The goal of this thesis is to design an attachment between a duct and a pod. A pod is what could be compared to an outboard engine for larger vessels. The pod is placed on the outside of the ship’s hull and can rotate 360 ̊. A duct is a large steel ring that can be placed around the propeller on the pod to increasethe propulsion force of the ship in lower velocities. The thesis is written for Rolls Royce AB in Kristinehamn.The demanded requirements of the design are: it must be easy to adapt to different sizes and shapes of pod houses, the duct must be able to bemounted and dismounted from the pod.The result from this thesis is that anupper and a lower attachment weredesigned. The upper attachment consists of two parallel steel plates which arebolted together with a screw plate that is welded to the pod house.The lower attachment is basically a wing with a streamlined profile which is bolted to a stay. Both attachments consist of bolted joints, two guide pins and bursting screws.The design is controlled by parameters in CAD, meaning that if you change the size of the duct the attachments automatically adapts themselves to the new dimensions.The total result from this thesis is a design suggestion and not an actual manufactured product.The design is dimensioned by a load analysis and strength calculations. The dimensions of the bolts are determined by basic load calculation and the guide pins aredimensioned to withstand the shear forces that occur.The conclusion of this thesis is that the developed attachment solution is a good way to solve this problem andthe personal recommendation is that it should be developed further.Further design work that needs to be done with the attachment is a more thorough analysis of the strength and especially with fatigue, since no account has been taken considering fatigue. / Målet med detta arbete är att konstruera en infästning mellan en dysa och en pod. En pod är vad som kan liknasen utombordsmotor för stora fartyg som sitter på utsidan av skrovet och kan rotera 360 ̊. En dysa är en stor stålring som kan sättas runt propellern, dysan ger förenklaten ökad framdrivningskraft och är lämplig för användning vid fartygstillämpningar där det handlar om låga hastigheter.Arbetet utförs på uppdrag av Rolls Royce AB i Kristinehamn.Kraven som ställdes på konstruktionen var att den skulle vara lätt att anpassa och att det skulle gå att montera och demontera dysan.Problemformuleringen för arbetet är att en ny infästning behövs för att kunna fästa dysan i poden.Resultatet av arbetet blev en övre och en nedre infästning på dysan. Den övre infästningen består av två parallella plåtar som skruvas ihop med en svetsad infästning på dysan. Den nedre infästningen består av en vinge med en strömningsvänlig profil som skruvas fast på ett stag. Båda infästningarna består av ett skruvförband, två styrpinnar och sprängskruv.Konstruktionen parametriserades i CAD så attomdysans storlekändrasså följer infästningarna med.Hela arbetet resulterade i ett konstruktionsförslag.Konstruktionen har dimensionerats med hjälp av en lastanalys och hållfasthetsberäkningarför att uppskatta infästningarnas nedböjning. Skruvförbanden har dimensionerats med enklare beräkningar och styrpinnarna har dimensioneratför att uppta skjuvspänningar.Slutsatsen som dras av arbetet är att konstruktionen är en lämplig lösning på problemet och rekommendationen är att det är värt att undersöka denna lösning vidare.Vidarearbete som behöver utföras är nogrannare dimensionering av konstruktionen, ingen hänsynhar tagits till utmattning eller vridning då det inte fanns tid inom arbetets tidsram för detta.

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.

Background: Intrahepatic cholangiocarcinoma (CCA) is a highly treatment-resistant malignancy of biliary epithelium with increasing global mortality. Histologically, CCA is characterised by a pronounced inflammatory stroma of tumour-associated myofibroblasts, macrophages, immune cells and a modified extracellular matrix (ECM). In other solid cancers, the stroma plays a tumour promoting role. The functional role of the stroma in CCA remains unclear. The origin and the proportional contribution to the stroma by haematopoietic and mesenchymal bone marrow (BM) -derived cells is not known in CCA. Intriguingly, reports suggest that mesenchymal stem cells (MSCs) may contribute to the epithelial compartment of malignant tumours. Furthermore, the Notch signalling pathway is known to play oncogenic and tumour suppressive roles in diverse neoplasms but its role in CCA remains unclear. Aims and Methods: The functional role of myofibroblasts and macrophages in the tumour stroma of CCA was investigated together with an analysis of the origin and contribution of BM-derived cells to the stromal and epithelial compartments of CCA. The Notch signalling pathway was studied as a potential signalling mechanism through which the stroma and malignant epithelial compartments of CCA may interact. Results: The thioacetamide rat model of CCA was optimised and found to display excellent histological congruence with human lesions. The tumour cellular microenvironment comprised of myofibroblasts, migratory macrophages and immune cells. During cholangiocarcinogenesis, progressive intrahepatic accumulation of inflammatory cells and proliferation of bipotential progenitor cells preceded the development of invasive CCA. In vitro, CCA lines were identified to contain a side population of stem cells. Adoptive transfer of BM from Enhanced Green Fluorescent Protein (EGFP) transgenic rats to wild type rats to establish chimeras was undertaken. In transplanted rats, persistent EGFP+ chimerism of both haematopoietic and mesenchymal stem cell compartments was established. In tumours, macrophages and neutrophils were overwhelmingly EGFP+ve, whereas myofibroblasts, fibroblasts and benign and malignant bile ducts were EGFP-ve. There was no evidence of cell fusion or EGFP silencing. These findings were confirmed in spontaneous breast, skin and colon tumours in EGFP+ chimeric rats not treated with TAA. In vitro studies to recapitulate the cellular and extracellular elements of the tumour niche identified that ECM components induce characteristic cell proliferation patterns dependent on the matrix component but do not appear to affect chemosensitivity. Bidirectional interaction between CCA cells and hepatic stellate cells (mediated by soluble factors) was identified. Furthermore, in direct co-culture, M2 polarised macrophages appear to enhance CCA cell proliferation compared to M1 macrophages. In considering the Notch pathway, Notch signalling components (particularly Notch3) and target genes were upregulated in human CCA specimens. Immunohistochemical analysis identified apparent distribution of Notch ligand on tumour stroma and Notch receptor subtypes on malignant epithelia. Although direct co-culture of CCA cells with myofibroblasts and M1/M2 polarised macrophages did not clearly demonstrate stromal:epithelial Notch pathway activation, this may have been a function of in vitro experimental limitations. Gamma-secretase inhibition downregulated the Notch pathway, reduced proliferation and appeared to enhance chemosensitivity of CCA cells in vitro. Conclusions: A stereotypical niche forms around CCA in developing and malignant lesions. There was no evidence of a BM-derived stem cell contribution to the epithelial component of CCA, breast, colon or skin malignancies. Haematopoietic but not mesenchymal components of the tumour stroma were of BM origin. Notch signalling is upregulated in CCA and appears to play a tumour promoting role in CCA; pathway inhibition represents a potential therapeutic target.

Orientador: Antônio José Maria Catâneo / Resumo: Objetivo: avaliar se a Mitomicina C (MMC) pode ampliar a taxa de sucesso da Dacriocistorrinostomia (DCR) externa ou endoscópica quando utilizada como tratamento adjuvante ao tratamento cirúrgico. Método: revisão sistemática de ensaios clínicos randomizados referentes a DCR externa ou endonasal, comparando as cirurgias realizadas com e sem MMC. Foram pesquisadas as bases de dados PubMed, Embase e Cochrane Central Register of Controlled Trials, de Janeiro de 1990 até Maio de 2018. A síntese dos resultados seguiu o programa RevMan 5.3 para a realização das meta-análises. Resultados: foram encontrados 937 estudos, dos quais 25 artigos foram considerados para a meta-análise. Subgrupo DCR externa (12 estudos, OR 2,69, IC 95% 1,36 a 5,32; I²= 38%), subgrupo DCR endoscópica (13 estudos, OR 1,70, IC 95% 1,21 a 2,39; I²= 0%). Observou-se que o uso da MMC aumenta a chance de sucesso da DCR nas duas técnicas (OR 2,03, IC 95% 1,44 a 2,88; I²= 16%) Conclusão: A MMC pode melhorar ligeiramente a taxa de sucesso na DCR externa e endoscópica. / Abstract: Objective: to evaluate the success rate of external or endoscopic dacryocystorhinostomy (DCR) with mitomycin C (MMC) as an adjuvant therapy. Method: a systematic review of randomized clinical trials (RCT) was done to analyze if the success rate of external or endoscopic DCR can be improve using MMC as adjuvant therapy. The following databases were searched: PubMed, Embase and Cochrane Central Register of Controlled Trials, ranging from January 1990 to May 2018. The results were obtained using the RevMan software (v5.3) to perform meta-analysis. Results: Nine hundred and thirthy-seven studies were found, of which 25 were considered for meta-analysis. The external DCR group had 12 studies (OR 2,69; 95% CI 1,36-5,32; I2= 38%), while the endoscopic DCR group had 13 studies (OR 1,70; 95% CI 1,12-2,39; I2= 0%). It was observed that the use of MMC can increases the success rate of DCR using both techniques (OR 2,03; 95% CI 1,44-2,88; I2= 16%). Conclusion: The use of MMC as adjuvant treatment in the external or endoscopic DCR can slightly improve the success rate of both techniques. / Mestre

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Base teórica: A Síndrome Hepatopulmonar (SHP) caracteriza-se por uma tríade clínica que inclui doença hepática, anormalidades em trocas gasosas e presença de dilatações vasculares pulmonares, sendo a obstrução prolongada do ducto biliar utilizada em modelos experimentais de cirrose biliar secundária que leva a reações inflamatórias agudas, podendo, ainda, ocasionar alterações sistêmicas. Animais com cirrose induzida experimentalmente por ligadura de ducto biliar (LDB) apresentam aumento do estresse oxidativo no tecido hepático, mas a inter-relação com o tecido pulmonar ainda não está bem esclarecida. O estresse oxidativo desempenha papel central na patogênese e na progressão de doenças crônicas, e o uso de antioxidantes tem sido proposto como agente terapêutico para compensar danos sistêmicos e hepáticos. Em estudos em que a utilização da melatonina foi comparada com a utilização de outros antioxidantes conhecidos como as vitaminas C e E, tal componente mostrou-se mais eficiente na redução do estresse oxidativo/nitrosativo, demonstrando que a melatonina atua como potente antioxidante. Objetivo: A presente tese teve como objetivo investigar o efeito do antioxidante Melatonina sobre o tecido pulmonar no modelo experimental de Síndrome Hepatopulmonar por ligadura de ducto biliar. Métodos: O trabalho está dividido em três partes: Parte I- Artigo de revisão intitulado “Hepatopulmonary Syndrome: Oxidative Stress and Physical Exercise,” enviado para publicação no periódico European Medical Journal; Parte II- Artigo original intitulado “Efeitos da melatonina sobre o tecido pulmonar no modelo experimental de síndrome hepatopulmonar”, enviado para o periódico Jornal de Pneumologia; Parte III- Artigo original intitulado “Efeitos da melatonina sobre o tecido hepático e pulmonar em animais com síndrome hepatopulmonar por ligadura do ducto biliar”, a ser enviado para o periódico Digestive Diseases and Sciences. Foi utilizado o modelo de cirrose biliar secundária, induzida pelo modelo de ligadura de ducto biliar (LDB), com ratos machos Wistar, pesando ± 300 gramas, divididos em quatro grupos: CO (controle), CO+MLT, SHP e SHP+MLT. Os animais foram tratados com MLT a partir do 15° dia e com LDB até o 28° dia. No 29° dia, mediante administração de fármacos anestésicos, foram coletados sangue, fígado, pulmão e fêmur. Resultados: Nos artigos II e III, ao se avaliar as transaminases, observou-se aumento significativo nas enzimas ALT, AST e FA no grupo SHP quando comparadas aos grupos controles, o que determina a presença de doença hepática. Posteriormente à administração da 7 melatonina, houve diminuição significativa do grupo SHP+MLT quando comparado ao grupo SHP na avaliação da lipoperoxidação, atividade das enzimas Catalase (CAT), superóxido dismutase (SOD) e glutationa Peroxidade (GPx), bem como na de Nitritos/Nitratos (NO2/NO3) e no sistema inflamatório, quando da avaliação da expressão da Óxido Nítrico Sintase Induzível (iNOS) e da região p65 do fator de transcrição nuclear-kB (NF-kB). Também houve diferença na gasometria arterial e na histologia do tecido pulmonar pelas técnicas de Hematoxilina e Eosina e Picrosírius, especialmente no diâmetro dos vasos pulmonares. Ao avaliar o dano ao DNA pelo Ensaio Cometa, foi observado aumento do índice e da frequência de dano no grupo SHP, com redução após o uso da MLT. Conclusão: Os resultados sugerem efeito protetor da melatonina no tecido hepático e pulmonar, no modelo de Síndrome Hepatopulmonar por ligadura de ducto biliar. / Background: Hepatopulmonary syndrome (HPS) is clinically characterized by a combination of liver disease, gas exchange abnormalities, and pulmonary vascular dilation. Prolonged biliary duct obstruction was used in experimental models of secondary biliary cirrhosis leading to acute inflammatory reactions and possibly to systemic changes. Animals with cirrhosis experimentally induced by biliary duct ligation (BDL) showed increased oxidative stress in liver tissue, but the lung tissue has not been investigated yet. Oxidative stress has a key role in the pathogenesis and progression of chronic diseases, and antioxidants have been proposed as therapeutic agents to compensate for systemic and liver damages. In studies comparing melatonin with other known antioxidants such as vitamins C and E, melatonin was found to be more efficient in reducing oxidative/nitrosative stress, showing that it is a potent antioxidant. Objective: The present doctoral dissertation aimed to investigate the effect of the antioxidant melatonin on lung and liver tissues in an experimental model of BDL-induced HPS. Methods: This dissertation is divided into three parts: Part I consists of the review article entitled “Hepatopulmonary Syndrome: Oxidative Stress and Physical Exercise,” which was submitted to the European Medical Journal; Part II consists of the original article entitled “Efeitos da melatonina sobre o tecido pulmonar no modelo experimental de síndrome hepatopulmonar”, which was submitted to Jornal de Pneumologia; and Part III consists of the original article entitled “Effects of melatonin on liver and lung tissues of animals with bile duct ligation-induced hepatopulmonary syndrome”, which will be submitted to Digestive Diseases and Sciences. A model of secondary BDL-induced biliary cirrhosis was used in male Wistar rats weighing ± 300 g and divided into four groups: control group (CG), CG+MLT, HPS, and HPS+MLT. Animals were treated with MLT or vehicle from day 15 to day 28 after BDL. On day 29, blood, liver, lung, and femur samples were collected after the administration of anesthetic drugs. Results: As for transminases, papers II e III found that there was a significant increase in enzymes ALT, AST e FA in the HPS group compared with the control groups, which indicates the presence of liver disease. The administration of melatonin led to a significant decrease in the HPS group compared with the control groups with regard to the following variables: lipid peroxidation; catalase, superoxide dismutase, and glutathione peroxidase activity; nitrite/nitrate ratio; and inducible nitric oxide synthase and p65 region of nuclear factor kappa B. There were also differences in blood gas analysis and histology of lung tissues as assessed by hematoxylin and eosin (HE) and picrosirius red staining, especially in the diameter of pulmonary vessels. The comet assay revealed an increase the index and frequency of DNA damage in the HPS group compared with the control groups, but these parameters were reduced with the use of melatonin in the HPS+MLT group. Conclusion: Results suggest that melatonin has a protective effect in liver and lung tissues using a BLD-induce HPS model.

A exploração visual dos dutos biliares é de relevante interesse clínico, pois fornece informação relacionada com a Atresia Biliar (AB). A AB é uma doença cujas causas ainda permanecem desconhecidas e que eventualmente leva a um transplante de fígado ou, nos casos mais avançados da doença, leva a óbito do paciente. A única evidência física conhecida até agora da existencia de AB é a obstrução das vias biliares. No entanto, o estudo desta doença tem sido limitado pela incapacidade de analisar o duto biliar de pacientes em estágios precoces da doença e muito pouco se sabe sobre a estrutura interna do duto biliar. Nos últimos anos, a microscopia confocal, uma técnica que permite a obtenção de conjuntos de dados 3D de amostras biológicas, tem sido utilizada em experiências médicas para estudar a estrutura interna e anatômica dos dutos biliares. Neste trabalho, é objetivo apoiar o estudo dessas estruturas através da visualização volumétrica de imagens dos dutos biliares. É proposto um pipeline de fluxo de dados capaz de processar e "renderizar"conjuntos de dados de imagens confocais utilizando o VTK (do inglês The Visualization ToolKit). O pipeline foi construído em duas etapas principais e consecutivas. Uma primeira etapa tem o objetivo de remoção de ruído e realce das estruturas relevantes por meio de filtragem no domínio da freqüência e difusão anisotrópica. O conjunto de dados assim pré-processado é usado com técnicas diretas de visualização de volumes baseadas em funções de transferência para exibir as estruturas dos dutos biliares. Os resultados mostram que a visualização volumétrica em conjunto com um pré-processamento adequado das imagens confocais permite evidenciar as regiões de interesse nos dutos biliares e melhora detalhes que são dificilmente visualizados nos dados originais. / The visual exploration of bile ducts in the liver is of relevant clinical interest, as it provides information related to the Biliary Atresia, a disease of unknown origin, which eventually leads to a liver transplant or ultimately to death. The only physical known evidence of biliary atresia is the obstruction of the bile ducts. However, the study of this disease has been limited by the inability to observe the bile duct in patients at early stages of the disease. Moreover, very little is known about the internal structure of the bile duct. In recent years, confocal microscopy, a technique that allows to obtain 3D image datasets from biological samples, has been used in medical experiments for studying the anatomical internal structure of bile ducts. We are interested in supporting the study of these structures through volumetric visualization of bile ducts images. In this work, we propose a data flow pipeline capable of processing and rendering datasets of confocal images using The Visualization ToolKit - VTK. The pipeline was built as two consecutive stages. We propose a first stage for denoising and enhancing the relevant structures of sample based on filtering in the frequency domain and anisotropic diffusion. We use the dataset preprocessed in this way for applying a direct volume rendering technique in a second stage based on transfer functions to visualize the bile duct structures. Our results have shown that volumetric visualization together with an adequate pre-processing of the confocal images allow experts to visualize the regions of interest in the bile ducts, improving details that are hardly visualized in the original data.

Base teórica: A Síndrome Hepatopulmonar (SHP) caracteriza-se por uma tríade clínica que inclui doença hepática, anormalidades em trocas gasosas e presença de dilatações vasculares pulmonares, sendo a obstrução prolongada do ducto biliar utilizada em modelos experimentais de cirrose biliar secundária que leva a reações inflamatórias agudas, podendo, ainda, ocasionar alterações sistêmicas. Animais com cirrose induzida experimentalmente por ligadura de ducto biliar (LDB) apresentam aumento do estresse oxidativo no tecido hepático, mas a inter-relação com o tecido pulmonar ainda não está bem esclarecida. O estresse oxidativo desempenha papel central na patogênese e na progressão de doenças crônicas, e o uso de antioxidantes tem sido proposto como agente terapêutico para compensar danos sistêmicos e hepáticos. Em estudos em que a utilização da melatonina foi comparada com a utilização de outros antioxidantes conhecidos como as vitaminas C e E, tal componente mostrou-se mais eficiente na redução do estresse oxidativo/nitrosativo, demonstrando que a melatonina atua como potente antioxidante. Objetivo: A presente tese teve como objetivo investigar o efeito do antioxidante Melatonina sobre o tecido pulmonar no modelo experimental de Síndrome Hepatopulmonar por ligadura de ducto biliar. Métodos: O trabalho está dividido em três partes: Parte I- Artigo de revisão intitulado “Hepatopulmonary Syndrome: Oxidative Stress and Physical Exercise,” enviado para publicação no periódico European Medical Journal; Parte II- Artigo original intitulado “Efeitos da melatonina sobre o tecido pulmonar no modelo experimental de síndrome hepatopulmonar”, enviado para o periódico Jornal de Pneumologia; Parte III- Artigo original intitulado “Efeitos da melatonina sobre o tecido hepático e pulmonar em animais com síndrome hepatopulmonar por ligadura do ducto biliar”, a ser enviado para o periódico Digestive Diseases and Sciences. Foi utilizado o modelo de cirrose biliar secundária, induzida pelo modelo de ligadura de ducto biliar (LDB), com ratos machos Wistar, pesando ± 300 gramas, divididos em quatro grupos: CO (controle), CO+MLT, SHP e SHP+MLT. Os animais foram tratados com MLT a partir do 15° dia e com LDB até o 28° dia. No 29° dia, mediante administração de fármacos anestésicos, foram coletados sangue, fígado, pulmão e fêmur. Resultados: Nos artigos II e III, ao se avaliar as transaminases, observou-se aumento significativo nas enzimas ALT, AST e FA no grupo SHP quando comparadas aos grupos controles, o que determina a presença de doença hepática. Posteriormente à administração da 7 melatonina, houve diminuição significativa do grupo SHP+MLT quando comparado ao grupo SHP na avaliação da lipoperoxidação, atividade das enzimas Catalase (CAT), superóxido dismutase (SOD) e glutationa Peroxidade (GPx), bem como na de Nitritos/Nitratos (NO2/NO3) e no sistema inflamatório, quando da avaliação da expressão da Óxido Nítrico Sintase Induzível (iNOS) e da região p65 do fator de transcrição nuclear-kB (NF-kB). Também houve diferença na gasometria arterial e na histologia do tecido pulmonar pelas técnicas de Hematoxilina e Eosina e Picrosírius, especialmente no diâmetro dos vasos pulmonares. Ao avaliar o dano ao DNA pelo Ensaio Cometa, foi observado aumento do índice e da frequência de dano no grupo SHP, com redução após o uso da MLT. Conclusão: Os resultados sugerem efeito protetor da melatonina no tecido hepático e pulmonar, no modelo de Síndrome Hepatopulmonar por ligadura de ducto biliar. / Background: Hepatopulmonary syndrome (HPS) is clinically characterized by a combination of liver disease, gas exchange abnormalities, and pulmonary vascular dilation. Prolonged biliary duct obstruction was used in experimental models of secondary biliary cirrhosis leading to acute inflammatory reactions and possibly to systemic changes. Animals with cirrhosis experimentally induced by biliary duct ligation (BDL) showed increased oxidative stress in liver tissue, but the lung tissue has not been investigated yet. Oxidative stress has a key role in the pathogenesis and progression of chronic diseases, and antioxidants have been proposed as therapeutic agents to compensate for systemic and liver damages. In studies comparing melatonin with other known antioxidants such as vitamins C and E, melatonin was found to be more efficient in reducing oxidative/nitrosative stress, showing that it is a potent antioxidant. Objective: The present doctoral dissertation aimed to investigate the effect of the antioxidant melatonin on lung and liver tissues in an experimental model of BDL-induced HPS. Methods: This dissertation is divided into three parts: Part I consists of the review article entitled “Hepatopulmonary Syndrome: Oxidative Stress and Physical Exercise,” which was submitted to the European Medical Journal; Part II consists of the original article entitled “Efeitos da melatonina sobre o tecido pulmonar no modelo experimental de síndrome hepatopulmonar”, which was submitted to Jornal de Pneumologia; and Part III consists of the original article entitled “Effects of melatonin on liver and lung tissues of animals with bile duct ligation-induced hepatopulmonary syndrome”, which will be submitted to Digestive Diseases and Sciences. A model of secondary BDL-induced biliary cirrhosis was used in male Wistar rats weighing ± 300 g and divided into four groups: control group (CG), CG+MLT, HPS, and HPS+MLT. Animals were treated with MLT or vehicle from day 15 to day 28 after BDL. On day 29, blood, liver, lung, and femur samples were collected after the administration of anesthetic drugs. Results: As for transminases, papers II e III found that there was a significant increase in enzymes ALT, AST e FA in the HPS group compared with the control groups, which indicates the presence of liver disease. The administration of melatonin led to a significant decrease in the HPS group compared with the control groups with regard to the following variables: lipid peroxidation; catalase, superoxide dismutase, and glutathione peroxidase activity; nitrite/nitrate ratio; and inducible nitric oxide synthase and p65 region of nuclear factor kappa B. There were also differences in blood gas analysis and histology of lung tissues as assessed by hematoxylin and eosin (HE) and picrosirius red staining, especially in the diameter of pulmonary vessels. The comet assay revealed an increase the index and frequency of DNA damage in the HPS group compared with the control groups, but these parameters were reduced with the use of melatonin in the HPS+MLT group. Conclusion: Results suggest that melatonin has a protective effect in liver and lung tissues using a BLD-induce HPS model.

A exploração visual dos dutos biliares é de relevante interesse clínico, pois fornece informação relacionada com a Atresia Biliar (AB). A AB é uma doença cujas causas ainda permanecem desconhecidas e que eventualmente leva a um transplante de fígado ou, nos casos mais avançados da doença, leva a óbito do paciente. A única evidência física conhecida até agora da existencia de AB é a obstrução das vias biliares. No entanto, o estudo desta doença tem sido limitado pela incapacidade de analisar o duto biliar de pacientes em estágios precoces da doença e muito pouco se sabe sobre a estrutura interna do duto biliar. Nos últimos anos, a microscopia confocal, uma técnica que permite a obtenção de conjuntos de dados 3D de amostras biológicas, tem sido utilizada em experiências médicas para estudar a estrutura interna e anatômica dos dutos biliares. Neste trabalho, é objetivo apoiar o estudo dessas estruturas através da visualização volumétrica de imagens dos dutos biliares. É proposto um pipeline de fluxo de dados capaz de processar e "renderizar"conjuntos de dados de imagens confocais utilizando o VTK (do inglês The Visualization ToolKit). O pipeline foi construído em duas etapas principais e consecutivas. Uma primeira etapa tem o objetivo de remoção de ruído e realce das estruturas relevantes por meio de filtragem no domínio da freqüência e difusão anisotrópica. O conjunto de dados assim pré-processado é usado com técnicas diretas de visualização de volumes baseadas em funções de transferência para exibir as estruturas dos dutos biliares. Os resultados mostram que a visualização volumétrica em conjunto com um pré-processamento adequado das imagens confocais permite evidenciar as regiões de interesse nos dutos biliares e melhora detalhes que são dificilmente visualizados nos dados originais. / The visual exploration of bile ducts in the liver is of relevant clinical interest, as it provides information related to the Biliary Atresia, a disease of unknown origin, which eventually leads to a liver transplant or ultimately to death. The only physical known evidence of biliary atresia is the obstruction of the bile ducts. However, the study of this disease has been limited by the inability to observe the bile duct in patients at early stages of the disease. Moreover, very little is known about the internal structure of the bile duct. In recent years, confocal microscopy, a technique that allows to obtain 3D image datasets from biological samples, has been used in medical experiments for studying the anatomical internal structure of bile ducts. We are interested in supporting the study of these structures through volumetric visualization of bile ducts images. In this work, we propose a data flow pipeline capable of processing and rendering datasets of confocal images using The Visualization ToolKit - VTK. The pipeline was built as two consecutive stages. We propose a first stage for denoising and enhancing the relevant structures of sample based on filtering in the frequency domain and anisotropic diffusion. We use the dataset preprocessed in this way for applying a direct volume rendering technique in a second stage based on transfer functions to visualize the bile duct structures. Our results have shown that volumetric visualization together with an adequate pre-processing of the confocal images allow experts to visualize the regions of interest in the bile ducts, improving details that are hardly visualized in the original data.