INTRODUÇÃO: O tratamento da fibrose cística (FC) envolve o uso de medicamentos fornecidos através de nebulizadores e seu funcionamento adequado é essencial. OBJETIVOS: Avaliar o desempenho de nebulizadores a jato utilizados por pacientes com FC e comparar dois manômetros para avaliação dos compressores. MÉTODOS: Estudo descritivo, transversal, de avaliação dos nebulizadores usados pelos pacientes com FC da Associação Mineira de Assistência a Mucoviscidose. Os pacientes trouxeram compressores (Proneb Ultra®) e nebulizadores (Pari LC plus®) para avaliação. O desempenho do compressor foi avaliado por medidas de pressão operacional através dos manômetros PARI PG101® (PARI GmbH, Starnberg, Alemanha) e FSA analógico (Famabras, Itaquaquecetuba, Brasil).As variáveis de eficiência do nebulizador foram: O débito de volume nebulizado (DVN), taxa de oferta da medicação (TOR) e volume residual (VR) foram calculados por diferenças de peso de cada nebulizador após 10 minutos de nebulização de solução salina (2,5ml). O diâmetro médio de massa da partícula (DMM) foi calculado através da equação proposta por Standaert et al. A análise estatística incluiu o pacote R (v.2.15) e MINITAB, com alfa=0,05. O coeficiente Kappa foi calculado para avaliar concordância de valores entre os equipamentos, e curva ROC construída para calcular o valor aferido no manômetro FSA com melhor sensibilidade/especificidade, utilizando o manômetro PARI PG101® como referência. A associação entre valores de pressão, DVN, TOR e VR foi calculada pela correlação de Spearman. RESULTADOS: Avaliados 146 sistemas com tempo mediano de uso de 32(12-60) meses±36 meses. Cinquenta e sete (39%) não funcionaram adequadamente, com valores pressóricos inferiores à metade da referência. Os sistemas com funcionamento inadequado comprometeram as variáveis de eficiência dos nebulizadores A concordância entre os diferentes métodos de avaliação de acordo com a classificação; com funcionamento adequado e inadequado através do coeficiente Kappa foi 0,81(IC95%- 0,65-0,97), p<0,001. Na avaliação da sensibilidade e especificidade foi observado o ponto de corte de 23,5 PSI no manômetro FSA mostrou sensibilidade=99% e especificidade=79% (p<0,001). Houve associação significativa entre DVN, VR e pressões aferidas. CONCLUSÕES: Uma proporção significativa dos sistemas de nebulização não funcionou adequadamente. As variáveis de eficiência da nebulização estavam comprometidas indicando que a pressão gerada no compressor é um aspecto crítico na eficiência do tratamento. O método alternativo da avaliação dos compressores se apresentou adequado para ser utilizado nos compressores utilizados no tratamento da FC. / INTRODUCTION: Cystic fibrosis (CF) treatment of involves the use of medications supplied through nebulizers and their proper functioning is essential. OBJECTIVES: To evaluate the performance of jet nebulizers used by CF patients and to compare two pressure gauges Compressors evaluation. METHODS: This was a descriptive, cross - sectional study of the nebulizers used by patients with CF of the Mucoviscidosis Care Association of Minas Gerais. The patients brought compressors (Proneb Ultra®) and nebulizers (Pari LC plus®) for evaluation. The performance of the compressor was evaluated by operating pressure measurements using PARI PG101® manometers (PARI GmbH, Starnberg, Germany) and analog FSA (Famabras, Itaquaquecetuba, Brazil). The variables of efficiency of nebulization under study were: nebulizer delivery volume (NDV), drug output rate (DOR), and residual volume (RV), which were calculated by weighing each nebulizer before nebulization and 10 minutes after nebulization using a saline solution (2.5 mL). The mass median diameter (MMD) was calculated using the equation proposed by Standaert et al. Statistical analysis included the package R (v.2.15) and MINITAB, with alpha = 0.05. The Kappa coefficient was calculated to evaluate agreement of values between the equipment\'s, and ROC curve constructed to calculate the value measured in the FSA manometer with better sensitivity / specificity, using the PARI PG101® manometer as reference. The association between pressure values, NDV, DOR and RV was calculated by the Spearman correlation. RESULTS: We evaluated 146 systems with a median time of use of 32 (12-60) months ± 36 months. Fifty-seven (39%) did not function properly, with pressure values lower than half the reference. The systems with inadequate functioning compromised the efficiency variables of the nebulizers. The agreement between the different evaluation methods according to the classification; with adequate and inadequate functioning through the Kappa coefficient was 0.81 (95% CI -0.65-0.97), p <0.001. In the evaluation of sensitivity and specificity, the cut-off point of 23.5 PSI on the FSA manometer showed sensitivity = 99% and specificity = 79% (p <0.001). There was a significant association between NDV, DOR, RV and measured pressures. CONCLUSIONS: A significant number of the nebulizer systems were ineffective. The variables of nebulization efficiency were compromised, which indicated that the pressure generated by the compressor was a critical aspect for treatment efficiency. The alternative method of compressors evaluation was suitable for use in CF treatment routine.

The bronchiectatic lung is a diseased state in which the airways are chronically damaged and dilated. This state is found in the clinical entities of cystic fibrosis and non-cystic fibrosis bronchiectasis. These are two highly relevant chronic suppurative lung diseases in which an understanding of the microbiology of these patients is considered key to appropriate management. This has traditionally been via the use of traditional culture techniques. However, with the development of molecular methodologies, the previously perceived wisdom is being challenged. In both cystic fibrosis and non-cystic fibrosis bronchiectasis, Pseudomonas aeruginosa is considered the most significant pathogen. In CF there has been considerable concern surrounding the risk of transmission of Pseudomonas aeruginosa between patients on the basis of a significant quantity of research into this matter. In contrast, there has been very little research performed into the equivalent risk in non-cystic fibrosis bronchiectasis. In this thesis we describe an extensive single-centre epidemiological review of Pseudomonas aeruginosa spanning both these diseases. Via this we have shown evidence of cross-infection within a non-cystic fibrosis bronchiectasis cohort. This epidemiological review has included multiple genotyping methods including multilocus sequence typing and whole genome sequencing, As an extension of the epidemiological review, we have performed an in silico prediction of hypermutator status from the whole genome sequencing data to provide greater understanding of the likelihood of cross-infection, and have also demonstrated a culture-independent adaption of multilocus sequence typing for potential screening for cross-infection. In addition to Pseudomonas aeruginosa, we have also looked at the wider bacterial community in the lungs of patients with these two conditions via culture-independent techniques. We have shown that whilst Pseudomonas aeruginosa is often an important component, these are clearly complex communities. We have primarily investigated the cohort with non-cystic fibrosis bronchiectasis, but we have demonstrated associations between clinically-relevant markers and complexity of the bacterial communities within the lungs of both these cohorts of patients. Whilst we have used the gold-standard technique of 16S rRNA sequencing, we have also shown the validity of a simple and potentially more feasible profiling technique for standard clinical care. In summary, through the application of culture-dependent and independent molecular techniques, this research has shed light on the epidemiology of Pseudomonas aeruginosa within our respiratory cohorts, and the complexity and clinical relevance of the wider microbial communities within these patients. Such studies are essential if we are to advance our understanding of the bronchiectatic lung and optimise strategies for patient management.

Orientador: Vera Luiza Capelozzi / Coorientador: Claudia Aparecida Rainho / Banca: João Lauro Viana de Camargo / Banca: Thais Helena A. Thomaz Queluz / Banca: Alexandra Muxfeldt Ab'Saber / Banca: Rimarcs Gomes Ferreira / Resumo: A fibrose pulmonar é a base patológica de uma variedade de doenças crônicas incuráveis. A IL-17A, uma glicoproteína secretada de células Th17, é uma citocina pró-fibrótica relacionada recentemente à síntese de colágeno V e fibrose pulmonar. O remodelamento parenquimatoso pulmonar da matriz extracelular pelo colágeno I e V, apoptose e resposta Th17 foi estudado em camundongos Balb/c, C57/B6J selvagens e com knockout para o receptor A da IL-17; para determinar as vias fisiopatológicas que prolongam a fase tardia do processo fibrótico induzido por bleomicina e paraquat. Microscopia eletrônica, imunofluorescência, imunohistoquímica, detecção in situ da apoptose, morfometria, reconstrução tridimensional e reação em cadeia da polimerase em tempo real foram usados para avaliar a quantidade, estrutura e cadeias moleculares dos tipos de colágenos, apoptose e células imunes. Verificamos um aumento da síntese e secreção do colágeno V que promove a perpetuação da fibrose pulmonar de maneira IL-17A dependente. Além disso, observou-se que marcadores críticos da resposta Th17 como IL17, STAT3, TGF-β, IL-6, IL- 21, IL-23 e células T CD4+ foram significantemente aumentados em cepas susceptíveis a fibrose pulmonar e intensificadas na ausência do receptor A da IL-17. O aumento de marcadores Th17 resulta em um aumento de células T CD4+ através de uma resposta imune que efetivamente bloqueia a degradação do colágeno V, o qual contribui para o bloqueio da apoptose. Nosso estudo indica que o colágeno V participa da perpetuação da fibrose pulmonar por mecanismos IL-17 dependente e independentes, indicando o potencial alvo terapêutico do colágeno V e das vias de sinalização da resposta IL-17 no tratamento das doenças fibroproliferativas pulmonares / Abstract: Pulmonary fibrosis is the pathologic basis for a variety of incurable human chronic lung diseases. IL-17A, a glycoprotein secreted from IL-17- producing cells, has recently been shown to be a profibrotic cytokine involved in type V collagen synthesis and pulmonary fibrosis. Remodeling of the extracellular matrix by collagen I and V, cell death and Th-17 immune response were evaluated in Balb/c, wild and IL-17 receptor A knockout C57/B6J mice, to determine the pathways that prolong the late phase of the fibrotic process induced by bleomycin and paraquat. Electron microscopy, immunofluorescence, immunohistochemistry, in situ detection of apoptosis, morphometry, tridimensional reconstruction and a real-time PCR were used to evaluate the amount, structure and molecular chains of collagen types, apoptosis and immune cells. We verified increased synthesis and secretion of type V collagen that promoted the maintenance of pulmonary fibrosis in a IL-17A dependent manner. However, we observed that the critical Th17 markers, IL-17, STAT3, TGF-β, IL-6, IL-21, IL-23 and CD4+ T cells, were significantly increased in the fibrosis-susceptible strain and intensified in the absence of IL-17 receptor A. Increased Th17 markers resulted in an increase in CD4+ T cells in fibrotic lung tissue toward an immune response that effectively blocked degradation of collagen V, which contributes to block apoptosis. Our studies indicate that collagen V participates in the maintenance of pulmonary fibrosis in both Th-17 - dependent and -independent manners and that collagen V and the components of the Th-17 signaling pathway are potential therapeutic targets for the treatment of fibroproliferative lung diseases / Doutor

Introdução: As bronquiectasias não-fibrocísticas são uma doença supurativa crônica caracterizada pela dilatação anormal e irreversível de um ou mais brônquios e são a via final de uma grande variedade de doenças, embora possam não ter uma causa identificável. Elas levam ao comprometimento da função pulmonar, colonização bacteriana crônica das vias aéreas, infecções respiratórias de repetição, redução da tolerância ao exercício e piora na qualidade de vida, entre outras coisas. Existem poucos estudos na literatura que abordam os fatores relacionados ao prognóstico desses pacientes. Objetivos: O objetivo deste estudo é avaliar a taxa de mortalidade e os fatores relacionados à morbidade e à mortalidade de uma coorte de pacientes com bronquiectasias não-fibrocísticas durante um seguimento de 6 a 8 anos e testar a habilidade dos escores Bronchiectasis Severity Index (BSI) e FACED de predizer a mortalidade dos pacientes na nossa coorte. Materiais e métodos: Trata-se de um estudo prospectivo de uma coorte de 70 pacientes com bronquiectasias não-fibrocísticas que foram originalmente recrutados de Maio de 2008 a Agosto de 2010. O estudo original forneceu os dados necessários para a classificação de gravidade da doença segundo os escores BSI e FACED e todos os dados usados para esse propósito foram coletados na avaliação inicial. Após o cálculo dos escores, os pacientes foram separados em diferentes grupos de acordo com a gravidade da doença. Nós também avaliamos os prontuários de todos os pacientes para determinar o número de hospitalizações por exacerbações após a avaliação inicial, o desfecho a longo prazo para cada paciente e a causa do desfecho quando apropriado. Os desfechos foram definidos como favoráveis e desfavoráveis (sobreviventes submetidos a transplante pulmonar e óbito por todas as causas) e foram determinados até 1° de março de 2016. Resultados: Dos 70 pacientes, 27 (38,57%) haviam morrido e 1 (1,43%) sido submetido ao transplante pulmonar. Análise de sobrevivência demonstrou que o tempo médio para a ocorrência dos desfechos desfavoráveis foi de 74,67 ± 4,00 meses (IC 66,82 – 82,52). A principal causa de óbito foi a exacerbação infecciosa aguda das bronquiectasias (60,7% dos óbitos). Na nossa coorte, o modelo de risco proporcional de Cox identificou a idade (p=0,035; HR 1,04; IC 1,01 – 1,08), o VEF1 % do previsto (p=0,045; HR 0,97; IC 0,93 – 0,99) e a Pemax (p=0,016; HR 0,97; IC 0,94 – 0,99) como preditores independentes de desfechos desfavoráveis. A maior parte dos pacientes (44,3%) foi classificada como tendo doença grave pelo escore BSI e 97,2% como tendo doença leve ou moderada (48,6% cada) pelo escore FACED. De maneira geral, o escore FACED foi um melhor preditor de desfechos desfavoráveis na nossa população de pacientes (log-rank test, FACED p = 0,001 e BSI p = 0,286). A análise da curva ROC demonstrou que ambos os escores foram similares na predição de desfechos desfavoráveis (área sob a curva BSI 0,65; FACED 0,66), mas nenhum deles foi um bom preditor para essa população específica de pacientes. Conclusão: Os pacientes da nossa coorte apresentaram maior comprometimento da função pulmonar e uma taxa de mortalidade mais alta do que o previamente reportado na literatura. A principal causa de óbito foi a exacerbação infecciosa aguda da doença. A idade mais avançada, o VEF1 % do previsto e uma Pemax mais baixa foram os fatores independentemente associados aos desfechos desfavoráveis. Os escores FACED e BSI não foram bons preditores de mortalidade para este grupo de pacientes, contrastando com os dados disponíveis na literatura até o momento, portanto outros estudos incluindo um maior número de pacientes são necessários para validar o uso deles na nossa população. / Background: Non-cystic fibrosis bronchiectasis is a chronic suppurative disease characterized by an abnormal and irreversible dilation of one or more bronchi. It is the final pathway of a large number of diseases, although it can be present without an identifiable cause. It leads to impaired lung function, chronic bacterial colonization, recurrent respiratory tract infections, reduced exercise tolerance and poor quality of life, among other things. There are few studies about prognostic factors in these patients. Objectives: The goal of this study is to assess the mortality rates and the factors related to the morbidity and mortality on a cohort of patients with non cystic fibrosis bronchiectasis during a 6 to 8-year follow-up and to test the ability of the Bronchiectasis Severity Index (BSI) and FACED scores in predicting mortality in our cohort. Materials and methods: This was a prospective cohort analysis of 70 patients with non-cystic fibrosis bronchiectasis who were originally recruited from May 2008 to August 2010. The original study records provided the necessary data for the determination of the disease severity scores (BSI and FACED) and all the data used for that purpose were collected at baseline. After the calculation of the scores, patients were separated into different groups according to disease severity. We also reviewed the records of all patients to determine the number of hospitalizations for exacerbations after baseline, the long-term outcome for each patient and the cause of the outcome when appropriate. Outcomes were defined as favorable and unfavorable (survivors who underwent lung transplantation and death from all causes) and were determined as of March 1st, 2016. Results: Out of 70 patients, 27 (38.57%) had died and 1 (1.43%) had undergone lung transplantation by the end of the study. Survival analysis demonstrated that the mean time for the occurrence of an unfavorable outcome was 74.67 ± 4.00 months (CI 66.82 – 82.52). The main cause of death among non-survivors was an acute infectious exacerbation of bronchiectasis (60.7% of the deceased). In our cohort, the multivariate Cox proportional hazard model analysis identified age (p=0.035; HR 1.04; CI 1.01 – 1.08), FEV1 % of predicted (p=0.045; HR 0.97; CI 0.93 – 0.99) and MEP (p=0.016; HR 0.97; CI 0.94 – 0.99) as independent predictors of unfavorable outcomes. Most patients (44.3%) were classified as having severe disease when BSI was used and 97.2% as having a mild or moderate disease (48.6% each) when FACED was used. Overall the FACED score was better at predicting unfavorable outcomes in our population of patients (log-rank test, FACED p = 0.001 and BSI p = 0.286). AUC from the ROC analysis shows us that both scores are similar in predicting poor outcomes in our cohort (BSI 0.65; FACED 0.66), but they weren't good predictors for this specific population. Conclusion: Patients in our cohort had worst lung function and a higher mortality rate than previously reported and the main cause of death among them was an acute infectious exacerbation of bronchiectasis. Older age, lower FEV1 % of predicted and lower MEP were independently linked to the occurrence of poor outcomes. FACED and BSI scores were not accurate in predicting mortality in our cohort, contradicting the available data at the moment, so other studies including a greater number of subjects are needed to validate their use in our population.

Metabolic syndrome (MetS) is a conglomeration of several risk factors for cardiovascular disease, with obesity currently being one of the common causes of disability and death in the United States.1 Underlying the obesity, however, there is metabolic imbalance that could be exacerbating the issue of metabolic syndrome.2 Approximately 34% of adults over 20 years old matched the criteria for metabolic syndrome.3 The risk factors for cardiovascular disease (CVD) associated with metabolic syndrome can, over time, lead to severe CVDs, such as heart failure (HF).4 Metabolic syndrome can also lead to developing metabolic heart disease over time. Understanding the development of cardiac hypertrophy and fibrosis in diet-induced metabolic heart disease allow development of an early treatment of metabolic heart disease (MHD) and HF. This study looked at one potential mediator and its role in cardiac hypertrophy and fibrosis, follistatin-like 3 (FSTL3). FSTL3 is an extracellular antagonist of members of the TGF-β superfamily. The goal of our study was to determine the effect, if any, a knockout of FSTL3 would have on the development of cardiac hypertrophy and fibrosis after a high-fat, high-sucrose diet for five months. FSTL3 knockout mice were given a high-fat, high-sucrose (HFHS) diet for five months. These mice were then sacrificed and their hearts were analyzed for cardiac myocyte hypertrophy and interstitial fibrosis using histological methods. After five months on the HFHS diet, wild-type (WT) mice had cardiac hypertrophy. In FLRG KO mice the diet-induced cardiac hypertrophy was attenuated. WT HFHS-fed mice developed interstitial fibrosis, and FLRG KO HFHS developed more accentuated interstitial fibrosis than WT HFHS diet fed mice. This study is useful in suggesting that FTSL3 contributes to the pathogenesis of cardiac hypertrophy in MHD. FTSL3 may be a useful biomarker for cardiac hypertrophy in patients with suspected MHD, and may be a viable target for therapeutic interventions aimed at decreasing pathologic myocardial hypertrophy.

La fibrose radio-induite (FRI) est une complication tardive de la radiothérapie souvent associée à une réponse inflammatoire chronique et à un infiltrat de macrophages. Aujourd’hui, les macrophages sont pressentis comme des médiateurs cellulaires important dans le processus de fibrose mais leur rôle n’a jamais été étudié dans le contexte de la FRI. Dans une précédente étude nous avions montré que l’irradiation (IR) induit une polarisation M1 des macrophages cardiaques après irradiation de souris ApoE-/- et est associée un score de fibrose élevé, ce qui suggérait que la polarisation des macrophages pourrait contribuer à la fibrogénèse radio-induite. Afin de valider cette hypothèse, nous avons cherché à caractériser le rôle des macrophages dans la FRI en utilisant un modèle classique de fibrose pulmonaire chez la souris C57Bl/6 induit après IR thoracique à 16Gy. Nous avons caractérisé les phénotypes et la fonction des macrophages alvéolaires (MA) et interstitiels (MI). Durant la phase précoce, les résultats montrent une déplétion des MA accompagnée de la sécrétion de CXCL1, MCP-1 et de MCSF. Cette déplétion est suivie d’une repopulation suite au recrutement et à la prolifération des monocytes/macrophages d’origine médullaire. La nouvelle population de MA présente une polarisation hybride accompagnée d’une augmentation simultanée de la sécrétion de cytokines Th1 et Th2. Durant la phase tardive les MI présentent une polarisation de type M2 accompagnée d’une diminution des cytokines Th1 et d’une augmentation de cytokines Th2 dans le lysat tissulaire. Nous avons ensuite cherché à caractériser la contribution des MA hybrides vs MI M2 dans le processus de fibrose. Nous avons montré que contrairement au MA hybrides, les MI M2 étaient capables d’induire l’activation des fibroblastes in vitro et l’expression de TGF-β1. De plus, la déplétion des MA hybrides avec une administration intranasale de clodronate exacerbe la FRI et induit l’augmentation de l’infiltrat de MI M2. Ensuite, nous nous somme interrogés à la contribution du processus de fibrose dans la polarisation des macrophages. Après 24h de coculture entre fibroblastes irradiés et macrophages pulmonaires non irradiés, une sécrétion de cytokines telles que M-CSF et TIMP-1 qui pourraient stimuler l’activation des fibroblastes est observée. De plus, l’inhibition de la FRI avec de la pravastatine montre que l’inhibition de la fibrose est accompagnée d’une augmentation des MI M1 et d’une diminution des MI M2 dans le poumon. En résumé, nos résultats montrent une contribution opposée des Macrophages Alvéolaires et des Macrophages Interstitiels dans le processus de fibrose radio-induite ainsi qu’une contribution du processus de fibrose dans le type d’activation des Macrophages interstitiels formant ainsi une boucle d’activation fibrogénique chronique. / Radiation-induced fibrosis (RIF) is a delayed complication of radiotherapy often associated with chronic inflammatory process and macrophage infiltration. Nowadays, macrophages are suggested to be important cellular contributors to fibrogenic process, but their implication in the context of RIF has never been investigated. In a previous study we have shown that irradiation (IR) induced the polarization of cardiac macrophages into M1 in ApoE-/- mice and was associated with a high fibrosis score in ApoE-/- mice, suggesting that macrophage polarization could drive tissue sensitivity to ionizing radiation. This observation prompted us to investigate the role of macrophages in RIF using a classical experimental model of lung fibrosis developed in C57Bl/6 mice after 16Gy thorax-IR. We profiled both alveolar macrophages (AM) and interstitial macrophages (IM). During the acute phase we found AM depletion associated with CXCL1, MCP-1 and M-CSF secretion, followed by a repopulation phase mediated by recruitment and proliferation of monocytes/macrophages from the bone marrow. Interestingly, the newly recruited AM exhibited a yet never described hybrid polarization (M1/M2), associated with the up-regulation of both Th1 and Th2 cytokines. At delayed times points, IM were M2-polarized and associated with downregulation of Th1 cytokines and upregulation of Th2 cytokines in tissue lysates. These results suggest a differential contribution of hybrid AM vs M2 IM to fibrogenesis. Interestingly, in contrast to activated hybrid AM, activated M2 IM were able to induce fibroblast activation in vitro mediated by an enhanced TGF-β1 expression. Therefore, specific depletion of hybrid AM using intranasal administration of clodrosome increased RIF score and enhanced M2 IM infiltration. We next evaluated if the fibrogenic process can in turn affect macrophage polarization. Interestingly, after coculture of irradiated fibroblast with non-irradiated pulmonary macrophages, secretion of cytokines such as M-CSF and TIMP-1, which can stimulate macrophage activation, was observed. Furthermore, RIF inhibition using pravastatin treatment showed that fibrosis inhibition was associated with a decrease in M2 IM accompanied by an increase in M1 IM, but had no effect on polarization of AM. These present study shows a dual and opposite contribution of alevolar versus intertitial macrophages in RIF and the contribution of the fibrogenic process to IM polarization, resulting thereby in a chronical fibrogenic loop.

Metabolic heart disease (MHD) caused by obesity or diabetes is characterized by cardiac hypertrophy, diastolic dysfunction, and fibrosis - a maladaptive remodeling of the extracellular matrix. Though the influence of cardiac fibrosis on the left ventricular diastolic dysfunction has been reported, little is known about the cardiac-specific secreted autocrine, paracrine, or endocrine factors termed "cardiokines" in MHD. Transforming growth factor beta (TGF-b1) is a well-known inducer of cardiac fibrosis. However, the role TGF-b2 in mediating cardiac fibrosis has yet to be described. In addition, follistatin-like 3 (FSTL3), an extracellular inhibitor of activin A and myostatin, is found to be elevated in end-stage heart failure patients and obese individuals. FSTL3 has been suggested as a cardiokine, yet its role in MHD has not been established. To identify cardiokines induced by MHD, two relevant mouse models were employed in this study: the high-fat high sucrose (HFHS) diet feeding model and the cardiomyocyte-specific Fatp1 overexpressing transgenic mouse model. Interstitial fibrosis was observed in both models, accompanied by fibrotic gene expression and anti-fibrotic miR-29 suppression. It was found that Tgf-b1 and Tgf-b2 mRNA were upregulated by 85% and 76%, respectively, in the non-myocytes of 1-month HFHS-fed mice, while Fstl3 was increased by 30% in the myocytes. In contrast, in the FATP1 transgenic animals, Tgf-b2 and Fstl3 were elevated by 3.8-fold and 1.9-fold in the myocytes while Tgf-b1 remained unchanged compared to control animals. The in vitro results tested in NIH3T3 and primary fibroblast cultures indicate that both TGF-b1 and TGF-b2 exerted profibrotic effects via activation of SMAD proteins and collagen synthesis, but FSTL3 did not. Plasma samples collected from patients with metabolic syndrome showed increased FSTL3 levels with strong correlations with cardiac hypertrophy and impaired diastolic function. Overall, this study has demonstrated that TGF-b1 and TGF-b2 are the key profibrotic cardiokines induced in MHD. The study has also revealed the role of FSTL3 as a biomarker for LV hypertrophy induced in MHD. The results presented here should facilitate the development of better diagnosis and treatment for this disease in the future.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease associated with the worldwide spread of obesity. NASH predisposes development of fibrosis and hepatocellular carcinoma (HCC), but has no approved therapy due to incomplete understanding of the pathogenesis. Notch signaling normally specifies cell fate during development, but here we investigate how this pathway becomes dysregulated in NASH and contributes to fibrosis and cancer. In the first study, we show that hepatocyte Notch activity tracks with disease severity and treatment response in NASH patients, and is similarly increased in a mouse model of diet-induced NASH and liver fibrosis. Different genetic models demonstrate causatively that hepatocyte Notch induces liver fibrosis via secretion of the fibrogenic factor Osteopontin that activates hepatic stellate cells (HSCs), while pharmacologic inhibition of hepatocyte Notch could ameliorate NASH-associated fibrosis. In the second study, we research how hepatocyte Notch activation leads to HCC in mice on NASH diet. Transcriptomic analysis reveals nerve growth factor (NGF) as a Notch target gene in hepatocytes, and the abundance of hepatocyte NGF precursor protein (proNGF) is uniquely associated with HCC. We provide evidence that proNGF may facilitate HCC growth and expansion in a non-cell autonomous manner by inducing HSC deactivation and fibrosis remodeling. In summary, hepatocyte Notch maladaptively contributes to fibrogenesis and possibly HCC expansion by directly signaling to HSCs at different stages of NASH progression, and could be an accessible target for treatment of NASH-associated liver pathologies.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide health problem which includes steatosis (triglyceride accumulation alone), non-alcoholic steatohepatitis (NASH, with liver inflammation), fibrosis, cirrhosis and hepatocellular carcinoma. Liver fibrosis, which is a reversible response, is the final phase of most chronic liver disease and is characterized by accumulation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). Glucocorticoids (GCs) regulate many aspects of metabolism involved in NAFLD. Also, GCs limit HSC activation in vitro. Tissue GC levels are regulated by 11β- hydroxysteroid dehydrogenase-1 (11β-HSD1) which converts inactive 11- dehydrocorticosterone (DHC) into active corticosterone. Previous studies demonstrate that 11β-HSD1 deficiency improves fatty liver in obesity models, but the role of 11β-HSD1 in mechanisms involved in the progression and/or resolution of hepatic injury is largely unknown. I hypothesized that 11β-HSD1 modulates fibrotic and inflammatory responses during hepatic injury and/or the resolution phase. First I sought to address if the levels of 11β-HSD1 during different models of liver injury are dysregulated. In mice, 11β-HSD1 was down-regulated in choline deficient diet (CDD) induced steatosis, methionine and choline deficient diet (MCDD) induced NASH, carbon tetrachloride (CCL4) induced liver fibrosis and thioacetamide (TAA) induced liver fibrosis. In CCL4 injured livers, the down regulation of 11β- HSD1 was observed around the scar area. To test if 11β-HSD1 plays a key role in modulating liver inflammation and fibrosis responses in NAFLD and liver fibrosis I used initially11β-HSD1 knockout (KO) mice. 11β-HSD1 KO showed higher HSC activation only in the High fat feeding model but not in CDD and MCDD models. In the CCL4 injury model, despite reduced hepatocellular injury, 11β-HSD1 KO mice showed enhanced collagen deposition during peak injury and increased fibrotic gene expression during the early resolution phase although unaltered inflammatory markers during both peak injury and resolution. To further dissect cell-specificity on the effect of 11β-HSD1, I repeated the CCL4-injury model using the hepatocyte-specific 11β-HSD1 KO (Alb-HSD1). Alb-HSD1 mice did not show increased susceptibility to fibrosis compared to control littermates suggesting that the 11β- HSD1 possibly modulates fibrotic response by affecting HSC function. To mechanistically address how GCs inhibit HSC activation in vitro I studied the effects of 11β-HSD1 on HSC in vitro. 11β-HSD1 expression was down-regulated during ‘spontaneous’ HSC activation, and 11β-HSD1 deficiency enhanced susceptibility to activation. The GC (11-DHC)’s inhibitory effect on HSC activation was reversed by 11β-HSD1 inhibition. Finally, to address the clinical relevance of 11β-HSD1 in hepatic injury and/or resolution a selective 11β-HSD1 inhibitor, UE2316, was used. UE2316 induced a pro-fibrotic phenotype in ob/ob mice and CCL4-treated C57BL/6 mice, but had no effect when administered only during injury resolution. In conclusion, 11β-HSD1 deficiency causes increased activation of HSCs following diet and chemical injury and promotes liver fibrosis. Effects of 11β-HSD1 inhibitors, which are a potential treatment for metabolic syndrome, are perhaps offset by adverse outcomes in liver.

Orientador: José Vicente Tagliarini / Resumo: CAMPOS, C. S. M. 2018. Avaliação da incidência e evolução das manifestações das doenças nasais em pacientes portadores de fibrose cística. 2018. Dissertação (Mestrado) – Faculdade de Medicina de Botucatu, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, 2018. Introdução: A fibrose cística é uma doença originada da mutação do gene responsável pela codificação da proteína CFTR. A ausência ou defeito dessa proteína leva a um transporte irregular de sal e água em células mucosas respiratórias e de glândulas exócrinas podendo resultar em diversas manifestações clínicas. As alterações otorrinolaringológicas frequentes são rinossinusite crônica e polipose nasal. Objetivo: Avaliar a incidência e a evolução de doenças nasais em pacientes portadores de fibrose cística com exame videonasolaringoscópico documentado em prontuário do período de agosto de 2015 a agosto de 2017. Casuística e Métodos: Realizou-se a coleta de dados de 65 pacientes acompanhados no ambulatório de fibrose cística do Hospital das Clínicas de Botucatu. Foram incluídos os pacientes que possuíam exames videonasolaringoscópicos realizados durante o período de acompanhamento e que concordaram em participar do estudo. Realizou-se um estudo de coorte com avaliação dos dados epidemiológicos, presença de comorbidades, achados nos exames videonasolaringoscópicos, positivação de cultura de orofaringe e resposta ao tratamento clínico. Resultados: Foram analisados 51 pacientes. A mutação delta F508 esteve pr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: CAMPOS, C.S.M. 2018. Evaluation of the incidence and evolution of the manifestations of nasal diseases in patients with cystic fibrosis. 2018. Thesis (Master) – Botucatu Medical School, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, 2018. Introduction: Cystic fibrosis is a disease originated from the mutation of the gene responsible for the coding of the CFTR protein. The absence or defect of this protein leads to an irregular transport of salt and water in respiratory mucous cells and exocrine glands, resulting in several clinical manifestations. Frequent otorhinolaryngological changes are chronic rhinosinusitis and nasal polyps. Objective: To evaluate the incidence and evolution of nasal diseases in patients with cystic fibrosis with a videonasolaryngoscopic exam documented in medical records from August 2015 to August 2017. Casuistry and Methods: Data were collected from 65 patients followed in the cystic fibrosis outpatient clinic Botucatu Medical School. Patients who had videonasolaryngoscopic examinations performed during the follow-up period and who agreed to participate in the study were included. A cohort study was conducted with epidemiological data, presence of comorbidities, findings in videonasolaryngoscopic examinations, oropharyngeal culture positivity and response to clinical treatment. Results: Fifty-one patients were analyzed. The delta F508 mutation was present in 20 patients. The comorbidities evaluated were: bronchiectasis (n = 39), p... (Complete abstract click electronic access below) / Mestre