肺癌是導致男性死亡的最常見原因以及是排在乳腺癌和結腸癌之後的導致女性死亡的第三大原因。雖然肺癌如此嚴重，但是如今治疗肺癌仍然是一个挑战。現今對肺癌的治療主要集中在化學治療和靶點藥物治療，但是由於這些治療有著很大的副作用和低治愈率，尋找其他的醫學替代方法十分迫切。甘草是其中最常用的中藥，它常常用作食品工業中的甜味劑。以往的研究表明，甘草具有多種的生物活性。但是甘草提取物對於肺癌的治療卻是十分匱乏的。 / 本論文主要目的是評價甘草提取物以及其中的有效成份對非小型肺癌細胞株A549 的影響，以及其作用的機理。我們的數據表明，甘草的乙酸乙酯（EAL）成份比甘草的乙醇提取物有著比較強的抑制癌細胞的作用。另外，對甘草的五個單體進行的測試中發現lico-3 是最具有抑制肺癌作用的。利用高效液相色譜法對甘草活性成份分析表明，lico-3 是EAL中的其中一個單體。 / 乳酸脫氫酶滲漏（LDH）的檢測結果以及异硫氰酸荧光素-碘化丙啶（FITC-PI）雙染的結果表明，EAL 能夠引起肺癌細胞的凋亡現象而非壞死現象。實驗結果表明由EAL引起的A549細胞凋亡是跟Bcl-2家族及Caspase家族有關係，同時EAL還能夠抑制Akt途徑從而導致細胞的死亡。 / 致肺癌細胞死亡的原因進行進一步研究表明，EAL還能夠引起抑制細胞週期的運作，停留在G2/M 時期。這可能是由於EAL引發了p53與p21的上調作用從而抑制了細胞的生長與增殖。 / 實驗結果說明了EAL引起的肺癌細胞株A549的凋亡作用是跟多重細胞通路有關， 同時表明了EAL是具有抗擊肺癌作用的潛能，能夠作為治療肺癌的藥物。 / Lung cancer is the most common cause of cancer death in men and third in women followed by breast cancer and colon cancer, yet treatment of lung cancer remains a challenge. Current treatments including chemotherapy and targeted drug treatment come with side-effects and low successful rate. Alternative medicine for treatment of lung cancer is warranted. Glycyrrhiza uralensis (Gan-Cao), commonly called “licorice, is one of the most commonly used herbs in traditional Chinese medicine (TCM). It is also used as flavoring and sweetening agents in many of food products. Previous studies have indicated that licorice exhibits a variety of biological activities. However, anticancer effects of licorice extract on lung cancer remain unclear. / In this study, we evaluated effects of licorice extract and its chemical components on human lung cancer cell line A549, and studied its mode of action. Our results showed the ethyl acetate fraction of licorice (EAL) was more effective in inhibition of A549 cell growth followed by ETL (IC₅₀: 50μg/mL). Moreover, among the five compounds tested, lico-3 was more potent compound. The HPLC analysis of the active fraction indicated that lico-3 was one of the compounds distributed in the EA fraction. / The results of LDH assay and FITC-PI co-staining method suggested low concentration of EAL can trigger apoptosis but not necrosis. The experimental findings show that EAL induce apoptosis in A549 cell lines involved in Bcl-2 family and caspase cascade. Also, EAL can arrest the Akt survival pathway in A549. Furthermore, the results indicate that EAL triggered G2/M phase arrest. The studies suggest EAL can up-regulate p53 and p21 to promote cell cycle arrest resulting in inhibition of proliferation. / Experimental results indicate that EAL is involved in multiple signal pathways to induce lung cancer cell death. The result suggests EAL is a potential candidate for lung cancer therapy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Yanling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 99-110). / Abstracts in Chinese. / Abstract --- p.III / 論文摘要 --- p.V / Acknowledgement --- p.VII / List of Contents --- p.VIII / List of Figures --- p.X / List of Tables --- p.XI / List of Abbreviations --- p.XII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Lung cancer --- p.1 / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Risk factors --- p.2 / Chapter 1.1.3 --- Types of lung cancer --- p.4 / Chapter 1.1.4 --- Stages and treatment of lung cancer --- p.5 / Chapter 1.1.5 --- Chemotherapy for lung cancer treatment --- p.8 / Chapter 1.2 --- Traditional Chinese Medicines --- p.11 / Chapter 1.2.1 --- Overview --- p.11 / Chapter 1.2.2 --- Licorice --- p.14 / Chapter 1.2.3 --- Chemical study of licorice --- p.16 / Chapter 1.2.4 --- Pharmacological activities of licorice --- p.16 / Chapter 1.3 --- Molecular mechanism of apoptosis --- p.21 / Chapter 1.3.1 --- Overview --- p.21 / Chapter 1.3.2 --- Bcl2 family --- p.21 / Chapter 1.3.3 --- Caspase pathway --- p.23 / Chapter 1.3.4 --- Akt pathway --- p.24 / Chapter 1.3.5 --- p53 protein --- p.26 / Chapter 1.3.6 --- Apoptosis and cancer --- p.27 / Chapter 1.4 --- Cell cycle --- p.29 / Chapter 1.4.1 --- Overview --- p.29 / Chapter 1.4.2 --- Cell cycle and p53 --- p.29 / Chapter 1.4.3 --- Cell cycle and cancer --- p.30 / Chapter 1.5 --- Aims of study --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Cell culture and treatment --- p.33 / Chapter 2.1.1 --- Cell line --- p.33 / Chapter 2.1.2 --- Chemicals and reagents --- p.34 / Chapter 2.1.3 --- Preparation of solutions --- p.34 / Chapter 2.2 --- Preparation of Licorice sample --- p.35 / Chapter 2.3 --- HPLC analysis --- p.35 / Chapter 2.3.1 --- Chemical and materials --- p.35 / Chapter 2.3.2 --- Instrumentation --- p.36 / Chapter 2.3.3 --- Preparation of Standard solutions --- p.36 / Chapter 2.3.4 --- Preparation of samples --- p.37 / Chapter 2.3.5 --- HPLC conditions --- p.37 / Chapter 2.3.6 --- Method validation --- p.37 / Chapter 2.4 --- Cell viable assay --- p.38 / Chapter 2.4.1 --- Samples preparation --- p.39 / Chapter 2.4.2 --- Procedure --- p.39 / Chapter 2.5 --- LDH assay --- p.40 / Chapter 2.5.1 --- Reagent preparation --- p.40 / Chapter 2.5.2 --- Procedure --- p.41 / Chapter 2.6 --- Annexin V assay --- p.41 / Chapter 2.6.1 --- Reagent --- p.42 / Chapter 2.6.2 --- Procedure --- p.42 / Chapter 2.7 --- Cell cycle study --- p.43 / Chapter 2.7.1 --- Chemicals and reagent --- p.43 / Chapter 2.7.2 --- Procedure --- p.44 / Chapter 2.8 --- Caspase3/7 Assay --- p.44 / Chapter 2.8.1 --- Reagent preparation --- p.45 / Chapter 2.8.2 --- Procedure --- p.46 / Chapter 2.9 --- Western blotting --- p.46 / Chapter 2.9.1 --- Reagent and antibodies --- p.46 / Chapter 2.9.2 --- Procedure --- p.50 / Chapter 2.9.3 --- Determination of protein concentration --- p.51 / Chapter 2.10 --- Data analysis --- p.51 / Chapter Chapter 3 --- Results --- p.52 / Chapter 3.1 --- Chromatographic conditions and HPLC identity conformation --- p.52 / Chapter 3.1.1 --- Linearity, limits of detection and quantification --- p.56 / Chapter 3.1.2 --- Reproducibility --- p.56 / Chapter 3.1.3 --- Analysis of ethyl acetate of licorice (EAL) using the validated method --- p.56 / Chapter 3.2 --- Licorice induces apoptosis in nonsmall cell lung carcinoma --- p.61 / Chapter 3.2.1 --- Cell viability assay --- p.61 / Chapter 3.2.2 --- LDH leakage assay --- p.71 / Chapter 3.2.3 --- Annexin V and PI staining --- p.73 / Chapter 3.3 --- Protein expression in EALinduced apoptotic cells --- p.75 / Chapter 3.3.1 --- Bcl2 family --- p.75 / Chapter 3.3.2 --- Activation of caspases by EAL treatment --- p.77 / Chapter 3.4 --- EAL could block Akt survival pathway --- p.79 / Chapter 3.5 --- EAL induces cell cycle arrest in nonsmall cell lung carcinoma --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Chemical analysis of licorice --- p.85 / Chapter 4.2 --- Licorice induced apoptosis but not necrosis on lung cancer cell A549 --- p.86 / Chapter 4.2.1 --- Licorice exhibits specific cytotoxicity to different cancer cells in vitro --- p.86 / Chapter 4.2.2 --- EAL induces cell death via apoptosis but not necrosis --- p.87 / Chapter 4.3 --- Growth inhibition by EAL inducing apoptosis --- p.89 / Chapter 4.3.1 --- EAL induces apoptotic cell death through modification of Bcl2 family --- p.89 / Chapter 4.3.2 --- EAL activate the caspase proteins --- p.90 / Chapter 4.4 --- Growth inhibition by EAL inducing survival pathway arrest --- p.92 / Chapter 4.5 --- Growth inhibition by EAL inducing cellcycle arrest --- p.94 / Chapter 4.6 --- General discussion --- p.96 / Reference --- p.99

With ever increasing advancements in imaging, there is an increasing abundance of images being acquired in the clinical environment. However, this increase in information can be a burden as well as a blessing as it may require significant amounts of time to interpret the information contained in these images. Computer assisted evaluation is one way in which better use could be made of these images. This thesis presents the combination of texture analysis of images acquired during the treatment of cancer with machine learning in order to improve radiotherapy. The first application is to the prediction of radiation induced pneumonitis. In 13- 37% of cases, lung cancer patients treated with radiotherapy develop radiation induced lung disease, such as radiation induced pneumonitis. Three dimensional texture analysis, combined with patient-specific clinical parameters, were used to compute unique features. On radiotherapy planning CT data of 57 patients, (14 symptomatic, 43 asymptomatic), a Support Vector Machine (SVM) obtained an area under the receiver operator curve (AUROC) of 0.873 with sensitivity, specificity and accuracy of 92%, 72% and 87% respectively. Furthermore, it was demonstrated that a Decision Tree classifier was capable of a similar level of performance using sub-regions of the lung volume. The second application is related to prostate cancer identification. T2 MRI scans are used in the diagnosis of prostate cancer and in the identification of the primary cancer within the prostate gland. The manual identification of the cancer relies on the assessment of multiple scans and the integration of clinical information by a clinician. This requires considerable experience and time. As MRI becomes more integrated within the radiotherapy work flow and as adaptive radiotherapy (where the treatment plan is modified based on multi-modality image information acquired during or between RT fractions) develops it is timely to develop automatic segmentation techniques for reliably identifying cancerous regions. In this work a number of texture features were coupled with a supervised learning model for the automatic segmentation of the main cancerous focus in the prostate - the focal lesion. A mean AUROC of 0.713 was demonstrated with 10-fold stratified cross validation strategy on an aggregate data set. On a leave one case out basis a mean AUROC of 0.60 was achieved which resulted in a mean DICE coefficient of 0.710. These results showed that is was possible to delineate the focal lesion in the majority (11) of the 14 cases used in the study.

Au cours de cette thèse, nous nous sommes intéressés à l’implication de la kallicréine-12 (KLK12), une protéase à sérine, dans la cancérogenèse pulmonaire. La KLK12 est connue comme étant surexprimée dans les tumeurs pulmonaires non à petites cellules mais son rôle dans la cancérogenèse n’est pas clairement établi. Nos travaux ont permis de lui attribuer un effet proangiogénique vis-à-vis des cellules endothéliales pulmonaires. En effet, la KLK12 peut stimuler la migration des cellules endothéliales pulmonaires en modifiant l’architecture de la matrice extracellulaire. D’autre part, elle est impliquée dans la régulation de la biodisponibilité de facteurs de croissance associés à l’angiogenèse tels que le Platelet-derived growth factor-B. De plus, nos travaux ont permis d’identifier une possible régulation de l’expression de la KLK12 par l’hypoxie, ce qui expliquerait sa surexpression dans les tumeurs pulmonaires et conforterait son implication dans l’angiogenèse. La confirmation in vivo des mécanismes d’action de la KLK12 pourrait permettre d’envisager son utilisation comme cible thérapeutique afin de réguler la néoangiogenèse tumorale. / In this thesis, we studied the involvement of the serine protease kallikrein-12 (KLK12), in lung carcinogenesis. The KLK12 is known to be overexpressed in non-small cell lung cancer but its role in carcinogenesis is not clearly established. Our work shows that it possesses a proangiogenic effect on pulmonary endothelial cells. Indeed, KLK12 stimulates lung endothelial cells migration notably by modulating the extracellular matrix architecture. On the other hand, KLK12 regulates the bioavailability of growth factors associated with angiogenesis such as plateletderived growth factor-B. In addition, our work has identified a possible regulation of the expression of KLK12 by hypoxia, which would explain its overexpression in lung tumors and would reinforce its involvement in angiogenesis. The confirmation in vivo of these mechanisms could help consider KLK12 as a therapeutic target for regulating tumor angiogenesis.

É feita a análise da evolução de provas funcionais ventilatórias de dois grupos de crianças clinicamente sadias, residentes no Município de Cubatão, examinadas inicialmente em dois estudos transversais realizados respectivamente em 1983 (verão) e em 1985 (inverno). O princípio epidemiológico que norteou essa pesquisa foi a realização de um estudo de Coorte Não-Controlada, ou mais apropriadamente, um Estudo Ecológico Longitudinal, onde esses dois grupos de crianças foram anualmente submetidos a exames espirométricos no período de 1987 a 1989, mantida a estrita sazonalidade, ou seja, o Grupo de 1983 foi sempre reexaminado no verão, enquanto que o Grupo de 1985 sempre no inverno. Aproximadamente 1.110 crianças, oriundas de todas as áreas do Município foram submetidas a provas de função pulmonar utilizando-se espirômetro seco, com especial ênfase na análise do comportamento da Capacidade Vital Forçada (CVF), do Volume Expiratório Forçado no Primeiro Segundo (VEF1,0), do \"Peak-Flow\" ou Pico de Fluxo Expiratório (PFE), e do Fluxo Médio Expiratório Forçado (FMF25-75 por cento ), todos considerados como indicadores sensíveis às influências dos níveis de poluição atmosférica registrados em Cubatão. As informações concernentes à poluição do ar foram obtidas através dos relatórios de pesquisas efetuadas pela Companhia de Tecnologia de Saneamento Ambiental do Estado de São Paulo (CETESB) e envolveram a análise das estimativas de emissões das indústrias prioritárias de Cubatão, das concentrações horárias de material particulado (MP), dióxido de enxofre (SO2) e do ozona (O3) registradss nas estações amostradoras de Vila Parisi, Cubatão-Centro e Vila Nova no período de Novembro de 1983 a Dezembro de 1989. Também integrou o contexto de análise, a avaliação das principais metas do Programa de Controle de Poluição do Ar instituído pela CETESB na região a partir de 1984, com o objetivo de se verificar se as reduções pretendidas nos níveis de poluição do ar se fizeram acompanhar de melhorias nas provas funcionais pulmonares das crianças estudadas. Verificou-se que os episódios agudos de poluição do ar, principalmente devidos ao material particulado, mostraram algum decréscimo. No entanto, aqueles devidos a oxidantes fotoquímicos, representados pelos níveis de ozona, tiveram progressivo incremento no período de 1984 a 1988, tanto em relação ao número de episódios, como na persistência e gravidade dos mesmos. Os resultados espirométricos indicaram que em ambos os grupos de crianças a função pulmonar mostrou decrementos significativos, com perdas em tomo de 5 a 8 por cento ao ano, em média, quando comparados com os valores inicialmente registrados por ocasião dos estudos transversais. Apesar das reduções terem sido observadas em todas as variáveis espirométricas, os maiores decrementos verificaram-se principalmente nas medidas de fluxo (PFE e FMF25_75 por cento ), e coincidiram com o ano em que foram assinalados os mais graves episódios de poluição devida a oxidantes fotoquímicos. Os bairros Cota 95, Cota 200, Jardim das Indústrias e Vila Natal, localizados na área de influência do núcleo industrial químicofpetroquímico sobressairam-se como os mais críticos em função dos piores resultados espirométricos apresentados ao longo de todo o estudo. Pôde-se concluir que apesar da função pulmonar das crianças ainda não ter atingido níveis considerados como patológicos, mesmo registrando valores inferiores aos limites da normalidade nas variáveis de fluxo no ano de 1988, verificou-se que de ano para ano a função pulmonar vem decrescendo progressivamente indicando que esta vai sendo afetada pela exposição as poluentes da região e que as medidas de controle da poluição instituídas em Cubatão não tiveram a eficácia esperada. Continuando nessa mesma trajetória evolutiva, isso poderá acarretar sérios e irreparáveis prejuízos à saúde da população. É necessário, portanto, a adoção de urgentes medidas para a melhoria da qualidade do ar em Cubatão nos anos futuros, no intuito de prevenir sérios agravos à Saúde Pública. / The purpose of this study was to investigate the effects of exposure to air pollution on lung function of children. Two groups of clinically healthy children, living at different areas of the city of Cubatão, who had been submitted to lung function tests in a previous cross-sectional study (1983 summertime and 1985 wintertime) were annually reexamined using the same type of test during the period 1987 to 1989. On epidemiological basis this is a longitudinal study, where every individual of the two groups of children was measured at each ocasion, always in the same season of the previous examination. About 1,110 children from all areas of the city were examined once a year using dry spirometer, with special emphasis on FVC, FEV 1,0, PEF and FMF25-75 per cent measurements, since they were considered as those which could be most affected by the local air pollution. Information regarding the conditions of air pollution were obtained from the CETESB\'s written reports. They include the estimate emissions of the main industries of the region; the hourly concentrations of particulate matter, sulfur dioxide and ozone measured in the sampling stations located in Vila Parisi, Cubatão-Centro and Vila Nova during the October/1983 to December/1989 period and also the results of the Source Control Strategy introduced by CETESB since 1984. It was observed that the acute episodes of air pollution due to PM were reduced in number. However, those due to photochemicaf oxidants, mainly represented by ozone leveis, were progressively more frequent during the period from 1984 to 1988; both their number and their severity increased during this period. Spirometric results shows that in both groups of children the lung function was significantly impaired, with losses of 5 to 8 per cent per year, when compared with the initial values of the cross-sectional studies. Despite the fact that all lung function parameters decline, the flow (PFE and FMF2s-7s per cent ) values were those most affected and closely related to the most serious episodes of air pollution due to photochemical oxidants mainly those observed in 1988. Children living in the areas of Cota 95, Cota 200, Jardim das Indústrias and Vila Natal, situated in the nearest area of influence of the chemical and petrochemical industries, presented worst spirometric results than children living in other places of Cubatão. The lung function of the examined children did not attain pathological values, despite the fact of presenting the worst results in flow parameters during the year of 1988. It was observed that their lung function is progressively impaired year by year, a clear indication of the progressive effects of air pollution on their lung function, and if such impairment continues there will carne the time when lung function will probably reach a pathologicallevel. It is concluded that there is an urgent need of improving the quality of air of Cubatão to avoid serious Public Health problems in a quite near future.

As estrongiloidíases são parasitoses intestinais causadas por várias espécies do gênero Strongyloides e apresentam distribuição cosmopolita. O objetivo deste estudo foi investigar a interferência do tratamento diário com dexametasona no ciclo pulmonar durante a infecção por Strongyloides venezuelensis em ratos. Investigamos o efeito do tratamento com glicocorticóides na migração de eosinófilos, mastócitos e macrófagos no parênquima pulmonar. Demonstramos ainda, como os efeitos do tratamento diário com a dexametasona atuam na formação do granulomas. Três principais aspectos foram encontrados: 1) Aumento da inflamação hemorrágica, provocado pela passagem das larvas para o espaço alveolar; 2) Significante redução da migração de eosinófilos e mastócitos no eixo axial pulmonar e, 3) Interferência crucial na migração de eosinófilos para os focos de passagem das larvas e, conseqüente, impedimento da organização do granuloma, sugerindo que a formação da rede de fibras reticulares deve ter um papel crucial no aprisionamento do parasita, favorecendo um melhor desempenho das células inflamatórias na eliminação do mesmo. Este trabalho mostrou que o uso de drogas com ação imuno-modulatória, tais como a dexametasona, pode interferir na morbidade no ciclo pulmonar durante a infecção por S. venezuelensis, contribuindo para revelar os mecanismos envolvidos na sua patogênese. / The aim of this study was investigate the interference of dexamethasone treatment in the pulmonary cycle of Strongyloides venezuelensis infection in rats. The immunomodulatory effects on the inflammatory process generated by the passage of the larvae into pulmonary parenchyma during their migration were analyzed. Three principal effects were found: 1) Increased alveolar hemorrhagic inflammation provoked by the passage of larvae into the alveolar spaces; 2) Significant decrease of eosinophil and mast cell migration to the axial septum of the lungs and 3) Impaired eosinophil migration to the parasite foci and deficient formation of the reticular fiber network, interfering with the granuloma organization. This study demonstrated that the use of drugs with immunomodulatory actions, such as dexamethasone, in addition to interfere with the morbidity from the pulmonary cycle of S. venezuelensis infection, can contribute to reveal the mechanisms involved in its pathogenesis.

Objetivo: Foi investigada a hipótese da hiperprolactinemia modular a resposta inflamatória alérgica pulmonar em ratos machos e em fêmas lactantes sem tratamento de domperidona. Métodos: Em ratos machos, a hiperprolactinemia foi de curta duração (5 dias) induzida pela domperidona (5,1 mg.kg-1 por dia, i.p). A resposta alérgica foi gerada por sensibilização e desafios inalatórios com ovoalbumina. Foi feita contagem de leucócitos totais e diferenciados do lavado bronco alveolar (BAL), lavado medular femoral (BFL) e sangue; a percentagem de produção de muco e colageno no pulmão, níveis de corticosterona e prolactina e citocinas TNF-α, IL-4, IL-6, IL-10, em explantes de pulmão e IFNg no BAL, foram medidos. Pela citometria foram avaliadaos os receptores de prolactina; Resultados: Hiperprolactinemia de curta duração feita antes do desafio inalatório disminuiu a resposta alérgica pulmonar na contagem de leucócitos no lavado broncoalveolar. Esse tratamento reduziu a celularidade no BFL e a percentagem de muco e aumentou a expressão de citocinas IL-4, IL-6, IL-10, TNFα e da expressão do IFNg. Níveis altos de prolactina diminuiram o número de eosinófilos ao pulmão no BAL. Pela citometria revelou-se que além de ter menor número de granulócitos migrados ao pulmão, estes apresentaram maior expressão do número de receptores por granulócito para prolactina no grupo tratado com domperidona. Alterações similares foram reveladas em fêmeas lactantes como foi a diminuição nos leucócitos do BAL, e no número de células do BFL. O tratamento profilático diminuiu a resposta alérgica tanto no grupo hiperprolactinêmico como no grupo veículo. O tratamento feito após o desafio inalatório não evidenciou alterações relevantes nas variáveis medidas. Conclusões: A hiperprolactinemia de curta duração, feita após a sensibilização e antes da inalação diminui a resposta inflamatória no pulmão em ratos. Os resultados deste estudo demonstram que a hiperprolactinemia induzida antes do desafio antigênico diminue a inflamação alérgica pulmonar. Assim, é provável que a prolactina endógena tenha um papel relevante como um imunomodulador da asma. Este estudo aponta a possibilidade futura do uso da domperidona para pacientes asmáticos. Durante a primavera muitas espécies de mamíferos têm seus filhotes e ocorre abundância de fatores alergenos no ar. Logo, um fator endógeno que favoreça a proteção de fêmeas durante a lactação, tal como a hiperprolactinemia, tem elevado valor adaptativo / Objective: It was investigated if hyperprolactinemia has modulatory actions on lung allergic inflammatory response in male rats. Lactating female rats were tested for pulmonary allergy as well. Methods: In male rats, short-term (5 days) hyperprolactinemia was induced by domperidone (5.1 mg.kg-1 per day, ip). Allergic response was generated by sensitization and inhalation challenge with ovalbumin. Total and differential leukocytes bronchoalveolar lavage (BAL), femoral medullary lavage (BFL) and blood; the percentage of collagen and mucus production in the lungs, plasma levels of corticosterone and prolactin cytokines and TNF-α, IL-4, IL-6, IL-10, IFNg explants lung and BAL, were measured. Flow cytometry was used to evaluate prolactin receptor; Results: Short-term hyperprolactinemia made before the inhaled challenge reduced the pulmonary allergic response in white blood cell counts in BAL. This treatment reduced the cellularity in BFL and the percentage of mucus and increased expression of cytokines IL-4, IL-6, IL-10, TNFa and IFNg expression. High prolactin levels decreased the number of eosinophils to the lung in BAL. There were fewer granulocytes migrated to the lung. These granulocytes showed higher expression prolactin receptors in hyperprolactinemia animals. Similar changes were revealed in lactating females. In these animals, there was a reduction in BAL leukocyte, and the number of cells BFL. Prophylactic treatment decreased the allergic response in both hyperprolactinemic and vehicle groups. The treatment made after inhalational challenge did not induce significant changes in the variables measured in this study. Conclusions: Short-term hyperprolactinemia, made after sensitization and before inhalation, decreases the inflammatory response in the lung of rats. The results of this study demonstrate that hyperprolactinemia, induced before antigen challenge, decreases pulmonary allergic inflammation. Thus, it is probable that the endogenous prolactin has an important role as an immunomodulator of asthma. This study points out the prospect of a future use of domperidone for asthmatic patients. For various mammalian species, parturition occurs during springtime. Pollen in the air might be an abundant allergic factor during springtime. Thus, protecting lactating females against this type allergy might have high adaptive value

Galectin-3 is a β-galactoside binding lectin expressed in tumour cells and macrophages and has been associated with increased malignancy in a variety of cancers. Previous work has shown that galectin-3 is an important regulator of macrophage function, promoting an alternative (M2) phenotype which potentiates chronic inflammation and fibrosis. Tumour associated macrophages (TAMs) adopt an M2 phenotype and are thought to promote tumour growth by down regulating T cell effector function and promoting angiogenesis. This project examines the hypothesis that host galectin-3 promotes lung cancer growth and spread. In order to test this hypothesis, Lewis Lung Carcinoma tumour growth and metastasis was investigated in strain matched mice either expressing or deficient in galectin-3. The Lewis Lung Carcinoma cell line (LLC1) is a spontaneous lung carcinoma line, derived from C57BL/6 mice, which readily forms tumours when transplanted. Furthermore, LLC1 cells were stably transfected with a Luciferase expressing vector in order to assist detection of tumour growth and metastasis in vivo. An orthotopic model of LLC1 growth suggested that galectin-3-/- animals do not support lung carcinoma growth and spread. This finding was confirmed by a subcutaneous model of cancer growth, where it was found that wild type animals display a higher proportion of macrophages expressing a prototypic M2 marker around tumour sites compared to galectin-3-/- animals. M2-promoting cytokine transcripts were also reduced in galectin-3-/- mice. Additionally, tumours of wild type mice were more invasive and presented more mature blood vessels compared to galectin-3-/- mice. To specifically address the role of recruited cells on tumour growth, metastasis and the inflammation profile around tumour sites, in relation to galectin-3 expression, bone marrow cells (BMCs) were transplanted from wild type to galectin-3-/- mice and vice versa. It was shown that galectin-3 positive BMCs restore the wild type phenotype of tumour growth in galectin-3-/- mice, while galectin-3 deficient BMCs impair tumour growth in wild type animals. Furthermore, macrophage ablation experiments demonstrated incapacity for tumour establishment in the absence of macrophages. A series of experiments investigating reported inhibition of galectin-3 by modified citrus pectin (MCP) via competitive inhibition did not provide conclusive results. MCP had no effect in vivo, but was able to inhibit LLC1 cell growth in vitro. Most importantly though, results were inconclusive as to whether galectin-3 binds MCP. Some ligand displacement was seen, but direct binding of the molecules could not be shown. In general, the results obtained demonstrate a strong pro-tumoural effect of galectin-3 on growth, tissue invasion and metastasis of LLC1 tumours via an increased proportion of Ym1-expressing macrophages around tumour sites. It was shown that macrophages are key cells for tumour initiation and that BMC phenotype in relation to galectin-3 expression determines the phenotype of tumour development in subcutaneous and orthotopic LLC1 models. Therefore, galectin-3 has a strong regulatory effect on tumour phenotype and could present a key target in the management of lung carcinomas.

Pulmonary fibrosis is a chronic and debilitating condition that proposes several challenges to both veterinary and medical clinicians. Despite considerable research, many fibrotic lung diseases remain elusive in terms of aetiology, pathogenesis and treatment. Furthermore, progress is hindered by the lack of a translatable animal model with durable and persistent fibrosis. Asinine Pulmonary Fibrosis (APF) is a spontaneous syndrome of aged donkeys with high prevalence (35%). No previous detailed characterisation of APF has been performed and disease diagnosis remains a challenge. APF was studied with regard to clinical, pathological and molecular features and the suitability of this condition as a model for a rare fibrotic lung disease in humans known as pleuroparenchymal fibroelastosis (PPFE) was assessed. In addition, target activatable optical imaging reagents for the real time detection of two key molecular markers of fibrosis: matrix metalloproteinases (MMPs) and lysyl oxidases (LOXF) were evaluated in spontaneous ex vivo models of fibrosis. Such reagents may be used alongside fibred confocal fluorescence microscopy (FCFM), a relatively noninvasive and cutting edge diagnostic tool, to detect and monitor fibroproliferation in animals and man. Whole lungs were collected from 32 aged donkeys at routine necropsy. Gross examination revealed pulmonary fibrosis in 19 donkeys (APF cases), while 13 (controls) had grossly normal lungs. HRCT images and histology sections were reviewed independently and blindly for each of the lungs. Ten of 19 APF lungs were categorised as being ‘consistent with’ PPFE according to previously defined histological and imaging criteria. All 10 PPFE-like lungs had marked pleural and subpleural fibrosis, predominantly within the upper lung zone, with accompanying intra-alveolar fibrosis and elastosis. An activatable Smartprobe for the detection of LOXF, TWB-219, was synthesised by The Bradley Group, Department of Chemistry (UoEDC). The probe was based on a tandem amine oxidation and β-elimination mechanism, resulting in signal amplification detected at the 488nm wavelength. The probe showed increased fluorescence in the presence of diamine oxidase as well as on incubation with aged human lung tissue cell-free homogenate as determined by a fluorescent plate reader. This signal amplification could be inhibited by β-aminopropionitrile, a recognised LOX inhibitor as well as by an in-house inhibitor specific to LOX. An evolutionary family of MMP probes with varying cleavage sequences and structures, synthesised by the UoEDC, was evaluated at each stage of progression with regard to signal to noise ratio, sensitivity and specificity. Probes were tested against recombinant enzymes from the MMP family as well as neutrophil elastase and plasmin. Signal amplification was also assessed on incubation with human and ovine ex vivo lung tissue. The final ‘lead’ MMP probe, SVC-186, was cleaved by MMP-2, -9 and -13. Signal amplification was also seen following incubation with both human and ovine tissue with significant inhibition in the presence of the pan- MMP inhibitor, marimastat. In conclusion, APF is an emerging condition of aged donkeys that shares key pathological and imaging features with human PPFE. Diagnosis of APF and other fibrotic lung conditions across species remains a challenge to veterinary and medical professionals. As such, optical imaging tools may provide dynamic, real time information on the presence and progression of fibroproliferation in the lung. TWB- 219 and SVC-186 produce a detectable increase in fluorescent signal at the 488nm wavelength when activated by LOXF and MMPs respectively. These probes have been shown to function in human ex vivo tissue as assessed by FCFM.

Pulmonary fibrosis is a common consequence of lung inflammation, leading to organ dysfunction and significant morbidity and mortality. Macrophages, through their diverse functions associated with polarisation status, play a role in lung homeostasis and alternatively activated (M2) macrophages have been associated with lung fibrosis. Prolyl hydroxylases (PHDs) are the main oxygen sensors and regulators of hypoxia inducible factors (HIFs). The PHD/HIF pathway is known to play a role in tissue inflammation and fibrosis, but their role in macrophage polarisation is not fully understood. Aim To study the role of the PHD/HIF pathway in macrophage polarisation and lung fibrosis, and specifically in Idiopathic Pulmonary Fibrosis (IPF). Hypothesis It was hypothesised that pan hydroxylase inhibition alters macrophage polarisation and modulates lung inflammation and fibrosis. Methods A combination of pharmacological (pan hydroxylase inhibitors DMOG and FG41) and genetic (HIF and PHD-null) tools were used to manipulate the PHD/HIF pathway. The bleomycin induced lung fibrosis model was used to define the effect of pan hydroxylase inhibition during the early, inflammatory or the late, fibrotic phase of this model. Murine bone marrow derived macrophages (BMDM), human monocyte derived macrophages and alveolar macrophages obtained from patients with lung fibrosis were used to study the effect of pan hydroxylase inhibition on macrophage polarisation. Bronchoalveolar lavage fluid (BALF) from patients was used to define the association between lung CCL18, an M2 associated chemokine, and disease progression in IPF. Results DMOG therapy during the early phase of the bleomycin model significantly reduced lung fibrosis at day 24. In contrast, late phase pan hydroxylase inhibition enhanced lung fibrosis at day 24. In both instances there was evidence of enhanced alveolar macrophage M2-like polarisation following pan hydroxylase inhibition. Reduced fibrosis after early pan hydroxylase inhibition was not a consequence of reduced acute lung inflammation or direct inhibition of collagen synthesis. In BMDM, pan hydroxylase inhibition resulted in an ‘augmented M2-like’ macrophage. Using LysM-Cre HIF-1α, HIF-2α and PHD-3 KO mice as well as chetomin, a potent inhibitor of HIF-1α and HIF-2α mediated gene expression, the HIF-dependent and HIF-independent polarisation markers were defined. PHD-3 deficiency was not sufficient to enhance M2 skewing. In contrast to murine BMDM, in human monocyte derived macrophages and alveolar macrophages from healthy volunteers and patients with interstitial lung disease including IPF, pan hydroxylase inhibition did not augment M2 polarisation and indeed significantly inhibited macrophage CCL18 expression. CCL18 studies in clinical BALF samples confirmed that CCL18 was elevated in the lungs of patients with IPF and other ILDs compared to controls. However, baseline BALF CCL18 concentrations did not correlate with disease severity or with disease progression, suggesting this is not a useful biomarker in IPF. Further, a unique study of serial BAL in IPF patients showed no association between 12-month change in CCL18 and disease progression over the same period. Indeed CCL18 concentrations mostly fell over 12 months in patients that did progress, strongly suggesting that CCL18 does not play a major pathogenic role in IPF. Concluding, it was shown that in both BMDM and murine lung pan hydroxylase inhibition promoted an ‘augmented M2-like’ polarisation. Pharmacological pan hydroxylase inhibition during the late fibrotic phase of injury enhanced fibrosis but it is not known if there was a causal association between M2 macrophages and lung fibrosis. Similarly, the functional relevance of finding enhanced M2 polarisation observed during early DMOG therapy, which subsequently resulted in attenuated fibrosis, is not known. In human macrophages, pan hydroxylase inhibition unexpectedly attenuated CCL18 production, a chemokine associated with an M2-like phenotype in man whilst other M2 markers were unchanged. However, there was no evidence to support a pathogenic role for CCL18 in IPF, and therefore there is little potential for using pan hydroxylase inhibition to target CCL18 and treat IPF.

Objetivo: O câncer de pulmão é a primeira causa de morte relacionada ao câncer quando considerados ambos os sexos. Os grandes esforços para a redução do tabagismo e para a introdução do filtro de cigarro mudaram a epidemiologia do câncer de pulmão. Em países desenvolvidos, a ascensão do adenocarcinoma e o declínio do epidermoide são de notório conhecimento. Outra característica é o aumento da incidência da doença entre mulheres. Um entendimento melhor da atual epidemiologia do câncer de pulmão é necessário para o desenvolvimento de estratégias de saúde pública de prevenção, diagnóstico e tratamento. Métodos: Análise retrospectiva de todos os pacientes com CPNPC tratados com ressecção pulmonar entre 1986 e 2010 em um hospital universitário do Sul do Brasil. As análises foram divididas em três períodos: 1986-1990, 1991-2000 e 2001-2010. O mesmo grupo de patologistas realizou o diagnóstico, e os estágios foram atualizados para a nova classificação da IASLC, 7ª edição. Todas as análises foram realizadas utilizando o programa SAS, versão 13. Resultados: Foram estudados 817 pacientes submetidos à ressecção pulmonar por CPNPC entre 1986 e 2010. Setenta por cento eram homens, média de idade de 61,4 anos, 44,2% carcinoma epidermoide e 40% adenocarcinoma, 26,7% estágio IIIA. A proporção de mulheres apresentou um aumento de 22% no primeiro período para 36% na última década. A idade média no momento da cirurgia era de 52,7 anos para mulheres e 57,3 para homens no primeiro período, e 60,1 para mulheres e 63,9 para homens no último período (p<0.001). A proporção de carcinoma epidermoide modificou de 49,1% inicialmente para 38,7% no último período (p=0.017). Em comparação, a prevalência do adenocarcinoma cresceu de 35,4% para 39,6% e, mais recentemente, para 41,2%. Em relação ao número total de pessoas acometidas pela doença, mulheres com adenocarcinoma representavam 9,4% no primeiro período, 12,5% no segundo e 16,8% no último período. Pacientes com estágio IIIA representavam 27,9% na última década. O tipo de cirurgia predominante foi a lobectomia. A pneumonectomia foi o procedimento cirúrgico em 21,9%, 18,8% e 16,8% dos casos em cada período, em ordem crescente, respectivamente (p<0.03). Conclusão: Neste estudo de pacientes no Sul do Brasil, a análise de gênero demonstrou que a taxa de câncer de pulmão entre as mulheres está aumentando nas últimas três décadas, mas ainda não chegou a ultrapassar a taxa masculina. A proporção de adenocarcinoma em mulheres aumentou. O significativo declínio da quantidade proporcional de pneumonectomia provavelmente reflete a mudança da indicação e técnica cirúrgica. A idade média de pacientes submetidos a tratamento cirúrgico aumentou tanto para homens quanto para mulheres, mas não alcançou a média de países desenvolvidos de 71 anos. A mudança da proporção do tipo histológico e de mulheres está de acordo com os dados de países desenvolvidos. / Objective: Lung cancer is the leading cause of cancer-related death worldwide when considering both genders. The great effort to reduce smoking and to introduce the usage of cigarette filter has changed lung cancer epidemiology. In developed countries, the increasing incidence of adenocarcinoma and the decrease of squamous cell carcinoma are well known. Other characteristic reported is the rising number of women with the disease. Better understanding of current lung cancer epidemiology is necessary for the appropriate design of public health strategies for prevention, diagnosis and treatment. Methods: Retrospective analysis of all patients with non-small cell lung cancer (NSCLC) treated with lung resection between 1986 and 2010 in a university hospital of Southern Brazil. Analysis was divided in three periods: 1986-1990, 1991-2000 and 2001-2010. The same pathology group performed histological diagnosis and all staging was updated according to the new IASLC, 7th edition. All analyses were performed using the SAS program, version 13. Results: We studied 817 patients who underwent lung resection for NSCLC from 1986 to 2010. Seventy percent were males, average age 61.4 years old, 44.2% squamous cell carcinoma and 40% adenocarcinoma, 26.7% stage IIIA. The female proportion increased from 22% in the first period to 36% in the last decade. Mean age at surgery treatment was 52.7 years old for women and 57.3 years old for men in the first period, and 60.1 for women and 63.9 for men in the last period (p<0.001). The proportion of squamous cell changed from 49.1% initially to 38.7% in the last period (p=0.017). In comparison, the adenocarcinoma prevalence increased from 35.4% to 39.6% and, most recently, to 41.21%. Of the total NSCLC patients, females with adenocarcinoma represented 9.4% in the first period, 12.5% in the second and 16.8% in last period. Patients with stage IIIA represented 27.9% in the last decade. Lobectomy was the predominant type of surgery. Pneumonectomy was the surgical procedure in 21.9%, 18.8% and 16.8% of the cases in each period, respectively (p<0.03). Conclusions: In this cohort of patients in Southern Brazil, gender analysis shows that rates of lung cancer in females are rising over the last three decades, but have not surpassed men rates. The proportion of adenocarcinoma in females has increased. The significant decrease of pneumonectomy rates probably reflects changes on surgical management techniques and indication. The mean age of patients undergoing surgical treatment has increased for both men and women, but has not reached the average age reported in developed countries, 71 years old. The histological and gender findings for lung cancer are in accordance with the data of developed countries.