented and an alveolar mechanic study is undertaken. Through direct quantitative assessment of alveoli during inflation and deflation, recruitment and de-recruitment of alveoli is quantitatively measured. Based on the empirical data obtained in this study, a new theory on alveolar mechanics is proposed. Finally, a longitudinal mouse lung cancer study utilizing the imaging techniques described and developed throughout this thesis is presented. Lung tumors are identified, tracked and analyzed over a 6-month period using a combination of micro-CT, micro-PET, micro-MRI, LSCM, CBCM, LIMA and H&E histology imaging. The growth rate of individual tumors is measured using the micro-CT data and traced back to the histology using the LIMA system. A significant difference in tumor growth rates within mice is observed, including slow growing, regressive, disappearing and aggressive tumors, while no difference between the phenotype of tumors was found from the H&E histology. Micro-PET and micro-MRI imaging was conducted at the 6-month time point and revealed the limitation of these systems for detection of small lesions ( < 2mm) in this mouse model of lung cancer. The CBCM imaging provided the first high-resolution live pathology of this mouse model of lung cancer and revealed distinct differences between normal, suspicious and tumor regions. In addition, a difference was found between control A/J mice parenchyma and Urethane A/J mice ‘normal’ parenchyma, suggesting a 'field effect' as a result of the Urethane administration and/or tumor burden. In conclusion, a comprehensive murine lung cancer imaging study was undertaken, and new information regarding the progression of tumors over time has been revealed.

Mechanical ventilation has been a major therapy used by physicians in support of surgery as well as for treating patients with reduced lung function. Despite its many positive outcomes and ability to maintain life, in many cases, it has also led to increased injury of the lungs, further exacerbating the diseased state. Numerous studies have investigated the effects of long term ventilation with respect to lungs, however, the connection between the global deformation of the whole organ and the strains reaching the alveolar walls remains unclear. The walls of lung alveoli also called the alveolar septum are characterized as a multilayer heterogeneous biological tissue. In cases where damage to this parenchymal structure insist, alveolar overdistension occurs. Therefore, damage is most profound at the alveolar level and the deformation as a result of such mechanical forces must be investigated thoroughly. This study investigates a three-dimensional lung alveolar model from generations 22 (alveolar ducts) through 24 (alveoli sacs) in order to estimate the strain/stress levels under mechanical ventilation conditions. Additionally, a multilayer alveolar tissue model was generated to investigate localized damage at the alveolar wall. Using ANSYS, a commercial finite element software package, a fluid-structure interaction analysis (FSI) was performed on both models. Various cases were simulated that included a normal healthy lung, normal lung with structural changes to model disease and normal lung with mechanical property changes to model aging. In the alveolar tissue analysis, strains obtained from the aged lung alveolar analysis were applied as a boundary condition and used to obtain the mechanical forces exerted as a result. This work seeks to give both a qualitative and quantitative description of the stress/strain fields exerted at the alveolar region of the lungs. Regions of stress/strain concentration will be identified in order to gain perspective on where excess damage may occur. Such damage can lead to overdistension and possible collapse of a single alveolus. Furthermore, such regions of intensified stress/strain are translated to the cellular level and offset a signaling cascade. Hence, this work will provide distributions of mechanical forces across alveolar and tissue models as well as significant quantifications of damaging stresses and strains.

The alveolar epithelium is a key functional component of the air-blood barrier in the lung. Comprised of two morphologically distinct cell types, alveolar epithelial type I (ATI) and type II (ATII) cells, effective repair of the alveolar epithelial barrier following injury appears to be an important determinant of clinical outcome. The prevailing view suggests this repair is achieved by the proliferation of ATII cells and the transdifferentiation of ATII cells into ATI cells. Supplemental oxygen and mechanical ventilation are key therapeutic interventions in the supportive treatment of respiratory failure following lung injury, but the effects of hyperoxia and mechanical deformation in the injured lung, and on alveolar epithelial repair in particular, are largely unknown. The clinical impression however, is that poor outcome is associated with exposure of injured (repairing) epithelium to such iatrogenic ‘hits’. This thesis describes studies investigating the hypothesis that hyperoxia & mechanical deformation inhibit normal epithelial repair. The in vitro data presented demonstrate that hyperoxia reversibly inhibits the transdifferentiation of ATII-like cells into ATI-like cells with time in culture. Whilst confirming that hyperoxia is injurious to alveolar epithelial cells, these data further suggest the ATII cell population harbours a subpopulation of cells resistant to hyperoxia-induced injury. This subpopulation of cells appears to generate fewer reactive oxygen species and express lower levels of the zonula adherens protein E-cadherin. Using a panel of antibodies to ATI (RTI40) and ATII (MMC4 & RTII70) cell-selective proteins, the effect of hyperoxia on the phenotype of the alveolar epithelium in a rat model of resolving S. aureus-induced lung injury was investigated. These in vivo studies support the view that, under normoxic conditions, alveolar epithelial repair occurs through ATII cell proliferation & transdifferentiation of ATII cells into ATI cells, with transdifferentiation occurring via a novel intermediate (MMC4/RTI40-coexpressing) immunophenotype. However, in S. aureus-injured lungs exposed to hyperoxia, the resolution of ATII cell hyperplasia was impaired, with an increase in ATII cell-staining membrane and a reduction in intermediate cell-staining membrane compared to injured lungs exposed to normoxia alone. As hyperoxia is pro-apoptotic and known to inhibit ATII cell proliferation, these data support the hypothesis that hyperoxia impairs normal epithelial repair by inhibiting the transdifferentiation of ATII cells into ATI cells in vivo. The effect of mechanical deformation on alveolar epithelial cells in culture was investigated by examining changes in cell viability following exposure of epithelial cell monolayers to quantified levels of cyclic equibiaxial mechanical strain. In the central region of monolayers, deformation-induced injury was a non-linear function of deformation magnitude, with significant injury occurring only following exposure to strains greater than those associated with inflation of the intact lung to total lung capacity. However, these studies demonstrate for the first time that different epithelial cell phenotypes within the same culture system have different sensitivities to deformation-induced injury, with spreading RTI40-expressing cells in the peripheral region of epithelial cell monolayers and in the region of ‘repairing’ wounds being injured even at physiological levels of mechanical strain. These findings are consistent with the hypothesis that alveolar epithelial cells in regions of epithelial repair are highly susceptible to deformation-induced injury.

The kinetics and mechanism of accumulation for liposomal ciprofloxacin (Lipo-CPFX) into the rat alveolar macrophage NR8383 cells were studied in vitro, in comparison to unformulated ciprofloxacin (CPFX). Upon incubation with CPFX or Lipo-CPFX, cellular drug accumulation was determined from the cell lysates or efflux was from the extracellular media by fluorescence-HPLC. The accumulation for Lipo-CPFX reached the asymptotic values at ≥ 2 hours, which was a result of uptake and efflux. The uptake appeared to be due to liposomes, mediated via cellular energy-independent mechanism like lipid fusion. In contrast, the efflux appeared to be due to ciprofloxacin, partly cellular energy-dependent, and involve probenecid-sensitive multidrug resistance proteins (MRPs). Overall, Lipo-CPFX enabled greater drug accumulation into the NR8383 cells than CPFX. This logically suggests a greater potential to treat respiratory infections especially caused by bacteria resistant to phagocytic killing.

Orientador: Jose Ricardo de Albergaria Barbosa
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-03T23:42:34Z (GMT). No. of bitstreams: 1 FreireFilho_FranciscoWagnerVasconcelos_D.pdf: 1185958 bytes, checksum: d471fe34fa012fb2c3725a04c776609d (MD5) Previous issue date: 2004
Resumo: O objetivo deste trabalho foi comparar dois sistemas de distratores osteogênicos alveolares justa-ósseos de 9mm de abertura máxima de fabricação nacional. Foram analisadas duas marcas comerciais (Grupo I e Grupo II), através das avaliações dimensionais dos distratores e parafusos, da resistência à tração dos distratores e da resistência à flexão e à torção dos parafusos. Na avaliação dimensional foram utilizados dez distratores de cada fabricante e quarenta parafusos, sendo dez de cada tamanho oferecido, 1,3 x 5mm e 1,3 x 7mm do grupo I e 1,5 x 5mm e 1,5 x 7mm do grupo II. Os dados foram submetidos ao teste dos postos assinalados de Wilcoxon para os distratores e o teste de Friedman para os parafusos. No teste de resistência à tração foram utilizados dez distratores de cada marca comercial e os resultados submetidos ao teste t Student. No teste de resistência à flexão foram utilizados quarenta parafusos e aplicados os testes F e de Tukey, ao nível de significância de 5%. No teste de resistência à torção, foram utilizados dez parafusos de cada fabricante, sendo do tipo 1,3 x 5mm do grupo I e 1,5 x 5mm do grupo II e os resultados submetidos ao teste t Student. Os distratores do grupo II apresentaram menor variação das mensurações realizadas, porém não houve diferença estatística entre os parafusos de ambas as marcas. Os distratores do grupo II foram estatisticamente mais resistentes à tração. Os parafusos de 1,5x5mm do grupo II foram os mais resistentes à flexão e à torção
Abstract: The aim of this study was to perform a comparative analysis between two different alveolar distractions devices, of 9mm length, built by manufactured in Brazil. These two different devices were provided by companies (group I and group II). The analysis consisted of a macroscopic assessment from the distraction devices and its screws, followed by a traction resistance of the devices and torsion and bending resistance of the screws. Were used, for macroscopic assessment, ten distraction devices and forty screws, which included ten screws of every length offered by each company. Data was submitted to the Wilcoxon test for devices and Friedman test for screws. Ten distraction devices from each company were used for the traction resistance, and its results were submitted to the Student t test. Forty screws were used for the bending resistance. Values were compared trough F and Tukey test, with 5% significance. For the torsion resistance, ten screws from each company were used. The 1.3 X 5mm screws from group I and the 1.5 X 5mm from group II were chose to realize this test, and its results were submitted to the Student t test. Devices from group II presented less variation of its measurements, but there were not any statistical difference between the screws. Group II devices were tatistically more resistant to traction. Screws 1.5x5mm, produced by group II, were more resistant to bending and to torsion
Doutorado
Cirurgia e Traumatologia Buco-Maxilo-Faciais
Doutor em Clínica Odontológica

THESIS SUMMARY:

GENERAL AIM

The general aim of this thesis was to study the generation of contractile force by human alveolar epithelial cells in culture in response to biochemical or mechanical stimuli using traction microscopy.

SPECIFIC AIMS

1. To implement a traction microscopy setup to measure the contractile force generated by human alveolar epithelial cells in culture.
1.1. To implement and validate a software to determine the deformation field induced by adhered cells on the elastic substrate, following previously described algorithms.
1.2. To implement and validate a software to determine the traction field induced by adhered cells and other contractility parameters, following previously described algorithms.
1.3. To implement a software to determine the contour of an adhered cell from a brightfield or phase contrast image of the cell.

2. To study the contractile response of human alveolar epithelial cells in response to thrombin.
2.1. To determine the gel substrate conditions and gel fabrication procedure which enable suitable cell culture and optimal detection of traction forces exerted by human alveolar epithelial cells. These gel conditions include: concentration of polyacrilamide gel components to provide optimal gels stiffness; concentration of fluorescent beads to optimally compute gel deformation; and suitable gel coating to enable cell attachment.
2.2. To determine the gel elastic properties (Young's modulus) by atomic force microscopy.
2.3. To measure the time-course of the contractile response to thrombin challenge.
2.4. To study the distribution of contractile forces exerted by adhered cells before and after thrombin stimulation.
2.5. To measure actin polymerization and reorganization induced by thrombin challenge.
2.6. To study the role of the actin cytoskeleton in the contractile response to thrombin by pre-treatments with cytochalasin D.
2.7. To study the role of pathways signalling MLC phosphorilation in the contractile response to thrombin by pre-treatments with ML7 and Y-27632.

3. To study the contractile response of human alveolar epithelial cells subjected to stretch.
3.1. To determine a suitable gel substrate that firmly attaches to a flexible membrane, allowing biaxial stretch application (max ~15%) and cell culture.
3.2. To determine the gel elastic properties (Young's modulus) of the gel at different strain levels by atomic force microscopy.
3.3. To implement and validate a stretching device to apply controlled biaxial and uniform strains to cultured cells and simultaneously measure contractile forces by deforming the gel substrate to which they are adhered.
3.4. To adapt the existing traction microscopy algorithms and software to allow computation of large bead displacements (~20 μm) and corresponding stretch fields.
3.5. To measure contractile forces exerted by human alveolar epithelial cells before, during and after being subjected to a stepwise deformation of up to 11.5% linear strain.
3.6. To assess the role of actin polymerization in the contractile response to stretch.
3.7. To assess the role of actomyosin crossbridges attachment or detachment in the contractile response to stretch.
3.8. To assess temporal changes in cell contractility after stretch release.
"Estudi de la contracció de cèl·lules epitelials alveolars en resposta a estímuls inflamatoris i de deformació mitjançant microscopia de tracció"

TEXT:

L'epiteli alveolar forma una barrera cel·lular semipermeable entre l'espai alveolar i l'interstici del pulmó, permetent l'intercanvi gasós a la vegada que restringeix el pas de líquid, macromolècules i cèl·lules cap a l'alvèol. El trencament de la monocapa, degut a la formació de forats entre cèl·lules adjacents, pot donar lloc a l'augment de la permeabilitat i l'entrada de líquid a l'alvèol, característics del dany pulmonar agut. La integritat de la monocapa epitelial es regeix per un equilibri dinàmic de forces als punts d'unió cèl·lula-cèl·lula, i cèl·lula-matriu. Les forces en joc es divideixen en una component de tensió centrípeta i una component d'adhesió centrífuga. La component centrípeta es deguda a les forces de contracció generades activament per la maquinària contràctil cel·lular i el retrocés passiu degut a la deformació cíclica a la que es troben sotmeses les cèl·lules alveolars durant la respiració. Per tal de garantir la integritat de la monocapa, les forces d'adhesió han de ser capaces de contrarestar la tensió centrípeta. L'equilibri de forces als punts d'unió pot veure's compromès degut a estímuls inflamatoris o bé mecànics.

El projecte de tesi es centra en el paper de les forces actives de contracció sobre la integritat de la monocapa alveolar en resposta a estímuls característics del dany pulmonar agut. Per tal d'estudiar aquesta component contràctil, el present projecte ha utilitzat la microscopia de tracció. Aquesta tècnica permet mesurar la força que cèl·lules adherents aïllades realitzen sobre el seu substrat, així com la seva distribució espaial i evolución temporal. La tècnica consisteix en cultivar cèl·lules adherents sobre substrats elàstics que contenen microesferes fluorescents. Comparant la posició de les microesferes quan la cèl·lula es troba adherida al substrat i un cop aquesta ha estat desenganxada amb tripsina, podem calcular la força que la cèl·lulaadherent realitzava sobre el substrat.

El projecte de tesi inclou dos estudis concrets, dedicats a dos estímuls característics de dany pulmonar agut als qual poden trobar-se sotmeses les cèl·lules epitelials alveolars. El primer estudi (secció 3) es centra en l'efecte que el mediador inflamatori trombina provoca sobre la realització de forces contràctils per part de cèl·lules alveolars epitelials. El segon estudi (secció 4) es centra en la resposta contràctil de cèl·lules alveolars epitelials a l'aplicar deformacions externes, simulant les condicions de respiració mecànica que requereixen molts pacients amb dany pulmonar agut.

Diversas estratégias de ventilação mecânica que estabelecem limites na pressão e volume intratorácicas têm sido propostas para pacientes com síndrome de angustia respiratória aguda (SARA). Estas recomendações são baseadas na observação de que a ventilação mecânica com volume corrente excessivo ou pressão positiva expiratória final (PEEP) insuficiente pode ocasionar lesões pulmonares graves, decorrentes de superdistensão de unidades alveolares. O objetivo do atual estudo foi aplicar manobras de recrutamento alveolar e PEEP em pulmões submetidos à lesão pulmonar aguda (LPA) através da administração de HCl . Foram utilizados 24 suínos Landrace - Largewhite, do sexo feminino, pesando entre 25 e 35 Kg. Após a anestesia os animais foram submetidos à ventilação com volume controlado (6 a 8 ml/Kg) e foram randomizados em 4 grupos: GI (6 animais não submetidos a LPA e tratados com PEEP progressivo de 5, 10, 15 e 20 cmH2O e regressivo de 20 a 5 cmH2O); GII (6 animais não submetidos a LPA e tratados com PEEP progressivo de 5, 10, 15 e 20 cmH2O e regressivo de 20 a 5 cmH2O associado a 3 manobras de recrutamento consecutivas, com pressão de 30 cmH2O antes de cada alteração do PEEP); GIII (6 animais submetidos a 1 hora de LPA por HCl e tratados como GI) e GIV (6 animais submetidos a 1 hora de LPA por HCl e tratados como GII). A mecânica respiratória e oxigenação foram avaliadas a cada 20 minutos, acompanhando cada alteração do PEEP. A LPA foi observada através de severas alterações na oxigenação e mecânica respiratória. A administração de MR associada a PEEP foi capaz de restaurar os valores controle, porém, os elevados valores de PEEP e CPAP foram acompanhados de significantes alterações hemodinâmicas quando comparadas com os animais que não foram submetidos a LPA. O derrecrutamento alveolar ocorreu provavelmente quando os valores de PEEP foram retornados para 5cmH2O. As lesões pulmonares foram uniformes nos animais que foram submetidos ao HCl, evidenciadas pela presença de necrose, hemorragia, congestão e infiltrado de células inflamatórias no interstício e nos alvéolos. O modelo experimental de lesão pulmonar aguda foi adequado para estudar MR seguidas por PEEP, pois apresentou importantes alterações dos valores de oxigenação e complacência, observado 1 hora após a instilação do HCl.Os valores de PEEP de 5cmH2O foram incapazes de manter o recrutamento no momento final do estudo, enquanto que os valores de PEEP de 10cmH2O foram suficientes para restabelecer a oxigenação com mínima alteração hemodinâmica. A complacência não melhorou após as manobras. Futuros estudos são necessários para confirmar os resultados obtidos, especialmente para mostrar que a manutenção do PEEP de 10cmH2O é suficientes para manter o recrutamento após as MR
Different mechanical ventilation strategies which define limits of intrathoracic pressures and volumes are being proposed for patients with acute respiratory distress syndrome (ARDS). These recommendations are based on observations that mechanical ventilation with excessive tidal volumes or insufficient values of positive end expiratory pressure (PEEP) can cause severe lung injury due to overinflation. The aim of the present study was to apply recruitment maneuvers (RM) and PEEP in lungs submitted to acute lung injury (ALI) due to the administration of hydrochloride acid. Twenty four female Landrace – Largewhite pigs, weighing 25 to 35 Kg were used. After anesthesia, animals were submitted to volume controlled mechanical ventilation (6 to 8ml/kg) and were randomly allocated in four groups of 6 animals each: GI animals without ALI and treated with progressive values of PEEP (5, 10, 15 and 20 cmH2O) or regressive (20 to 5 cm H2O); GII animals without ALI and treated with progressive values of PEEP (5, 10, 15 and 20 cmH2O) or regressive (20 to 5 cm H2O) plus 3 consecutive recruitment maneuvers with 30 cmH2O; GIII animals submitted to 1 hour of ALI and treated as GI; GIV animals submitted to 1 hour of ALI and treated as GII. Parameters of respiratory mechanics, ventilation and oxygenation were measured each 20 minutes according to the change of the PEEP values. ALI could be observed by the severe changes of oxygenation and respiratory mechanics noted. The use of RM and PEEP were able to restore control values. Nevertheless, application of high values of PEEP and CPAP were accompanied by significant hemodynamic changes which could be evidenced in animals without ALI. Derecruitment probably occurred when PEEP value reached 5 cmH2O. The lung lesions were uniform in the HCL-injured animals and consisted of necrosis, hemorrhage, congestion, and inflammatory cells infiltration that involved both the interstitium and the alveoli. The experimental model of lung injury was adequate to the study of RM followed by PEEP since significant changes of the oxygenation and compliance values could be observed 1 hour after acid instillation. PEEP values of 5cmH2O were incapable to maintain recruitment at the end of the observation period, while 10 cmH2O were sufficient to promote the reestablishment of oxygenation index with minimal hemodynamic changes. Compliance did not improve during the maneuvers. Further studies are necessary to confirm the results obtained, especially to show that the maintenance of a PEEP value of 10 cmH2O are sufficient to maintain recruitment after the RM

Francisella tularensis is a facultative intracellular pathogen and is the etiological agent of tularemia. One key aspect to the success of Francisella as a pathogen is ability of the organism to establish infection with a low inoculum, as few as 10 colony forming units (cfu). Essential to this process is the Francisella pathogenicity island (FPI). Several studies have been performed to understand how the FPI is regulated; however, the working model is not complete, as the signals important for regulation are unknown. Additionally, the mechanisms of the proteins MigR, TrmE, and CphA, which are important for activation of the FPI, are unknown. I initiated the study of this regulatory system by measuring the ability of various cellular stresses to activate an iglA-lacZ reporter. I identified that amino acid starvation and growth in basic pH activated expression of the reporter in both LVS and Schu S4. By combining these two stresses I was able to induce iglA-lacZ reporter expression in an additive manner. As it was previously demonstrated that ppGpp is important for stabilization of the regulatory complex that transcribes FPI genes, I demonstrated by TLC that both amino acid starvation and basic pH effected iglA-lacZ expression by increasing ppGpp. Due to the importance of ppGpp in FPI expression and because MigR, TrmE, and CphA each appear to be involved in a metabolic process: fatty acid metabolism (migR) t-RNA modification (trmE) and amino acid storage (cphA), I had hypothesized that the effect on these mutations were due to decreased levels of the small alarmone ppGpp. I compared ppGpp accumulation of LVS mutants in migR, trmE, and cphA to the parent strain and observed that loss of these genes resulted in reduced ppGpp. To better understand the importance of ppGpp synthesis in F. tularensis pathogenesis, I compared the phenotypes of these strains in primary human macrophages and two immortalized epithelial cell lines. These experiments demonstrated that although each of these strains had reduced ppGpp, there were cell line specific growth phenotypes. Mice infected with these strains survived suggesting tight regulation of the FPI is required for virulence. When similar mutations were characterized in the Schu S4 background these mutations retained their regulatory role; however, mutation of migR did not significantly decrease virulence in mice. As my data demonstrated that there are different challenges that Francisella must overcome to successfully replicate within cells, I developed an in vitro model to study the interactions of F. tularensis with human alveolar type II cells (AT-II). Interestingly, Schu S4 internalizes and replicates in these recently immortalized human AT-II cells whereas, LVS internalizes, but replicates poorly within these cells. Finally, to better understand the role of AT-II cells in vivo, I performed Transmission Electron Microscopy (TEM) of infected mice. These data confirmed that Schu S4 infected both alveolar macrophages and AT-II cells. Together, this work contributes to the understanding of how Francisella adapts to various environments by modulating virulence gene expression and highlights differences between virulent Schu S4 and LVS, which may partially contribute to virulence differences observed between strains.

Les nanomatériaux carbonés (NMC) sont très utilisés dans le monde industriel et leurs applications, nombreuses, sont en plein développement. L’absence de réglementation pour leur préparation et leur emploi fait qu’il est nécessaire comme pour tous les nano-objets, de déterminer le risque qu’une exposition fait courir à l’Homme et d’adapter la législation en conséquence. Une meilleure connaissance de leur potentiel toxique est donc nécessaire. Les difficultés de plus en plus grandes pour utiliser les modèles animaux, rend nécessaire le développement d’études avec des lignées cellulaires au sein desquelles les macrophages ont une place prépondérante. Ces NMC sont très légers et forment facilement des aérosols et les modèles préférés sont les macrophages alvéolaires. Cependant il n’existe pas à l’heure actuelle de lignées de macrophages alvéolaires humains à la différence de cellules de rat. Le sujet de ma thèse porte sur l’étude de la réponse macrophagique aux NMC et la compréhension des effets de leurs caractéristiques physiques et chimiques sur leur transcriptome. Les NMC étudiés sont les nanotubes de carbone (NTC) multi feuillets, les NTC mono feuillets, le noir de carbone et l’oxyde de graphène. Nos résultats montrent que tous les NMC étudiés déclenchent une réaction inflammatoire dans les cellules NR8383 et les cellules THP-1 différenciées, et certains d’entre eux induisent une cytotoxicité importante. La taille, la fonctionnalisation et la forme contrôlent les mécanismes de toxicité induits par les NMC. Des NTC de tailles similaires altèrent des voies de signalisation identiques, une fonctionnalisation par des groupements amines produit un stress des lysosomes tandis que la fonctionnalisation par des groupements carboxyle entraine un stress du réticulum endoplasmique (RE). Les nanotubes induisent une désorganisation du cytosquelette plus importante que les nanoparticules sphériques. Nous avons également mis en évidence une accumulation de lipides chez les cellules NR8383 suite à un stress du RE induit par le Mitsui-7, un NTC multi feuillet. Le même NTC induit aussi une fusion de ces macrophages. La formation de ces cellules spumeuses et des cellules géantes à multi-noyaux sont des évènements clés entrainant la formation de granulomes. Les résultats obtenus présentent un support important pour la compréhension des effets des NMC montrant une certaine toxicité non négligeable de point de vue moléculaire. Cette toxicité est dépendante des caractéristiques physiques et chimiques de ces nanomatériaux. Ainsi, en se basant sur ce type de données, on pourra s’orienter vers une fabrication safe-by-design pour limiter les risques liés à leur exposition
Carbon nanomaterials (CNM) are widely used in the industrial world and they have many applications. The absence of legislation controlling their preparation and uses makes necessary, as for all nano-objects, the study of their toxicity in order to determine the risk of human exposure and to adapt legislation accordingly. Therefore, a better knowledge of their toxic potential is necessary. The increasing difficulties in using animal models make necessary the development of studies using cell lines especially macrophages that play a predominant role. These CNM are very light and form easily aerosols, reason why the preferred models for toxicity studies are alveolar macrophages. However, there are no human alveolar macrophage lines currently but rat cells exist. The subject of my thesis is to study macrophages response to CNM and the understanding of the effect of their physical and chemical characteristics on the transcriptome. The CNM studied are multiwall carbon nanotubes (CNT), single wall CNT, carbon black and graphene oxide. Our results show that all CNM studied trigger an inflammatory reaction in NR8383 and differentiated THP-1 cells, also some of them induce cytotoxicity. Size, functionalization and form control CNM toxicity mechanisms: CNT with similar size alter identical signaling pathways, amino group functionalization produces lysosomal stress, whereas functionalization with carboxyl groups causes reticulum endoplasmic (RE) stress, nanotubes induce cytoskeleton disorganization more than spherical nanoparticles. Otherwise, we identified lipid accumulation in NR8383 cells due to RE stress induced by Mitsui-7, a multiwall CNT. There was also a fusion of these macrophages. The formation of these foam cells and giant multi-nucleus cells are key events leading to granulomas formation. The results obtained are an important support for understanding CNM effects, showing some significant toxicity at molecular level. This toxicity is dependent on the physical and chemical characteristics of these nanomaterials. Thus, based on this type of data, we can move towards a safer manufacture to avoid the risks associated with their exposure

Mais de 60% dos pacientes com Doença Pulmonar Obstrutiva Crônica (DPOC) podem apresentar hiper-responsividade brônquica. Entretanto, não se sabe se, além das vias aéreas, o tecido pulmonar periférico também apresenta uma resposta exagerada a um agonista na DPOC. No presente estudo foi investigado o comportamento mecânico in vitro e as alterações estruturais e inflamatórias do tecido pulmonar periférico de 10 pacientes com DPOC comparados com 10 controles não fumantes. Foram realizadas medidas de resistência (R) e elastância (E) de fatias pulmonares em situação basal e após desafio com Acetilcolina. Também foram analisados no tecido alveolar as densidades de neutrófilos, eosinófilos, macrófagos, mastócitos e linfócitos CD8+ e CD4+, além do conteúdo de células positivas para -actina de músculo liso, fibras elásticas e colágenas. Os valores de R após o tratamento com Acetilcolina (RACh) e a porcentagem de aumento de resistência (%R) foram significativamente maiores no grupo DPOC comparado ao grupo controle (p0,03). O grupo DPOC também apresentou densidade de macrófagos (p=0,04) e linfócitos CD8+ (p=0,017) significativamente maior e conteúdo de fibras elásticas significativamente menor (p=0,003) comparado ao grupo controle. Foi observada uma correlação positiva significativa entre a %R e a densidade de eosinófilos e linfócitos CD8+ (r=0,608, p=0,002; e r=0,581, p=0,001, respectivamente), e também uma correlação negativa significativa entre a %R e a relação VEF1/ CVF (r=-0,451, p<0,05). Concluímos que a resposta colinérgica de fatias de parênquima pulmonar está aumentada em pacientes com doença pulmonar obstrutiva crônica e parece estar relacionada tanto à densidade de eosinófilos e de linfócitos CD8+ no tecido alveolar quanto ao grau de obstrução determinado pela prova de função pulmonar.
Up to 60% of COPD patients can present airway hyperresponsiveness. However, it is not known whether the peripheral lung tissue also presents an exaggerated response to agonists in COPD. In this study we investigated the in vitro mechanical behavior and structural and inflammatory changes of peripheral lung tissue of 10 COPD patients and compared to 10 non-smoking controls. We measured resistance (R) and elastance (E) of lung strips at baseline and after acetylcholine (ACh) challenge. We further assessed the alveolar tissue density of neutrophils, eosinophils, macrophages, mast cells and CD8+ and CD4+ cells, and the content of -smooth muscle actin+ cells, elastic fibers and collagen fibers. Values of R after ACh treatment (RACh) and percent increase of tissue resistance (%R) were significantly higher in COPD group compared to controls (p0.03). There was a significantly higher density of macrophages (p=0.04) and CD8+ cells (p=0.017) and a lower elastic fiber content (p=0.003) in COPD group compared to controls. We observed a significant positive correlation between %R and eosinophil and CD8+ cells density (r=0.608, p=0.002; and r=0.581, p=0.001, respectively), and also a negative correlation between %R and FEV1/FVC (r=-0.451, p<0.05). We conclude that the cholinergic responsiveness of parenchymal lung strips is increased in COPD patients and seems to be related to alveolar tissue eosinophilic and CD8 lymphocytic inflammation and also to the degree of airway obstruction at pulmonary function test.

Etude du vieillissement de polymeres renforces ou non de fibres de verre ou de mousses syntactiques immerges dans l'eau sous des pressions de 0 a 300 bars. Adsorption d'eau, proprietes mecaniques, eclatement des microspheres dans les mesures syntactiques

博士
國立臺灣大學
毒理學研究所
85
Mercury compounds of both natural and industrial origin are wide- spread environmental pollutants. Organic mercury compounds have been involved in large-scale poisoning episodes. Human exposure to mercurials occurs in the production of batteries, in chloralkali plants, gold mining, dentistry, and the use of grain fungicides. Although the primary targets of mercury compounds have been identified as the nervous systems and the kidney, toxic effects on other organ systems have also been reported. Of particular concern are the effects of mercury compounds on the immune system, which include effects of methylmercury (MeHg) on T cells, B cells, and natural killer cells. Although mercury compounds are considered to be immunosuppressive agents, their effects on the alveolar macrophages are still not clear. The important function of alveolar macrophages has been recognized to be a mediator of a variety of essential biologic activities, particularly in the acute inflammatory response and tissue repair. Therefore we attempted to study the effect of MeHg on rat alveolar macrophages. The Mechanism of MeHg-induced Apoptosis of Alveolar Macrophages First of all, through the use of a scanning electron microscope, we have found that alveolar macrophages treated with 10 mM of MeHg for 24 hrs showed a decrease of surface microvilli, and those treated with 15 m M of MeHg underwent deformity and subsequent cell death. To investigate their death patterns, we aspirated DNA from alveolar macrophages and analyzed them by gelelectrophoresis. We discovered that the DNA ladder phenomenon became more obvious as the MeHg increased in concentration. When we intracellularly applied 5 mM EGTA to eliminate calcium ions, we observed a decrease of the ladder phen omenon. Zinc at 1 mM had a similar inhibitory effect. Moreover, an apoptosis peak was observed on flow cytometry analysis of DNA stained with propidium iodide. Alveolar macrophages stained with Hoechst 33342 demonstrated apoptotic bodies induced by MeHg. From the above data, we know that MeHg can induce a typical apoptosis in alveolar macrophages. As we moved on to study the mechanism of apoptosis as induced by MeHg in alveolar macrophages, we discovered that MeHg could increase the intracellular calcium ion concentration and decrease the pH in alveolar macrophages. To find out which endonuclease was responsible for the MeHg-induced DNA fragmentation of alveolar macrophages, we aspirated the nuclear proteins of alveolar macrophages and test them under different pH values and in conditions with or without calcium ions, and discovered that the endonuclease activity was calcium-dependent without relations to pH values. Therefore, MeHg first increased the calcium level, which in turn increased the endonuclease activity and induced DNA fragmentation and apoptosis. Study on MeHg-induced Necrosis and Apoptosis of PMNs Mediated by Calcium and Acid Mercury is not only an environmental pollutant, but a drug with clinical use as well. The neutrophils is a kind of phagocytic cell that plays the role of a scavenger in human bodies, so it has a greater chance of exposure to MeHg. By evaluating the cytotoxicity of MeHg on PMNs, we can better understand the actions of MeHg. Addition of MeHg of 10 mM to the PMNs increased the intracellular calcium ion concentration and induced a drop in pH, which reached a trough at the 100th second before gradually returning to a neutral value. When the extracellular calcium was eliminated by 5 mM EGTA, the first phase disappeared, but the second phase remained. The L-type calcium channel blocker verapamil was able to block the first phase. These results testified that the first phase of calcium ion increase induced by MeHg was mediated by a clacium channel, while the second phase resulted from the calcium ion release from the intracellular calcium ion storage pool. When we used propidium iodide (PI) to investigate the MeHg-induced neutrophils death, we discovered that the cytotoxicity of 15 mM MeHg augmented as the extracellular calcium ion concentration increased. On the contrary, when we lowered the extracellular calcium ion level with 5 mM EGTA, the toxicity of MeHg lessened significantly. Therefore, the MeHg toxicity is closely associated with the amount of influx of extracellular calcium ions. Moreover, we found out that neutrophils treated with 10 mM MeHg for 24 hrs manifested chromatin condensation, apoptotic bodies and the formation of DNA ladder. Therefore, neutrophils apoptosis was closely related to the decrease of intracellular pH caused by MeHg in a concentration-dependent fashion. Another finding that supports this view is that MeHg, EGTA and zinc can all cause DNA ladders, and they can also induce intracellular acidification. Among them, MeHg decreased the intracellular pH in a concentration-dependent manner. When we analyzed the influence of pH and calcium on the endonuclease activity by its ability to cut plasmid DNA, we found out that the endonuclease was not activated either with or without calcium at neutral pH, but the activation took place in acidic solutions at pH below 6.5 whether or not there was any calcium present. Therefore, MeHg activates an acid-sensitive endonuclease within neutrophils, which in turn induces neutrophils apoptosis. The Influence of MeHg on the Immune Functions of Alveolar Macrophages and the Mechanism of Cellular Signal Transduction The influence of MeHg on the immune functions of alveolar macrophages were approached in three aspects. When not stimulated, alveolar macrophages produces minimal NO and TNF-a, which make analysis very difficult. For this reason, we studied the influence of MeHg on the LPS-induced NO and TNF-a production, and found out that MeHg had a significant inhibitory effect on both of them. As we investigated the possible mechanism, we discovered that LPS increased NO and TNF-a by increasing the activity of receptor tyrosine kinase, which could be inhibited by genistein. H7 and staurosporine could also inhibit the production of LPS-NO, but H7 and H89 was unable to inhibit the production of LPS-TNF-a, which could be inhibited by W7 (20mM). An interesting finding was that the inhibition of c-AMP (1 mM) was far greater than the production of LPS-TNF-a. In our study of the inhibitory effect of MeHg on LPS-NO and LPS-TNF-a by intercepting the signal transduction, we found out that H89 (PKA inhibitor) could antagonize the inhibition of MeHg on LPS-NO, but genistein, H7 and staurosporine failed to manifest this inhibitory effect. The inhibitory effect of MeHg on LPS-TNF-a could be antagonized by H89 in a concentration- dependent manner. H7 also had the antagonistic effect, while genistein and staurosporine failed to show any influence at all. As we moved further to study the mechanism by which H89 antagonized the inhibitory effect of MeHg on LPS-NO and LPS-TNF-a, we found out it was related to MeHg-induced increase of intracellualr calcium, which in turn activated Ca-dependent adenylate cyclase, which then increased the activity of c- AMP-PKA. PKA induced the phosphorylation and subsequent inactivation of raf-1, leading to the MeHg-induced inactivation of ERK. Finally we were able to prove that MeHg indeed produced its inhibitory effect via this signal transduction pathway, making NOS protein significantly lower than the LPS control group. In our study of the mechanism by which MeHg inhibited the phagocytic activity of alveolar macrophages (FITC-latex bend uptake), we found out that genistein, H-7 and staurosporine had no influence at all, but W-7 at 50 mM significantly enhanced the inhibitory effect of MeHg. It was therefore inferred that MeHg might via the Ca2+-activated calmodulin kinase pathway. MeHg-induced Functional Low-conductance Calcium-dependent Potassium Currents As we use the patch-clamp techniques to study the membrane current, we discovered that MeHg could open a potassium channel. MeHg generated by a pressure-injector induced an outward current Io(MeHg) at -40 mV. The removal of extracellular calcium or verapamil reduced its amplitude, and intracellular dialysis with 5 mM EGTA completely inhibited this outward current. Intracellular dialysis with heparin (5 mg/ml) also significantly reduced the amplitude. Therefore, this outward current was calcium-dependent. It was completely blocked by potassium channel inhibitors quinine (0.2 mM) and 4-aminopyridine (1 mM). The MeHg action was also completely blocked by small-conductance potassium channel inhibitors such as apamin (1 mM) and dequalinium (0.5 mM). When we replace the potassium ions with cesium ions, this current was totally inhibited as well. These findings suggest that MeHg opens a Ca2+-dependent K+ channel. As we analyzed the single channel current, we found out that the conductance of this potassium channel was 12.0 pS and 8.1 pS when the pipette solution was 145 mM and 36 mM respectively. There has been no report so far as to the possible influence of this small-conductance K+ channel on the functions of alveolar macrophages. In our study, after we blocked the potassium channels of alveolar macrophages with potassium channel inhibitor dequalinium, we discovered that it had no significant influence on LPS-induced release of TNF-a and NO after 24 hrs of incubation, but it manifested a significant antagonistic effect on the MeHg inhibition of TNF-a and NO production. Another potassium channel blocker quinine had a similar antagonistic action. On the other hand, pretreatment with 1 mM dequalinium significantly inhibited MeHg-induced increase in intracellular calcium ion concentration. Therefore, by inhibiting the activation of this potassium channel, it was possible to alter the TNF-a inhibition caused by MeHg-induced intracellular calcium increase. In our study of the relationship between membrane currents and the mechanism by which MeHg caused cell death, we discovered that MeHg probably caused cellular apoptosis by increasing calcium ion level, and that the potassium channel activated by the increase of calcium ion was able to lower the toxicity of MeHg. The Influence of MeHg on the Proton Current When we tried to detect intracellular pH changes with BCECF, we discovered that MeHg induced a drop in intracellular pH and activated a proton channel. MeHg generated by a pressure-injector induced an outward current at +20 mV which was completely blocked by intracellular dialysis with 5 mM EGTA. Moreover, calcium ions given directly through the pipette also induced a calcium- dependent outward current, which increased in amplitude as the concentration of calcium ions increased. This current was not changed by a replacement of intra- and extracellular ions with Cs, Asp or NMDG-Asp. Both ion substitation experiments and their reversal potential distinctly dependent on the pHi suggest, this current is mainly caused by protons. As we observed the intracellular pH changes, we found that a bath application with 20 mM MeHg caused an immediate acidification of the cell, closely followed by its alkalization. In our attempt to detect the production of O2-, low-concentration of MeHg induced the production of O2-, while MeHg at a high concentration manifested an inhibitory effect. This proton current could be inhibited by such channel blockers as Cd2+ (1 mM), Zn2+ (1 mM) and DQ (10 mM). Calmodulin antagonist (W-7) and calmodulin kinase inhibitor KN- 93 can almost completely inhibit this H+ conductance. Therefore, we first discovered that the rat alveolar macrophages have a Ca2+- calmodulin-dependent proton conductance, which can be regulated by calmodulin-kinase. Zn2+, a blocker of this proton conductance, enhances the MeHg inhibition of the phagocytic activity and of the NO production, but has a reversing effect on the MeHg inhibition of TNF-a release. In conclusion, after we studied the mechanism of the toxicity of MeHg on rat alveolar macrophages, we found out that MeHg acted on the membrane channels and caused an influx of extracellular calcium. Moreover, it was able to induce a calcium release from the intracellular storage pool and therefore increased the intracellular calcium level. On one hand, this increase of calcium level activated calcium-dependent endonuclease leading to apoptosis. On the other hand, it induced calcium-dependent adenylate cyclase-Raf-1 phosphorylation, thus inhibiting the production of NO and TNF- a. In addition, the increase of intracellular calcium level activated Ca2+- calmodulin-kinase, which in turn induced the phosphorylation of membrane H+-channel protein and generated the H+-current. This explained why MeHg quickly lowered the intracellular pH but immediately returned it to a neutral value. The increase of intracellular calcium triggered the alteration of multifunctional signalling pathways of the cell. The opening of protective calcium- dependent potassium channels was also initiated by the MeHg- induced calcium increase.

碩士
國立臺北科技大學
製造科技研究所
105
Simplified drilling sequence which reducing the number of drills used in alveolar bone drilling in dental implant surgery has become a trend for the past few years. It is hoped that the simplified drilling sequence can reduce the surgery time but on the other hand the surgical difficulty and bone damage may also arise. In this study, the mechanical and thermal effects of three simplified sequences were investigated by using finite element analysis combining failure theory and heat transfer module, and the effects of different drill parameters were also investigated using parametric finite element analysis. In the verification experiment, the correlations between the drilling experiment test results and finite element analysis results of conventional sequence showed high to intermediate correlation level. The three simplified drilling sequences were divided into “one twist drill” (S1), “equal drill diameter increase” (S2) and “equal drilling cross-sectional area increase” (S3). In the parametric analysis of drill parameters, only the final drill model of conventional sequence was used to investigate the influences of friction coefficient and drill density. The drilling condition used in all analysis groups were, feed rate: 1 mm/s, cutting speed: 800 rpm, cutting depth: 10 mm and initial temperature: 25°C. The axial cutting reaction force, axial cutting torque and temperature change at depths of 3, 6, and 9 mm and distance of 1 mm from the hole wall were recorded in both experiment and analysis. The results showed that the conventional sequence had less thermal damage and breakage risk but the surgery time may be longer. Although the chances for drilling modification may be more for traditional sequence, however, the bone-implant interface may be less ideal. On the other hand, the simplified drilling sequences were found to have opposite effects when compared to the conventional drilling sequence. The simplified drilling group S2 and S3 can reduce the thermal damage as compared to sequence S1 where as sequence S3 has the least thermal damage. From the parametric analysis results, when reducing the friction coefficient could reduce the thermal damage. When all other parameters were set to be the same, higher drill density would result in more thermal damage. The results of this study can provide reference for dental drill manufacturers when developing new drill sets.

碩士
長庚大學
生物醫學研究所
98
Human pulmonary alveolar epithelial cells play an important role in the proliferation, migration, and inflammatory processes in the respiratory system. Several factors have been shown to trigger the mechanisms for the pathologenesis of airway diseases including asthma and chronic obstructive pulmonary disease (COPD). Although the factors concerning about the increased incidence of inflammatory responses are well known, however, the intracellular signaling pathways involved in the expression of inflammatory proteins are not completely recognized. Elevated levels of pro-inflammatory cytokines such as tumor necrosis factor- (TNF-) have been found in the airway fluids, which may induce up-regulation of cytosolic phospholipase A2 (cPLA2) implicated in the pathogenesis of inflammatory diseases. Although TNF- has been reported to activate all of mitogen-activated protein kinases (MAPKs) including p42/p44 MAPK, p38 MAPK, and JNK/SAPK, and transactivation of growth factor receptors and PI3K/Akt, the relationship between the activation of these signaling pathways and expression of cPLA2 or other genes remains largely unknown in human alveolar epithelial cells (HPAEpiCs). Therefore, whether activation of these MAPKs, growth factor receptors and PI3K/Akt pathways by TNF- linked to cPLA2 expression is needed determining in HPAEpiCs. In addition, it is of interest that many of the genes regulated by MAPKs are dependent on NF-B, AP-1, and p300 for transcription. These transcription factors have also been shown to be involved in cPLA2 gene expression at the transcriptional level in various cell types. Western blot and Real-time RT-PCR showed that in HPAEpiCs, TNF-α induced cPLA2 mRNA and protein expression in a time-dependent manner, which were attenuated by pretreatment with the inhibitors of ROS (NAC, APO, DPI), PDGF receptor (AG1296), PI3K (Wortmannine), and MEK1/2 (PD98059) or transfection with siRNA of p42. These results suggest that PDGFR transactivation participates in cPLA2 expression induced by TNF-α. Accordingly, TNF-α-stimulated phosphorylation of p38 MAPK and JNK were inhibited by pretreatment with NAC, APO, or DPI. TNF- induced cPLA2 expression was blocked by the selective inhibitors of AP-1 (Tanshinone IIA) and NF-B (Bay11-7082). Moreover, TNF-α-stimulated cPLA2 promoter activity was blocked by these selective inhibitors. In this study we investigated the effect of TNF-α induced cPLA2 expression at the transcriptional and translational levels, which were mediated through three independent pathways. First, TNF-α activated Jak2-dependent PDGFR transactivation, PI3K/Akt, p42/p44 MAPK, and p300/c-Jun/c-Fos/ATF2/AP-1 signalung pathway in HPAEpiCs. Secand, TNF-α-stimulated TNFR1 induced association of TRAF2, ASK-1 and p47phox, which promoted recruitment with ROS production, p38 MAPK phosphorylation, JNK1/2 phosphorylation resulting in AP-1 activation and cPLA2 expression and PGE2 release. In addation, TNF-α-induced ROS production which promoted recrument with NIK, IKK resulting in NF-B activation and cPLA2 expression and PGE2 release. These results provide new insights into the mechanisms of TNF-α action which may be therapeutic value in lung diseases.

碩士
長庚大學
生物醫學研究所
100
Respiratory problems such as asthma and chronic obstructive pulmonary disease (COPD) had been concerned issue of human airway diseases. Lipopolysaccharide (LPS), a key component of the outer membranes of Gram-negative bacteria, plays an important role in the induction of adhesion molecules expression and reactive oxygen species (ROS) generation in airway inflammatory diseases. However, the mechanisms by which up-regulation of intercellular adhesion molecule-1 (ICAM-1) induced by LPS may contribute to inflammatory responses or exert as a host defense in respiratory diseases are not completely understood. Here, we investigated the mechanisms of LPS-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that LPS induced ICAM-1 protein and mRNA expression, promoter activity, and the adhesion of THP-1 cells , as well as the phosphorylation of c-Src, PDGFR, EGFR, Akt, and p65, which were attenuated by pretreatment with an anti-TLR4 Ab or the inhibitor of MEK1/2 (U0126), JNK1/2 (SP600125), p38 MAPK (SB202190), c-Src (PP1), PDGFR (AG1296), EGFR (AG1478), PI3K (LY294002), NADPH oxidase [diphenylene iodonium chloride (DPI)], ROS (Edaravone), NF-B (Bay11-7082) or AP-1 (Tanshinone II A). LPS-induced TLR4, MyD88, TRAF6, c-Src, p47phox, and Rac-1 complex formation was revealed by immunoprecipitation using an anti-TRAF6 and antic-Src Ab, followed by Western blot against an anti-TLR4, anti-MyD88, anti-TRAF6, anti- p47phox, anti-Rac-1, or anti-c-Src Ab. These results demonstrated that LPS-induced ICAM-1 expression was mediated through TLR4/ MyD88/ TRAF6/ p47phox /Rac-1/ c-Src via EGFR, PDGFR/PI3K or MAPKs, in turn initiates NF-B activation, and ultimately induced ICAM-1 expression and the adhesion of THP-1 cells on HPAEpiCs. Moeover, in animal medol we also can find ICAM-1 expression and inflammation symptom.

碩士
長庚大學
生物醫學研究所
101
Human pulmonary alveolar epithelial cells play an important role in the proliferation, migration, and inflammatory processes in the respiratory system. Several factors have been implicated to trigger the mechanisms for the pathologenesis of airway diseases including asthma and chronic obstructive pulmonary disease (COPD). Human pulmonary alveolar epithelial cells (HPAEpiCs) play an important role in airway inflammatory processes. Several studies demonstrate that expression of adhesive molecules on the cell surface of epithelial cells plays a critical role in these inflammatory responses. Although the factors concerning about the increased incidence of inflammatory responses are well known, however, the intracellular signaling pathways involved in the expression of inflammatory proteins are not completely recognized. Sphingosine 1-phosphate (S1P), one of the lipid components has attracted much attention as a possible signaling mediator that regulates immune responses and inflammatory processes in the respiratory system. Although S1P has been shown to activated MAPKs, PI3K/Akt, PTK and other signaling molecules up-regulation of ICAM-1 in various cell types, however, the mechanisms underlying S1P-induced ICAM-1 expression in HPAEpiCs remain unknown. Thus, this study is to investigate the signaling pathways implicated in S1P-induced ICAM-1 expression in these cells. Our hypothesis is that up-regulation of ICAM-1 induced by S1P may contribute to inflammatory responses or exert as a host defense in respiratory diseases. We found that S1P induced ICAM-1 protein and mRNA expression, promoter activity, and the adhesion of THP-1 cells , as well as the phosphorylation of c-Src, PDGFR, EGFR, p42/p44, p38, Jnk1/2, Akt, PKCδ, PYK2, NADPH oxidase, ROS, p65 and c-Jun, which were attenuated by pretreatment with an anti-S1PR1, anti-S1PR3 Ab or the inhibitor of c-Src (PP1), PDGFR (AG1296), EGFR (AG1478), Erk1/2 MAPK (U0126), Jnk1/2 (SP600125), p38 MAPK (SB202190), PI3K (LY294002), PKCδ (Rottlerin), PYK2 (PF431396), NADPH oxidase (APO, DPI), ROS (Edaravone), NF-kB (Bay11-7082) or c-Jun (Tashinone IIA). These results demonstrated that S1P-induced ICAM-1 expression was mediated through S1PR1, S1PR3/Gi &; Gq protein via c-Src/EGFR and PDGFR/p42/p44 and p38/PI3K/Akt, or PKCδ / PYK2 / NADPH / ROS in turn initiates NF-kB (p65) and AP-1 (c-Fos and c-Jun) activation, and ultimately induces ICAM-1 expression and the adhesion of THP-1 cells in HPAEpiCs.

碩士
輔仁大學
基礎醫學研究所碩士班
102
Adiponectin is one of adipocyte-derived hormones. Regarding to reports, adiponectin has pro-inflammatory or anti-inflammatory effects in different organs. But in lung system, what the role of adiponectin is still not known. On the other hand, lung inflammation companied occurrence of various acute or chronic lung diseases, including chronic obstructive pulmonary disease, emphysema and idiopathic pulmonary fibrosis, acute respiratory distress syndrome and obstructive apnea. It is found that several genes such as cPLA2 and COX-2 express and cooperate mediating the occurrence and amplification of inflammatory responses. cPLA2 and COX-2 are up-regulated resulting in the increase of end-point product prostaglandins in response to the stimulation of exogenous stimuli or cytokines. However, it is not clear whether adiponectin contributes to lung diseases via modulating expression of cPLA2 and COX-2 and resulting in lung inflammation. Thus, in this work, how adiponectin regulated pulmonary inflammation and the related mechanisms will be established. Our hypothesis is that up-regulation of cPLA2 and COX-2 stimulated by adiponectin promote inflammatory responses in the lung. To addressing these questions, the experiments with pharmacological inhibitors were performed to investigate the roles of ROS, JAK/STAT, AMPK, PI3-K/Akt, Src and PKC in adiponectin-induced cPLA2 and COX-2 expression in lung alveolar type II cells. Several techniques such as Western blot, RT-PCR and cell fraction isolation assay were used to investigate the molecular interactions between signaling components. Our results proved that adiponectin induced cPLA2 and COX-2 expression throught adipoR1 and adipoR2. Moreover, pretreatment of N-acetyl-cysteine, rotenone, apocynin, Ro31-8220, Gö-6976, rottlerin, PP1, LY294002, wortmannin, BML-275, AG490, WP1066, STAT5 inhibitor, garcinol attenuated adiponectin-stimulated protein and mRNA expression of cPLA2 and COX-2. And adiponectin modulated accumulation of ROS and activation of mitochondria. Similarly, adiponectin regulated activation of PKC, c-Src, AMPK, JAK2 and STAT3. These results revealed that adiponectin-regualted expression of cPLA2 and COX-2 genes at least via AdipoR1, AdipoR2, ROS, mitochondria, NADPH oxidase, PKC, c-Src, PI3-K/AKT, AMPK, JAK/STAT and p300. These results will provide new insight into the mechanisms of adiponectin actions, supporting the hypothesis that adiponectin may contribute to inflammatory responses involved in the development of lung diseases. Increased understanding of signal transduction mechanisms underlying cPLA2 and COX-2 gene regulation will create opportunities for the development of anti-inflammation, anti-cancer and anti-metastasis therapeutic strategies.

碩士
長庚大學
生物醫學研究所
99
Staphlococcus aureus (S. aureus), the common Gram-positive bacteria, causes a wide range of human inflammatory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). These inflammatory responses are mediated by complex interactions between both circulating polymorphonuclear cells (PMNs) and the vascular endothelium. Several studies have indicated that expression of adhesive molecules on the cell surface of epithelial cells plays a critical role in these inflammatory responses. Although S. aureus has been shown to induce ICAM-1 or VCAM-1 expression in various cell types, the mechanisms underlying S. aureus-induced VCAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unknown. Western blot, Real time-PCR, and monocytes adhesion analyses showed that in HPAEpiCs, S. aureus induced VCAM-1 mRNA and protein expression in a time-dependent manner, which were attenuated by the inhibitors of c-Src (PP1), PDGF receptor (AG1296), PI3K (LY294002), JNK (SP600125), p38 MAPK (SB202190), MEK1/2 (U0126), AP-1 (Tanshinone IIA), NF-kB (Bay11-7082) and p300 (GR343), or transfection with siRNA of TLR2, MyD88, p42, p38, JNK1, c-Src, PDGFR, Akt, p65, c-Jun, ATF2, p300. These results suggest that PDGFR transactivation participates in VCAM-1 expression induced by S. aureus. Accordingly, S. aureus-stimulated phosphorylation of Akt, p38 MAPK, JNK and ERK1/2 was inhibited by pretreatment with PP1, AG1296, LY294002, SP600125, SB202190 or U0126. Taken together, in this study we investigated the effect of S. aureus induced VCAM-1 expression at transcriptional and translational levels. S. aureus-stimulated TLR2 induced association of MyD88 and c-Src, which mediated through two pathways. First, S. aureus activated c-Src, PI3K/Akt, ERK1/2 and JNK1/2. Second, S. aureus activated dependent PDGFR transactivation, p38 and JNK1/2. The two pathways both linked to AP-1, NF-B and p300, and induced VCAM-1 expression which led to monocytes adhesion. These results provide new insight into the mechanisms of S. aureus action, supporting that S. aureus may contribute to promote inflammatory responses involved in the development of respiratory diseases. Increased understanding of signal transduction mechanisms underlying VCAM-1 gene regulation creates opportunities for the development of anti-inflammation therapeutic strategie.

The insulin receptor ortholog EmIR of the fox-tapeworm Echinococcus multilocularis displays significant structural homology to the human insulin receptor (HIR) and has been suggested to be involved in insulin sensing mechanisms of the parasite’s metacestode larval stage. In the present work, the effects of host insulin on Echinococcus metacestode vesicles and the proposed interaction between EmIR and mammalian insulin have been studied using biochemical and cell-biological approaches. Human insulin, exogenously added to in vitro cultivated parasite larvae, (i) significantly stimulated parasite survival and growth, (ii) induced DNA de novo synthesis in Echinococcus, (iii) affected overall protein phosphorylation in the parasite, and (iv) specifically induced the phosphorylation of the parasite’s Erk-like MAP kinase orthologue EmMPK1. These results clearly indicated that Echinococcus metacestode vesicles are able to sense exogenous host insulin which induces a mitogenic response. To investigate whether EmIR mediates these effects, anti-EmIR antibodies were produced and utilized in biochemical assays and immunohistochemical analyses. EmIR was shown to be expressed in the germinal layer of the parasite both on the surface of glycogen storing cells and undifferentiated germinal cells. Upon addition of exogenous insulin to metacestode vesicles, the phosphorylation of EmIR was significantly induced, an effect which was suppressed in the presence of specific inhibitors of insulin receptor-like tyrosine kinases. Furthermore, upon expression of EmIR/HIR receptor chimera containing the extracellular ligand binding domain of EmIR in HEK 293 cells, a specific autophosphorylation of the chimera could be induced through the addition of exogenous insulin. These results indicated the capability of EmIR to sense and to transmit host insulin signals to the Echinococcus signaling machinery. The importance of insulin signaling mechanisms for parasite survival and growth were underscored by in vitro cultivation experiments in which the addition of an inhibitor of insulin receptor tyrosine kinases led to vesicle degradation and death. Based on the above outlined molecular data on the interaction between EmIR and mammalian insulin, the parasite’s insulin receptor orthologue most probably mediates the insulin effects on parasite growth and is, therefore, a potential candidate factor for host-parasite communication via evolutionary conserved pathways. In a final set of experiments, signaling mechanisms that act downstream of EmIR have been analyzed. These studies revealed significant differences between insulin signaling in Echinococcus and the related cestode parasite Taenia solium. These differences could be associated with differences in the organo-tropism of both species
Der orthologe Insulinrezeptor EmIR des Fuchsbandwurmes Echinococcus multilocularis weist signifikante strukturelle Homologie zum humanen Insulinrezeptor (HIR) auf. Es wurde schon seit geraumer Zeit vermutet, dass EmIR an den Mechanismen beteiligt sein könnte, die es dem Metacestoden Larvenstadium des Parasiten erlauben Insulin zu detektieren. In dieser Arbeit wurden die Effekte von Wirtsinsulin auf Echinococcus Metacestoden-Vesikel und die vermutete Interaktion zwischen EmIR und Insulin von Säugern mittels biochemischer und zellbiologischer experimenteller Ansätze untersucht. Die exogene Zugabe von humanem Insulin zu in vitro kultivierten Parasitenlarven hatte folgende Effekte: (i) das Überleben und das Wachstum des Parasiten wurde signifikant stimuliert; (ii) die DNA de novo Synthese in Echinococcus wurde induziert; (iii) die generelle Proteinphosphorylierung des Parasiten wurde beeinflusst; (iv) die Phosphorylierung der orthologen Erk-like MAP Kinase, EmMPK1, des Parasiten wurde spezifisch induziert. Diese Beobachtungen zeigen deutlich, dass Echinococcus Metacestoden-Vesikel exogenes Insulin des Wirtes detektieren können und dass dieses Insulin einen mitogenischen Effekt auf den Parasiten hat. Um zu untersuchen, ob diese Effekte durch EmIR vermittelt werden, wurden anti-EmIR Antikörper hergestellt und in biochemischen experimentellen Ansätzen und immunohistochemischen Analysen eingesetzt. Es konnte gezeigt werden, dass EmIR in der Germinalschicht des Parasiten expremiert wird, sowohl an der Oberfläche von Glykogen-Speicherzellen als auch von undifferenzierten Germinalzellen. Nach der Zugabe von exogenem Insulin konnte eine signifikante Zunahme der Phosphorylierung von EmIR festgestellt werden. Diese Stimulierung konnte durch die Zugabe eines spezifischen Inhibitors für Insulinrezeptor-ähnliche Tyrosinkinasen unterdrückt werden. Desweiteren konnte mittels der Expression eines chimären EmIR/HIR-Rezeptors, der die extrazelluläre Ligandenbindungsdomäne von EmIR enthielt, in HEK293 Zellen gezeigt werden, dass die Zugabe von exogenem Insulin eine spezifische Autophosphorylierung der Chimäre induziert. Diese Ergebnisse bezeugen die Fähigkeit von EmIR Insulin-abhängige Signale des Wirtes einerseits zu detektieren und andererseits an die Echinococcus Signalwege weiter zu leiten. Die Bedeutung von Insulin-Signalmechanismen für das Überleben und das Wachstum des Parasiten konnte durch in vitro Kultivierungsexperimente aufgezeigt werden. Die Zugabe eines Inhibitors spezifisch für Insulinrezeptor Tyrosinkinasen verursachte die Degradation und den Tod der Metacestoden-Vesikel. Basierend auf den dargelegten molekularen Daten bezüglich der Interaktion zwischen EmIR und Insulin von Säugern erscheint es sehr wahrscheinlich, dass der orthologe Insulinrezeptor des Parasiten die Effekte von Insulin auf das Wachstum des Parasiten vermittelt. Aus diesem Grund ist EmIR ein potentieller Kandidat für die Kommunikation zwischen Wirt und Parasiten mittels evolutionär konservierten Signalwegen. Die Signalmechanismen unterhalb von EmIR wurden in abschließenden Experimenten untersucht. Diese offenbarten deutliche Unterschiede in der Weiterleitung von Insulin induzierten Signalen zwischen Echinococcus und dem verwandten parasitären Zestoden Taenia solium. Diese Unterschiede könnten mit dem unterschiedlichen Organtropismus beider Arten in Verbindung stehen

Introdução: Esta revisão narrativa examina artigos de particular interesse sobre o tema: complicações anestésicas do nervo alveolar inferior. Objetivos: O objetivo desta revisão narrativa é determinar o que pode levar a complicações anestésicas do nervo alveolar inferior, o seu tratamento e prevenção. Materiais e Métodos: As buscas foram realizadas nos websites SCIELO, B-ON e PUB MED, utilizando as seguintes palavras-chave: "anasthesia, complications alveolar inferior”, com o objetivo de angariar e debater o máximo de informação sobre este tema. Resultados e Conclusões: Os resultados obtidos revelam a importância de reconhecer a anatomia do nervo alveolar inferior e sua envolvência, do tipo de técnicas anestésicas e material utilizável sendo assim capaz de diminuir a probabilidade de erros e complicações intra e pós anestésicas. Devendo também reconhecer estas complicações caso ocorram e como as solucionar.
Introduction: This narrative review examines articles of particular interest for anesthetic complications of the lower aleveolar nerve. Objectives: The objective of this narrative review is to determine how to approach anesthetic complications of the lower aleveolar nerve, treatment, prevention. Materials and Methods: The searches were carried out on the websites SCIELO, B-ON and PUB MED, using the following keywords: "anasthesia,lower alveolar complications", in order to gather and discuss as much information as possible on this topic. Results and Conclusions: The results obtained reveal the importance of recognizing the anatomy of the lower alveolar nerve and its involvement, the type of anesthetic techniques and material that can be used, thus being able to reduce the probability of intra- and post-anesthetic errors and complications. It should also recognize these complications if they occur and how to solve them.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, with two predominant histologic subtypes: embryonal and alveolar. These histologies display distinct clinical courses, and despite refinements in dose and duration of multimodality therapy, the 5-year overall survival of patients diagnosed with metastatic RMS remains <30%. Thus, there is an urgent need to define novel targets for therapeutic intervention. Interrogation of cancer cell signal transduction pathways that regulate the pathogenic behaviours of tumor cells has been successful in defining targets in numerous tumor types. These have ultimately yielded clinically-relevant drugs that have improved the disease-free and overall survival of patients diagnosed with cancer. Work contained in this thesis describes the interrogation of several potential targets for inhibition in RMS. Interruption of RMS cell proliferation, survival and apoptosis is examined through disruption of the protein kinase integrin-linked kinase (ILK) and the nuclear receptor estrogen-receptor β. ILK, in particular, is demonstrated to have dual competing functions through the regulation of c-jun amino-terminal kinase (JNK) signaling: an oncogene in alveolar, and a tumor suppressor in embryonal RMS. These findings are recapitulated in other tumor cell lines, indicating that expression levels of JNK1 correlate with ILK function in a broad spectrum of tumor types. Furthermore, interruption of rhabdomyosarcoma cell migration as a surrogate marker of metastasis is examined through disruption of the stromal-cell derived factor 1α/chemokine (CXC)receptor 4 signaling network, as well as through cooperative interactions between ILK and the mammalian target of rapamycin. Finally, we demonstrate that the insulin-like growth factor pathway is a potential target for therapeutic inhibition, which also distinguishes tumors of embryonal and alveolar histology. These studies provide a rationale for the development of novel agents, as well as the use of established drugs targeting these pathways in rhabdomyosarcoma.

博士
臺北醫學大學
臨床醫學研究所
99
Lipopolysaccharide (LPS), a Gram-negative bacterial outer membrane component, is one of the major causes of septic shock with acute lung injury. Pulmonary alveolar epithelial type II cells have highly specialized functions for synthesizing and secreting surfactant proteins (SPs) to participate in the physiological and pathophysiological regulation of sepsis-induced acute lung injury. Alterations in the levels of surfactant components in the lungs during inflammation are quite complex. Toll-like receptors (TLRs) that play important roles in innate immunity can transduce pathogen-triggered signals to regulate certain inflammation-related gene expressions through the activation of transcription factors. In contrast to low concentration of LPS-induced physical activity, intratracheal instillation of a high concentration of LPS in mice directly caused the death of bronchial epithelial cells. Thus, LPS may have pathophysiological and toxic effects on affecting the alveolar type II epithelial cells. The purpose of this research was aimed to evaluate the molecular mechanisms of LPS-induced cell apoptosis and surfactant proteins biosynthesis using human lung carcinoma type II epithelium-like A549 cells as the experimental model. Firstly, the study was aimed to evaluate the apoptotic effect of toxic concentration of LPS in A549 cells and the possible mechanisms. Exposure of A549 cells to clinical concentration (1~10 ng/ml) of LPS did not affect cell viability, but toxic dose (1~10 μg/ml) of LPS decreased cell viability in concentration- and time-dependent manners. In parallel, LPS concentration- and time-dependently induced apoptosis of A549 cells. LPS only at a high concentration of 10 μg/ml caused mildly necrotic insults to A549 cells. Exposure of A549 cells to LPS increased the levels of cellular nitric oxide and reactive oxygen species (ROS). Pretreatment with N-acetylcysteine, an antioxidant, significantly lowered LPS-caused enhancement of intracellular ROS in A549 cells and simultaneously attenuated the apoptotic insults. Treatment of A549 cells with LPS caused significant decreases in the mitochondrial membrane potential and biosynthesis of adenosine triphosphate. LPS triggered the release of cytochrome c from the mitochondria to the cytoplasm. Activities of caspases-9 and -6 were augmented following LPS administration. Consequently, exposure of A549 cells to LPS induced DNA fragmentation in a time-dependent manner. Pretreatment of A549 cells with N-acetylcysteine significantly ameliorated LPS-caused alterations in caspase-9 activation and DNA damage. Secondly, we attempted to evaluate the signal-transducing mechanisms of LPS-caused regulation of SP-A biosynthesis in A549 cells. Exposure of A549 cells to clinical concentration (1 ng/ml) of LPS increased SP-A protein and mRNA production in concentration- and time-dependent manners without affecting SP-D mRNA production. Clinical concentration of LPS time- dependently induced TLR2 mRNA expression and increased phosphorylation of mitogen-activated protein kinase (MEK) 4 & c-Jun NH2 terminal kinase 1 (JNK1) and augmented the translocation of c-Jun from the cytoplasm to nuclei without affecting c-Fos. Application of TLR2 small interference (si)RNA into A549 cells decreased the levels of this receptor and simultaneously inhibited LPS-induced SP-A mRNA and protein syntheses. After knocking-down the translation of MyD88 by RNA interference, the LPS-triggered MEK4 phosphorylation was attenuated. Application of TLR2 siRNA decreased LPS-enhanced phosphorylation of MEK4 and JNK1. Pretreatment of A549 cells with SP600125, an inhibitor of JNK1, significantly lowered LPS-induced c-Jun translocation and SP-A mRNA production. Taken together, this study has shown that toxic concentration of LPS specifically induces apoptotic insults to human alveolar epithelial cells through ROS-mediated activation of the intrinsic mitochondrion-cytochrome c-caspase protease mechanism and clinical concentration of LPS selectively induces SP-A gene expression possibly through TLR2-mediated sequential activation of MyD88-MEK4-JNK1-AP-1 in human alveolar epithelial A549 cells The LPS-induced apoptosis and sp-a gene expression in alveolar epithelial type II cells can indicate the status of Gram-negative bacteria-caused septic shock and acute lung injury. There are certain limitations in the present study, including A549 cells are derived from human lung carcinoma. The mechanisms of LPS-induced oxidative stress, cell apoptosis and production of SPs in A549 cells may be different from normal alveolar epithelial cells. In future studies, we will perform translational study to evaluate the signal-transducing effects of LPS on alveolar epithelial cells of animals and broncho-alveolar lavages with acute lung injury to validate our in vitro data.

L’ATP est bien connue pour son rôle de transporteur d'énergie à l’intérieur des cellules, mais en dehors de la cellule, elle agit en tant que molécule de signalisation extracellulaire. En se liant aux récepteurs purinergiques, l’ATP extracellulaire amorce la signalisation purinergique afin de réguler certains processus physiologiques et pathophysiologiques. Dans les poumons, l’ATP stimule la sécrétion de surfactant et promeut la clairance mucociliaire. Compte tenu du rôle critique de l’ATP extracellulaire dans les poumons, il est important de comprendre le mécanisme du relargage d’ATP cellulaire — la première étape de la signalisation purinergique. Parce que les forces mécaniques constituent le déclencheur principal du relargage d’ATP, cette thèse a pour but d’investiguer le(s) mécanisme(s) physiologique(s) et les sources cellulaires d’un tel relargage d’ATP mécanosensible. Cet ouvrage est divisé en trois parties : 1) Pour étudier les caractéristiques spatiales et temporelles du relargage d’ATP, j’ai développé une technique d’imagerie hautement sensible basée sur la bioluminescence de la luciférine-luciférase couplée avec un système de lentilles à grand champ de vision (WFOV, wide field of view) optimisant l’apport de lumière. Pour évaluer notre approche d’imagerie, j’ai soumis des cellules A549, dérivées d’un adénocarcinome pulmonaire humain, à un étirement ou un choc hypotonique de 50% pour déclencher un relargage d’ATP. J’ai démontré que notre technique nous permet de quantifier précisément la quantité et le taux (ou l’efflux) d’ATP s’échappant des cellules. Le WFOV constitue un outil essentiel utilisé dans les études décrites dans cette thèse pour déterminer le mécanisme et la source cellulaire du relargage d’ATP dans l’alvéole. 2) Afin d’examiner le mécanisme physiologique du relargage d’ATP induit par l’étirement dans les cellulaires alvéolaires primaires, j’ai déterminé les contributions individuelles des cellules alvéolaires de type 1 (AT1) en comparaison des cellules alvéolaires de type 2 (AT2). Pour ce faire, des cellules AT2 fraîchement isolées de poumons de rats ont été ensemencées sur une chambre flexible en silicone et cultivées jusqu’à sept jours, ce qui permettait aux cellules AT2 de se transdifférencier progressivement en cellules semblables aux cellules AT1. Le ratio des cellules alvéolaires (AT2:AT1), étant de 4:1 au jour 3, est devenu 1:4 au jour 7. La quantité d'ATP libérée diminuait avec le nombre décroissant de cellules AT2, les impliquant en tant que principale source pour le relargage d’ATP en réponse à un étirement. Alors que les modulateurs pharmacologiques des canaux d’ATP, carbenoxolone et probénécide, ne diminuaient pas la quantité d’ATP libérée, le BAPTA, un chélateur de calcium intracellulaire ([Ca2+]i), l’a significativement réduite. De même, ces trois modulateurs exercent des effets similaires sur les réponses calciques intracellulaires mesurées par le Fura-2, suggérant une connexion entre le relargage d’ATP et les niveaux de [Ca2+]i. 3) Pour explorer le rôle qu’ont les propriétés viscoélastiques de la membrane dans le relargage d’ATP mécanosensible, j’ai démontré qu’une déformation de 30% induisait un relargage d’ATP transitoire qui était accompagné d’une absorption d’iodure de propidium (PI, propidium iodide) chez des cellules AT2. Ceci est cohérent avec une rupture membranaire transitoire induite par une déformation, assez large pour le passage d’ATP et de PI. L’efflux d’ATP augmente aussi selon le taux de déformation, et la durée de déformation prolonge la demi-vie du relargage d’ATP. Donc, ces résultats fournissent des indices sur la manière dont l’étirement de la membrane viscoélastique peut mener au relargage d’ATP par un mécanisme alternatif impliquant une mécanoporation de la membrane cellulaire. Dans l’ensemble, ces résultats démontrent que le relargage d’ATP ne se produit pas à travers les canaux conduisant l’ATP mais plutôt par une mécanoporation transitoire de la membrane. D’autres études sur les dommages membranaires sont nécessaires pour mieux comprendre sa contribution dans le relargage d’ATP mécanosensible et les signaux de [Ca2+]i. De telles études élucideront la signalisation purinergique dans les organes qui sont constamment exposés à des contraintes physiques. Ceci pourrait suggérer des cibles/approches thérapeutiques pour moduler les impacts négatifs d’un relargage d’ATP excessif observés lors de certaines conditions pathologiques, telles que les lésions pulmonaires induites par la ventilation mécanique.
ATP is widely known to be an energy carrier within cells, but outside of the cell, it acts as an extracellular signaling molecule. Upon binding to purinergic receptors, extracellular ATP initiates the purinergic signaling to regulate certain physiological and pathophysiological processes. In the lungs, ATP stimulates surfactant secretion and promotes mucociliary clearance. Given the critical role of extracellular ATP in the lungs, it is important to understand the mechanism of cellular ATP release — the first step of purinergic signaling. Because mechanical forces constitute the primary trigger of ATP release, this thesis aims to investigate the physiological mechanism(s) and cellular sources of such mechanosensitive ATP release. This work is divided into three parts: 1) To study the spatial and temporal characteristics of ATP release, I developed a highly sensitive imaging technique based on luciferin-luciferase bioluminescence coupled with a custom-designed lens system, which combined a wide field of view (WFOV) and high light-gathering power. To evaluate our imaging approach, I subjected A549 cells, derived from human lung adenocarcinoma, to stretch or 50% hypotonic shock to trigger ATP release. I demonstrated that our technique allows us to precisely quantify the amount and the rate (or efflux) of ATP escaping from cells. The WFOV constitutes an essential tool used in the studies described in this thesis to determine the mechanism and cellular source of ATP release in the alveolus. 2) To examine the physiological mechanism of stretch-induced ATP release in primary alveolar cells, I determined the individual contributions of alveolar type 1 (AT1) in comparison with alveolar type 2 (AT2) cells. To this end, freshly isolated AT2 cells from rat lungs were seeded on a flexible silicone chamber and were cultured for up to seven days, which allowed AT2 cells to progressively transdifferentiate into AT1-like cells. The ratio of alveolar cells (AT2:AT1), being 4:1 on day 3, became 1:4 on day 7. The quantity of released ATP decreased with the decreasing numbers of AT2 cells, implicating them as the main source of ATP release in response to stretch. While pharmacological ATP channel modulators, carbenoxolone and probenecid, did not diminish the amount of ATP release, BAPTA, an intracellular calcium ([Ca2+]i) chelator, significantly reduced it. Likewise, these three modulators had similar effects on intracellular calcium responses measured by Fura-2, suggesting a connection between ATP release and [Ca2+]i levels. 3) To explore the role of membrane viscoelastic properties in mechanosensitive ATP release, I demonstrated that a 30% strain induced transient ATP release that was accompanied by uptake of propidium iodide (PI) in AT2 cells. This is consistent with a strain-induced transient membrane rupture, big enough for the passage of ATP and PI. ATP efflux also increases with strain rate, and hold time prolongs the half-life of ATP release. Thus, these results provide clues on how stretching of the viscoelastic membrane may lead to ATP release via an alternate mechanism involving transient mechanoporation of the cell membrane. Overall, these findings demonstrate that stretch-induced ATP release does not occur through ATP-conducting channels but rather a transient membrane mechanoporation. Further studies on membrane injury induced by strain are needed to better understand its contribution to mechanosensitive ATP release and [Ca2+]i signaling. Such studies will elucidate purinergic signaling in organs that are constantly exposed to physical stresses. This could suggest novel therapeutic targets/approach to modulate the negative impacts of excessive ATP release observed under certain pathological conditions, such as ventilator-induced lung injury.

La détection et la caractérisation des nanoparticules manufacturées (NPM) est l’une des premières étapes pour contrôler et diminuer leurs risques potentiels sur la santé humaine et l’environnement. Différents systèmes d’échantillonnage dans l’air existent pour l’évaluation d’une exposition aux NPM. Cependant, ils ne mesurent pas le risque potentiel de cette exposition à la santé humaine ni les mécanismes cellulaires qui en seraient responsables. Nos objectifs de recherche sont 1) Évaluer les effets de différents types de nanoparticules sur des cellules pulmonaires humaines et 2) Identifier de nouveaux mécanismes intracellulaires activés lors de l’exposition à divers types de NPM. Méthodologie: La lignée de cellules A549 a été utilisée. Trois types de NPM ont été étudiés (différentes concentrations et temps d’exposition): les nanoparticules de dioxyde de titane de type anatase (TiO2), les nanotubes de carbone simple paroi (NTCSP) et les nanoparticules de noir de carbone (NC). La viabilité cellulaire a été mesurée par le test MTS, le test PrestoBlue et le test d’exclusion du bleu de Trypan (uniquement pour les NTCSP). La mesure du stress oxydatif a été déterminée par la mesure des dérivés réactifs de l’oxygène (ROS) en utilisant l’essai DCFH-DA. L’activation d’une réponse anti-oxydative a été déterminée par la mesure de la forme réduite (GSH) et oxydée (GSSG) du glutathion, ainsi que du ratio GSH/GSSG (seulement avec NTCSP et TiO2). Résultats: Les trois nanoparticules ne semblent pas être toxiques pour les cellules A549 car il y a une diminution significative mais minime de la viabilité cellulaire. Cependant, elles induisent une augmentation du contenu intracellulaire en ROS qui est à la fois dépendante du temps et de la concentration. Aucun changement dans les concentrations de GSH et GSSG n’a été observé. En conclusion, nos données indiquent que la mesure de la viabilité n’est pas un critère suffisant pour conclure à la toxicité des NPM. La production de ROS est un critère intéressant, cependant il faudra démontrer l’activation de systèmes anti-oxydatifs pour expliquer l’absence de mortalité cellulaire suite à l’exposition aux NPM.
Detection and characterization of manufactured nanoparticles (NPs) is one of the first steps to control and reduce potential risks to human health and the environment. Various sampling schemes in air exist for the evaluation of exposure to NPs. However, they do not measure the potential risk of this exposure to the human health and the cellular mechanisms that are responsible. Our research objectives are 1) To evaluate the effects of different types of nanoparticles on human lung cells and 2) Identify new intracellular mechanisms activated during exposure to various types of NPs. Methodology: The cell line A549 was used. Three types of NPs were studied (different concentrations and exposure time): titanium dioxide nanoparticles of anatase (TiO2), the simple wall carbon nanotubes (SWCN) and black carbon nanoparticles (BC). Cell viability was measured by the MTS assay, the PrestoBlue assay and the Trypan blue due exclusion test (only for the SWCN). To investigate whether the NPs stimulated ROS generation in A549 cels, the intracellular ROS level was measured using the DCFH-DA assay. The potential induction of oxidative stress responses in cells when exposed to TiO2 and SWCN was determined by the quantification of the extracellular levels of reduced (GSH) and oxidized glutathione (GSSG) forms. Results: The three nanoparticles do not appear to be toxic to A549 cells because there is a significant but small decrease in cell viability. However, they induce ROS production which is both time and concentration dependent. No change in the concentrations of GSH and GSSG were observed. In conclusion, our data indicate that measuring the cell viability is not a sufficient criterion for concluding if the NPs are toxic. ROS production is an interesting criterion, however, we have to demonstrate the activation of anti-oxidative systems to explain the absence of cell death following exposure to the NPs.