Human epithelium-specific ETS transcription factor-1 (ESE-1), which is also known as E74-like factor-3 (Elf3) in mice, is strongly expressed in lung during fetal development and in certain lung cancers. The primary goal of the work presented in this thesis was to investigate whether ESE-1 is involved in regeneration of the injured lung epithelium by administering naphthalene to both wild-type (Elf3 +/+) and Elf3-deficient (Elf3 -/-) mice. However, optimal conditions for proper utilization of the naphthalene-induced lung injury model must first be established. Therefore, dose-response studies were initially conducted by administering three different doses of naphthalene to both male and female mice, as described in chapter 2. Although it is shown that the extent of naphthalene-induced Clara cell injury is dose-dependent in both male and female mice, female mice are more sensitive to naphthalene-induced injury than male mice independent of the dose. Furthermore, it is also demonstrated that these gender-dependent differences in naphthalene injury can subsequently influence downstream lung repair kinetics. In light of these findings, lung regeneration was examined in both sexes of both Elf3 +/+ and Elf3 -/- mice. As reported in chapter 3, the kinetics of bronchiolar epithelial cell proliferation and differentiation is delayed considerably in Elf3 -/- mice following naphthalene injury. Moreover, expression of transforming growth factor-beta type II receptor, which is a well-known transcriptional target gene of ESE-1 and is involved in the induction of epithelial cell differentiation, is significantly lower in the bronchiolar airway epithelium of Elf3 -/- mice as compared to Elf3 +/+ mice under steady-state conditions and during repair of naphthalene-induced damage. Collectively, these findings occur to a similar extent in both sexes of both Elf3 +/+ and Elf3 -/- mice, and suggest that ESE-1 plays an important role in regulating the kinetics of airway epithelial regeneration after acute lung injury.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. This thesis investigated the role of Syk in the development of the asthma phenotype in acute and chronic mouse models of allergic airways inflammation. Airway hyperresponsiveness (AHR) to methacholine and inflammation increased significantly in HDM-induced compared with the saline control mice. We demonstrated that in vivo inhibition of Syk by selective Syk inhibitors, and genetic deletion of Syk using conditional Syk knockout mice attenuated AHR despite of inflammatory cell influx in the lung. Histological analysis showed airway remodeling in the chronic model, which was attenuated to some degree by deletion of Syk. This study identified a role of Syk in airway hyperresponsiveness and remodeling without significantly affecting leukocyte recruitment in HDM model of airways disease. My results support the improvement of therapeutic strategies in asthma by targeting the Syk pathway.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. This thesis investigated the role of Syk in the development of the asthma phenotype in acute and chronic mouse models of allergic airways inflammation. Airway hyperresponsiveness (AHR) to methacholine and inflammation increased significantly in HDM-induced compared with the saline control mice. We demonstrated that in vivo inhibition of Syk by selective Syk inhibitors, and genetic deletion of Syk using conditional Syk knockout mice attenuated AHR despite of inflammatory cell influx in the lung. Histological analysis showed airway remodeling in the chronic model, which was attenuated to some degree by deletion of Syk. This study identified a role of Syk in airway hyperresponsiveness and remodeling without significantly affecting leukocyte recruitment in HDM model of airways disease. My results support the improvement of therapeutic strategies in asthma by targeting the Syk pathway.

Several epidemiological studies have revealed an association between particulate matter (PM) pollution and various health effects. Importantly, there is evidence to suggest that individuals with pre-existing respiratory disease, such as asthma, are more sensitive to elevated ground levels of particulate matter as compared to healthy subjects. Among the various sources of PM pollution, diesel powered vehicles have been identified as important contributors. The aim of this thesis was to investigate the airway effects of experimental chamber exposure to diesel exhaust (DE) in healthy and asthmatic subjects, focusing on airway responsiveness, airway inflammation and lung function. To achieve a comprehensive picture of the airway responses to DE, a number of different methods were used, including lung function measurements, methacholine inhalation tests, induced sputum and bronchoscopy. Each subject acted as his/her own control by being exposed both to filtered air and DE in a crossover design. Short term exposure to DE, at a particle concentration (PMi0) of 300 ug/m3, was associated with a clinically significant increase in bronchial hyperresponsiveness in asthmatic subjects. In accordance with the epidemiological data suggesting a 1-4 day lag effect for most health outcomes to PM pollution, the increase was detected one day after DE exposure, indicating a long lasting response to DE in asthmatic airways. Diesel exhaust induced a range of airway inflammatory changes as reflected in induced sputum, bronchoalveolar lavage and bronchial mucosal biopsies. In healthy subjects, DE exposure was associated with an increase in neutrophils and IL-6 in sputum, elevated levels of IL-8 and IL-6 in bronchial wash (BW), enhanced expression of IL-8 and GRO-a in the bronchial epithelium and with increases in P-selectin and VCAM-1 in the airway mucosa. In contrast, asthmatics responded with an increase in IL-6 in sputum and an enhanced expression of IL-10 in the bronchial epithelium following exposure DE. Thus, clear differences were identified between healthy and asthmatic subjects in the inflammatory response to DE. Airway epithelial cells constitute the first line of cellular defence towards inhaled air pollutants and increasing evidence suggests that these cells contribute markedly to the initiation of airway inflammatory responses. The bronchial epithelium was identified to have an important regulatory role in response to diesel exhaust, including the capacity to produce chemoattractant and immunoregulatory proteins associated with development of airway inflammation and bronchial hyperresponsiveness. Lung function measurements revealed that short-term exposure to DE induces an immediate bronchoconstrictive response in both healthy and asthmatic individuals, with significant increases in airway resistance (Raw) following DE exposure. This thesis also investigated the effects of a lower concentration of DE (PMio 100 ug/m3) than previously studied. It was shown that exposure to DE at a concentration corresponding to a PM level that may be encountered in busy traffic situations, was still associated with potentially adverse airway responses in healthy and asthmatic subjects. In summary, the results presented here indicate that short term exposure to diesel exhaust, at high ambient concentrations, has the potential to induce a range of biological events in the airways of healthy and asthmatic subjects. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 2002, härtill 4 uppsatser.</p> / digitalisering@umu

Asthma is a chronic disorder highlighted by intermittent airway inflammation and characterized by paroxysmal dyspnea and airway hyperresponsiveness (AHR). A key feature of severe asthma is the development of airway wall remodeling, which is thought to occur through repeated rounds of inflammation and tissue repair. Remodeling includes structural changes such as increased mass of airway smooth muscle (ASM), and excessive collagen deposition. ASM cells contribute to airway remodeling via the expression and secretion of extracellular matrix (ECM) proteins. This is particularly driven by inflammatory processes, which include mediators such as transforming growth factor (TGF)-β1 and damage associated molecular pattern (DAMP) proteins, such as high mobility group box 1 (HMGB1). HMGB1 is ubiquitously expressed as a non-histone DNA-binding protein that can regulate gene expression, but can also be released in response to stress to underpin inflammation and tissue repair. In this study we tested the hypothesis that extracellular HMGB1 induces signaling pathways that control responses linked to progression of airway inflammation, remodeling and hyperresponsiveness in human ASM cells. We used primary cultured ASM cells as well as hTERT-immortalized human ASM cells. With immunoblotting we demonstrate that exogenous HMGB1 (10 ng/mL) can induce rapid and sustained phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) that is comparable to that induced by a potent mitogen, platelet derived growth factor (PDGF-BB, 10 ng/mL). We also found that TGF-β1 (2.5 ng/mL) promotes the accumulation of secreted HMGB1 in culture medium in a time line concomitant with expression of ECM proteins, collagen and fibronectin, suggesting a role for HMGB1 in pro-fibrotic effects of TGF-β1. By lentiviral delivery, we induced stable expression of short hairpin RNA (shRNA) that silenced expression of endogenous HMGB1 or mammalian diaphanous 1 (mDia1), a cytoplasmic scaffold protein that is required for HMGB1-induced cell responses through one of its receptors, receptor for advanced glycation end products (RAGE). Immunoblot analyses revealed that silencing of mDia1 was associated with markedly decreased induction of p42/p44 MAPK phosphorylation by exogenous HMGB1. In HMGB1-silenced human ASM cells, we observed significantly reduced synthesis and secretion of collagen A1 and fibronectin in response to TGF-β1 (2.5 ng/mL, 0-48 hrs). However, exogenous HMGB1 was not sufficient to rescue ECM synthesis in response to TGF-β1 in HMGB1-silenced cells - this suggests that intracellular, but not necessarily secreted HMGB1, regulates ECM expression and secretion in response to TGF-β1. Consistent with this interpretation, exogenous HMGB1 alone was not sufficient to induce ECM synthesis or secretion in primary cultured ASM cells. In conclusion, we show that though in human ASM cells extracellular HMGB1 alone can activate MAPK signaling, likely via mDia1-dependent pathways involving RAGE. it is not capable of prompting ECM protein expression. Recombinanat exogenous HMGB1 does not appear to directly affect ECM synthesis, rather intracellular (nuclear) HMGB1 likely modulates activity of genes that are affected by TGF-β1. Overall, HMGB1 has potential to regulate tissue repair processes involving ASM through intracellular and extracellular mechanisms, thus our findings support further work to elucidate the role of HMGB1 in pathogenesis of obstructive airway disease.

The main goal of this research was the in vitro investigation of the stiffness response of contracted airway smooth muscles under different external oscillations. Living animal airway smooth muscle tissues were dissected from pig tracheas and stimulated by a chemical stimulus (acetylcholine). These tissues were then systematically excited with different external vibrations. The force change was recorded to reflect the muscle stiffness change under vibration. The static and dynamic stiffness of contracted airway smooth muscles in isometric contraction were determined before, during and after vibrations. A continuum cross-bridge dynamic model (the fading memory model) was modified to accommodate smooth muscle behaviour and dynamically describes the cross-bridge kinetics. A two-dimensional finite element model (FEM) was developed to simulate longitudinal and transverse vibrations of the tissue. An empirical equation, derived from the experiments, is incorporated into the FEM. The results indicate that the stiffness of active smooth muscles can be physically reduced using external vibrations. This reduction is caused by a certain physical position change between actin and myosin. The dynamic stiffness has the tendency of decreasing as the frequency and/or amplitude of external vibration increases. However, the static stiffness decreases with an increase in the frequency and amplitude of excitation until it reaches a critical value of frequency where no variation in stiffness is observed. It is postulated that the tissue elasticity and mass inertia are the main contributors to the dynamic stiffness while the actin-myosin cross-bridge cycling is the main contributor to the static stiffness.

Thesis (Ph.D.)--University of Western Australia, 2005.

Orientador: Francisco Jose´[UNESP] Teixeira Neto / Resumo: Girotto, C.H. Avaliação da máscara laríngea como alternativa a sonda endotraqueal para manutenção da anestesia inalatória sob ventilação espontânea em capivaras (Hydrochoerus hydrochaeris). 47 p. Dissertação (Mestrado) – Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, 2018. A intubação orotraqueal roedores é um procedimento de maior dificuldade que em outras espécies. Este estudo comparou o uso máscara laríngea humana (ML) com a sonda endotraqueal (Sonda-ET) para manter a patência da via aérea em capivaras anestesiadas sob ventilação espontânea. Seis capivaras (24-54 kg) foram contidas quimicamente com cetamina (7,2 ± 1,1 mg/kg), midazolam (0,16 ± 0,04 mg/kg) e acepromazina (0,03 ± 0,01 mg/kg) em duas ocasiões (intervalos ≥ 7 dias entre procedimentos). A anestesia foi mantida com isoflurano diluído em oxigênio durante 90-120 minutos sob ventilação espontânea. Durante cada anestesia, a patência da via aérea foi mantida aleatoriamente com a Sonda-ET ou ML. Tomografia computadorizada (TC) da faringe/laringe foi realizada em 3/6 animais com a ML e 2/6 animais com a Sonda-ET. A ANOVA de duas vias para medidas repetidas, teste t pareado ou de Wilcoxon foram utilizados para análise estatística (P < 0,05). A concentração de isoflurano expirado (ETiso), freqüência cardíaca (FC), pressão arterial média invasiva (PAM), pH arterial, pressão parcial de dióxido de carbono arterial (PaCO2) e pressão parcial de oxigênio arterial (PaO2) não diferiram entre os tratamentos. Os ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Abstract Girotto, C.H. Evaluation of a laryngeal mask as an alternative to orotracheal intubation for maintenance of inhalant anesthesia under spontaneous ventilation in capybaras (Hydrochoerus hydrochaeris). 47 p. Dissertation (MSc) – School of Medicine, São Paulo State University, Botucatu, 2018. Orotracheal intubation carries greater difficulty in rodents than in most domestic species. This study compared the human laryngeal mask (LMA) with an endotracheal tube (ETtube) for maintaining airway patency in anesthetized capybaras (Hydrochoerus hydrochaeris). Six capybaras (24–54 kg) were remote darted with ketamine (7.2 ± 1.1 mg/kg), midazolam (0.16 ± 0.04 mg/kg) and acepromazine (0.03 ± 0.01 mg/kg) in two occasions (≥ seven-day intervals). Anesthesia was maintained with isoflurane in oxygen for 90–120 min under spontaneous ventilation. During each anesthetic, the airway was randomly maintained with an ETtube or LMA. Computed tomography of the pharynx/larynx was performed in 3/6 animals and 2/6 animals with the LMA and ETtube, respectively. Data was analyzed with a two-way ANOVA for repeated measures, paired t-test or Wilcoxon´s signed-rank test. End-tidal isoflurane (ETiso), heart rate (HR), invasive mean arterial pressure (MAP), arterial pH, arterial carbon dioxide partial pressure (PaCO2), and arterial oxygen partial pressure (PaO2) did not differ between treatments. Median (lower–upper range) of ETiso values were 0.6 (0.5–1.5)% and 0.6 (0.4-0.9)% with the ETtube and LMA, r... (Complete abstract click electronic access below) / Mestre

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