Orientador: Antonio Fernando Ribeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T12:30:23Z (GMT). No. of bitstreams: 1 Enes_GiovanadaSilvaTavares_M.pdf: 802222 bytes, checksum: 877dccb29eaf785982eed2f02beb39c6 (MD5) Previous issue date: 2012 / Resumo: A Fibrose Cística é uma doença autossômica recessiva, sistêmica, hereditária, crônica e progressiva e pode levar à morte. São características da doença as secreções mucosas espessas e viscosas que obstrui os ductos das glândulas exócrinas e contribuem para o aparecimento de doença pulmonar obstrutiva crônica, insuficiência pancreática com má digestão e má absorção e conseqüente desnutrição secundária, além de níveis elevados de eletrólitos no suor. Por ser uma doença crônica, ela exige cuidados sistemáticos pela vida toda, e na maioria dos casos quem exerce a função de cuidadora é a mãe. Além de viver uma nova experiência de ser mãe, ela terá que conviver com a frustração dele ser doente.Com este estudo foi possível compreender a relação que mãe e filho doente crônico constroem desde o momento do diagnóstico e conhecimento do tratamento, permeados por sentimentos como culpa e solidão. Assim, essas mães renunciam suas próprias vidas em função do cuidado do filho. Cuidados esse compartilhado com uma equipe de saúde multiprofissional ainda deficitária. Apesar de ter sido avaliado por elas como positivo, as sugestões por melhorias também surgiram: como uma melhor articulação entre os serviços de saúde nos diversos níveis, uma maior divulgação da doença e o aumento do número de dias de atendimento. Outro aspecto importante encontrado foi sobre importância do papel do psicólogo não só na atuação com o paciente e a família durante todo o tratamento; mas também na necessidade de oferecer um espaço para que os profissionais de saúde despreparados pudessem compartilhar suas angústias e frustrações o que reflete diretamente na assistência prestada / Abstract: The Cystic Fibrosis is a disease systemic, hereditary, chronic and progressive and it can lead to the death. There are characteristic of the disease the thick and viscous mucous secretions what it obstructs the ducts of the exocrine glands and contribute to the appearance of chronic obstructive pulmonary disease, pancreatic insufficiency with bad digestion and bad absorption and consequent secondary malnutrition, besides elevated levels of electrolytes in the sweat. Because of being a chronic disease, she demands systematic cares for the life completely, and in most of the cases who plays the function of care is the mother. Besides surviving a new experience of being a mother, she will have to coexist in spite of the fact that his frustration to be doente.Com this study there were possible understood the relation what mother and chronic sick son build from the moment of the diagnosis and knowledge of the treatment, permeated by feelings as fault and solitude. So, these mothers renounce his lives themselves in function of the care of the son. Taken care this shared one with a team of still deficient multiprofessional health. In spite of having been valued by them like positive, the suggestions for improvements also appeared: like a better articulation between the health services in several levels, a bigger spread of the disease and the increase of the number of service days. Another considered important aspect was on importance of the paper of the psychologist not alone in the acting with the patient and the family during the whole treatment; but also in the necessity of offering a space so that the unprepared health professionals could share his anguishes and frustrations what thinks straightly about the given presence / Mestrado / Saude da Criança e do Adolescente / Mestre em Ciências

Abstract Idiopathic usual interstitial pneumonia (UIP) and bronchiolitis obliterans organizing pneumonia (BOOP) are fibrous pulmonary disorders in both of which there is newly formed connective tissue in distal air spaces. UIP is a progressive and usually fatal lung disease without any efficient treatment, while the prognosis of BOOP is good. In both diseases, an injury of the alveolar epithelium and its basement membrane (BM) leads to migration of fibroblasts and myofibroblasts into air spaces and production of extracellular matrix by these cells. In UIP, the newly formed intraluminal connective tissue lesions cause fusion of alveolar structures and interstitial remodeling, while in BOOP the newly formed connective tissue may resolve completely. One of the major aims of the research on pulmonary fibrosis is to define the mechanisms that lead to persistence of the newly formed connective tissue and thus to irreversible fibrosis in UIP. The aim of the present study was to compare the extent of capillarization, apoptotic activity and re-epithelialization of the newly formed connective tissue in BOOP and UIP. The number of capillaries per tissue surface area was measured. Furthermore, the expression of angiogenic growth factors vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF) was evaluated in the same areas, in addition to the expression of Flt-1 and Flk-1, which serve as receptors for VEGF. Apoptotic activity was analyzed using TUNEL-method, and the immunohistochemical expression of apoptosis regulating proteins bcl-2, mcl-1, and bax was studied. Finally, the extent of re-epithelialization was studied with the immunohistochemical and ultrastructural localization of laminin-5 γ2 chain, and the sites of synthesis of laminin-5 γ2 chain mRNA. In BOOP, an efficient repair process with good capillarization along with high expression of VEGF and bFGF, and orderly re-epithelialization of the newly formed connective tissue takes place after lung injury. The apoptotic activity of the newly formed connective tissue is also high, presumably leading to resolution of the intraluminal connective tissue in BOOP. In UIP, the newly formed connective tissue showed poor capillarization, inadequate re-epithelialization and low apoptotic activity. The results suggest disturbed or delayed repair process in UIP, contributing to irreversible interstitial fibrosis and remodeling.

Hepatitis B virus infection (HBV) is an important health problem worldwide, with a significant rate of chronic infection, which can lead to cirrhosis and hepatocellular carcinoma (HCC). Increased age is an important determinant of progression to cirrhosis and HCC, possibly because age is a crude measure of the duration of HBV infection. Increasing age is associated with changes in liver structure, blood flow and function and with reduced response to injury, impaired regeneration and increased mortality in acute liver disease. Age has been identified as a co‐factor in several chronic liver diseases including chronic hepatitis C infection (HCV). Available evidence suggests differential ageing of various intrahepatic cell types in different liver diseases and the ageing process may be more complex in the liver than originally thought. Telomeres are DNA structures located at the end of each chromosome, which protect the underlying coding DNA from breaks and fusions and shorten with increasing age. Both DNA damage and cell proliferation lead to progressive telomere shortening, which ultimately leads to cell cycle arrest and a state of replicative senescence. Persistent HCV and HBV infections lead to cell cycle arrest, providing a favourable environment for viral replication. Evidence suggests that progressive telomere shortening occurs with advancing stage of liver disease in HBV and specifically from cirrhosis through large cell dysplasia to small cell dysplasia and to HCC. Whether cell cycle arrest leads to a senescent‐like state or whether this is the result of oxidative stress is unknown. Unpublished data using cell cycle phase markers in chronic HBV infection reveal that hepatocytes, which support HBV replication, are arrested in G1, which is mediated by hepatocyte p21 expression. I hypothesise that: 1. In normal liver tissue, different cell types age at different rates and this is altered during disease; 2. Hepatocyte senescence plays a significant role in the natural course of chronic HBV infection and underlies HBV antigen expression. I developed and optimised large volume Q‐FISH methodology to measure telomere length and nuclear size in a variety of intrahepatic cell lineages. In normal liver tissue, cholangiocytes had longer telomeres compared with all other intrahepatic lineages over a wide age range. Hepatocytes did not show any age‐related telomere shortening, in contrast to Kupffer and hepatic stellate cells. In chronic HBV infection, all hepatocytes had shortened telomeres when compared to age and sex‐matched controls consistent with accelerated ageing. HBV replication was confined to those hepatocytes with longer telomeres, suggesting that HBV entry or replication is less efficient in older hepatocytes and compatible with the fall in serum HBV DNA and HBsAg titre seen with advancing age. There may be two populations of hepatocytes in chronic HBV infection; hepatocytes that are growth arrested with short telomeres not supporting HBV replication and biologically 'younger' hepatocytes with longer telomeres that do support HBV replication. The change in cellular HBV antigen localisation with disease progression is also explained by age related changes in HBV expression. Nuclear Hepatitis B core antigen expression (HBcAg), characteristic of the early immune tolerant phase of infection, was associated with the longest telomeres, while cytoplasmic HBcAg expression was associated with shorter telomeres. Furthermore, the total number of hepatocyte telomeres fell with increasing fibrosis stage. Hepatocyte nuclear size, a marker of senescence, increased as HBcAg expression shifted from nucleus to cytoplasm; and p21, another senescence marker, never co‐localised with HBcAg expression. These results suggest that the location and production of HBV antigens are related to increased functional hepatocyte age and the onset of cellular senescence.

Cystic Fibrosis is one of the most common genetic recessive diseases among Caucasians and is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene on chromosome 7. There are different classes of CFTR mutation, leading to differences in disease severity among patients. In addition to the CFTR genotype, secondary genetic factors, modifier genes, also influence CF phenotypes. Due to the dysfunction of CFTR protein and production of thickened mucus, bacterial infection in the lungs is favored and can lead to further clinical complications in CF patients. Pseudomonas aeruginosa is one of the most common bacteria detected among patients. The aim of this project was to investigate four candidate modifier genes, Factor B, Complement Factor 3, Toll-like Receptor 4 and Heme oxygenase-1, which might affect the status of Pseudomonas aeruginosa infection. A total of 22 single nucleotide polymorphisms (SNPs) were selected in these four genes and they were tested against five phenotypic traits, including age of diagnosis, FEV1% predicted andstandard deviation value, age of first Pseudomonas aeruginosa infection and Pseudomonas aeruginosa infection status. Among the selected SNPs, both case-control studies and family-based analysis were performed in order to establish any correlation between the genotypes and the phenotypes. In addition, haplotype analysis was performed to determine whether there was interaction between SNPs or whether there were unidentified SNPs in the vicinity of the selected ones that might contribute to the observed phenotypic traits. Among the 22 chosen SNPs, 13 of them were found to be significantly linked to one or more of the tested phenotypes. The three most significant associations were BF_2557 with lung function, HMOX1_9531 with lung function and BF_7202 with age of diagnosis. Several haplotypes were significantly associated with one of the five phenotypes. There was no evidence for the presence of unidentified SNPs or interaction between SNPs. Most of haplotype associations were likely due to the presence of a single SNP which was found to be significantly linked to the phenotype. Conclusively, both SNPs and haplotype analyses suggest that the four candidate genes are modifiers of disease severity in CF. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Introduction: Emerging pathogens (EP) in cystic fibrosis (CF) include organisms that have infected individuals with CF for many years e.g. Burkholderia multivorans and Mycobacterium abscessus and more recently identified potential pathogens in CF e.g. Pneumocystis jirovecii and Pandoraea spp. The clinical implications of infection with these organisms are emerging but much remains unknown. Current evidence suggests that infection with some EP is associated with a worse prognosis. This thesis aimed to investigate the epidemiology, prevalence and clinical impact of EP in adults with CF.Methods: (1) The prevalence of P. jirovecii was determined in adults attending Manchester Adult Cystic Fibrosis Centre (MACFC) who were clinically stable versus those experiencing an acute pulmonary exacerbation (PEx). (2) The prevalence of M. abscessus at MACFC was determined, isolates of M. abscessus were strain typed, and cross infection risk was assessed. The clinical impact of Gram-negative EP was assessed by: (3) assessing their prevalence and determining if any patients attending MACFC harboured identical strains and had opportunities for cross infection to occur, and by (4) following these patients longitudinally and comparing outcome with age, gender and FEV1 matched Pseudomonas aeruginosa infected controls. Results: (1) P. jirovecii was detected via sputum PCR in 10 (4.4%) of 226 samples tested from 111 patients. P. jirovecii was more likely to be detected in samples taken from an acute pulmonary exacerbation compared with samples taken from stable patient visits (7 (9.2%) of 76 exacerbations samples versus 3 (2%) of 150 stable visit samples, p = 0.033). (2) Prevalence of M. abscessus was stable at ≤3.6% from 2010 to 2015. 21 patients (91.3%) with a positive culture for M. abscessus since 2010 were infected with M. abscessus subsp abscessus. 2 clusters of 7 and 6 patients harboured strains with identical variable number tandem repeat profiles and some of these patients had opportunities for cross infection to occur. 28.6% of patients developed M. abscessus pulmonary disease, 38.1% were persistently culture positive with no related pulmonary disease, and 33.3% spontaneously cleared M. abscessus from their sputum. (3) Prevalence of Gram-negative EP ranged from 1.9% (Ralstonia spp.) to 6.2% (B. multivorans). Small numbers of patients shared strains of B. multivorans; Stenotrophomonas S. maltophilia and Achromobacter; Ralstonia and Pandoraea species. Epidemiological connections consistent with possible cross infection were found in patients infected with Pandoraea and Ralstonia species. (4) Patients with B. multivorans; S. maltophilia; Ralstonia spp. and Pandoraea spp. had higher antibiotic requirements than P. aeruginosa infected matched controls. B. multivorans; Achromobacter spp.; Ralstonia spp. and Pandoraea spp patients had median FEV1 (% predicted) values ≥10% (absolute) lower than the overall median FEV1.Conclusion: Prevalence of all EP investigated at MACFC was low. P. jirovecii was approximately 5 times more likely to be detected in patients with acute PEx compared with stable patients suggesting it may be a cause of PEx. Results suggest that some patients attending MACFC may have acquired infection with M. abscessus subsp abscessus, Pandoraea spp. or Ralstonia spp. through cross infection. Patient numbers are too small to establish this with certainty and a common environmental source is possible. Gram-negative EP other than Achromobacter spp. were associated with higher acute antibiotic requirements than P. aeruginosa matched controls suggesting these EP are associated with an increased risk of PEx. The fact that many Gram-negative EP were associated with lower median lung function may indicate that these EP cause accelerated lung function decline or that patients with more advanced disease are at most risk of acquiring EP.

<p>Liver fibrosis is the result of different types of chronic liver diseases, such as cholestatic liver disease and nonalcoholic steatohepatitis, among others. Fibrosis, if left unchecked, may progress to the point of cirrhosis – permanently affecting liver function detrimentally and potentially leading to development of hepatocellular carcinoma. Inflammatory response following tissue injury is vital for the initiation of fibrosis; chronic inflammation results in abnormal tissue healing and promotes a pro-fibrogenic response.</p> <p>Activins are cytokines that have been identified as members of the TGFβ superfamily of growth and differentiation factors. Activin A and B, in particular, have been identified as having roles in the pathophysiology of liver disease, but have not been investigated thoroughly. We treated mice with concanavalin A, a potent T-cell mitogen with liver specificity when administered intravenously, and characterized the resulting response to liver injury and how activin A and B are modulated during this acute inflammatory phase. We showed that activin B is highly increased in circulation following inflammation, as well as locally in the liver as well as the spleen. We then neutralized activin A and B via neutralizing antibodies in our concanavalin A-induced liver injury model to determine if inhibition of these ligands may confer protective effects during the acute inflammatory response in liver. Neutralization of either activin A or activin B protected hepatocytes, improved liver function, and significantly reduced circulating cytokines following concanavalin A administration. Finally, we determined whether inhibition of activin A or B might prevent or reverse the development of liver fibrosis after disease has been established. We induced liver fibrosis in mice via the hepatotoxin carbon tetrachloride, and then treated with neutralizing antibodies while still maintaining carbon tetrachloride administration. Neutralization of activin A and B markedly reduced liver fibrosis, protected hepatocytes, and improved liver function. Our findings implicate both activin A and B as major players in the acute inflammatory response to liver injury, as well as during chronic injury and fibrogenesis, and demonstrate the therapeutic potential of targeting these ligands for the treatment of fibrosis in chronic liver diseases.</p>

Background: Heart attack or myocardial infarction (MI) is a major cause of death worldwide. MI is generally attributed to the detrimental effects of myocardial ischemia/reperfusion (I/R) injury. I/R injury induces cell death and reduces heart function. To compensate, the heart remodels with an associated increase in cell death, fibrosis, and hypertrophy, which can further compromise heart function. Ubiquitin (UB) is an evolutionarily conserved protein. Our lab has shown that pre-I/R injury treatment with exogenous UB preserves heart function and reduces fibrosis 3-days post-I/R in mice. A major objective of this study is to analyze the long-term cardioprotective potential of UB post-I/R injury. Here the UB treatment was continued until 28 days post-I/R to include the entire remodeling period. To enhance the clinical applicability, UB treatment was started at the time of reperfusion. Methods: C57BL/6 mice (aged ~3 months) underwent myocardial I/R surgery. Mice were anesthetized and the left anterior descending coronary artery (LAD) was ligated for 45 minutes. The ligature was then removed for reperfusion. Mice were treated with UB (1µg/g body weight; intraperitoneal (IP) injection) or saline at the time of reperfusion; followed by 3-days of saline or UB IP treatment twice per day. The mice were then implanted with micro-osmotic pumps containing UB (1 μg·g−1·h−1) or saline to continue treatment 28-days post I/R. Mice were sacrificed at 28-days post I/R injury. Sham animals underwent the same surgery without LAD ligation. Heart functional parameters (percent ejection fraction and fractional shortening) were analyzed by echocardiography in a time-dependent manner (3, 7, 14 and 28 days post-I/R). Extracted hearts were embedded in paraffin. Heart sections (5µm) were stained with Mason’s Trichrome to measure fibrosis, TUNEL to measure apoptosis, and fluorescein-conjugated wheat germ agglutinin to measure hypertrophy. Index of fibrosis was quantified as a percentage of total left ventricular area, apoptosis was quantified as a percentage of the total number of nuclei, and hypertrophy was quantified by measuring the myocyte cross-sectional area. Major findings: 1) I/R+saline exhibited a significant decrease in the functional parameters of the heart at 3, 7, 14 and 28 days post-I/R vs sham (n=4-12). No significant decrease in heart function observed between I/R+UB vs sham, and heart function was significantly lower in I/R+saline compared to UB+I/R (n=7-12); 2) I/R surgery significantly increased fibrosis in the myocardium of I/R+saline vs sham. No significant difference was observed between UB+I/R and sham, and fibrosis was significantly lower in UB+I/R vs I/R+saline (n=4-6); 3) Apoptosis was significantly higher in I/R+saline vs sham (p4) Myocyte hypertrophy was significantly higher in I/R+saline vs sham (pConclusion: Long-term UB treatment has the potential to preserve heart function with effects on myocardial fibrosis, myocyte apoptosis, and hypertrophy following myocardial I/R injury.

The purpose of this study was to investigate the role of osteopontin (OPN) in diabetic hearts. Diabetes was induced in wild-type (WT) and OPN knockout (KO) mice by using streptozotocin (150 mg/kg) injection. Left ventricular (LV) structural and functional remodeling was studied 30 and 60 days after induction of diabetes. Induction of diabetes increased OPN expression in cardiac myocytes. Heart weight-to-body weight ratio was increased in both diabetic (D) groups. Lung wet weight-to-dry weight ratio was increased only in the WT-D group. Peak left ventricular (LV) developed pressures measured using Langendorff perfusion analyses were reduced to a greater extent in WT-D versus KO-D group. LV end-diastolic pressure-volume curve exhibited a significant leftward shift in WT-D but not in KO-D group. LV end-diastolic diameter, percent fractional shortening, and the ratio of peak velocity of early and late filling (E/A wave) were significantly reduced in WT-D mice as analyzed by echocardiography. The increase in cardiac myocyte apoptosis and fibrosis was significantly higher in the WT-D group. Expression of atrial natriuretic peptide and transforming growth factor-β1 was significantly increased in the WT-D group. Induction of diabetes increased protein kinase C (PKC) phosphorylation in both groups. However, phosphorylation of PKC-βII was significantly higher in the WT-D group, whereas phosphorylation of PKC-ζ was significantly higher in the KO-D group. Levels of peroxisome proliferator-activated receptor-γ were significantly decreased in the WT-D group but not in the KO-D group. Thus increased expression of OPN may play a deleterious role during streptozotocin-induced diabetic cardiomyopathy with effects on cardiac fibrosis, hypertrophy, and myocyte apoptosis.

Background: Osteopontin, also known as cytokine Eta-1, plays an important role in postmyocardial infarction remodeling by regulating collagen accumulation. Aldosterone promotes collagen synthesis and structural remodeling of the heart. The role of osteopontin in aldosterone-induced fibrosis and myocardial remodeling is unknown. Osteopontin expression and left ventricular structural and functional remodeling were determined in wild-type and osteopontin knockout mice after aldosterone infusion. Methods and Results: Immunohistochemical analyses showed increased interstitial osteopontin protein in the wild-type left ventricle after 7 days of aldosterone infusion. After 4 weeks of aldosterone infusion, heart rate was unchanged, and there were similar increases in blood pressure (BP) and heart-to-body weight ratio in both wild-type and knockout mice. Left ventricular end-diastolic diameter was significantly higher, whereas percent fractional shortening was significantly lower (P < .05) in knockout versus wild-type mice after 4 weeks of aldosterone infusion. Aldosterone infusion increased fibrosis and apoptosis (TUNEL-positive) in both wild-type and knockout mice. However, the increase in the extent of fibrosis and apoptosis was significantly lower in knockout hearts. Conclusions: Increased osteopontin plays an important role in the regulation of aldosterone-induced remodeling with effects on left ventricular dilation, fibrosis, and apoptosis.

Indiana University-Purdue University Indianapolis (IUPUI) / A burgeoning area of focus within liver disease research is centered on the concomitant muscle atrophy present in end stage liver disease patients which shows a correlation to severity of hepatic fibrosis and transplant survival outcomes. Of particular interest, nonalcoholic steatohepatitis (NASH) is a form of liver disease that is characterized as the hepatic manifestation of metabolic syndrome. If left untreated, the disease can progress to the state of cirrhosis and hepatocellular carcinoma requiring transplant. Concordant with increasing global prevalence of obesity, NASH is projected to become the leading cause for liver transplants by 2020. Due to a lack of therapeutic options, these patients represent a large unmet medical need in the western world. A major hurdle to therapeutic research is the lack of a quick, reproducible, and cost effective in vivo model that recapitulates the plethora of pathologies and their molecular underpinnings manifested by this disorder. Our studies attempted to validate and expand upon a two-hit model of NASH, which incorporated both the integral comorbidities associated with metabolic challenges of obesity along with liver injury. The two-hit model manifests not only the hepatic morphohistological characteristics of the disease, but also incorporates the obligatory muscle atrophy. To further elaborate on the potential direct link between liver and skeletal muscle and remove any confounding issues associated with the model, in vitro administration of hepatotoxins representing various pathologies associated with liver disease, were used to recapitulate the liver-muscle endocrine signaling that exists in vivo. Our data shows that a variety of hepatoxins can elicit hepatocellular damage which releases factors that inhibits myotube size in vitro. The two hit model also preserves many of conserved molecular underpinnings observed in clinical hepatic fibrosis. Of particular interest, the TGFβ superfamily has been demonstrated to play an important regulatory role in the progression of fibrosis in NASH patients. TGFβ, Activin A, and Follistatin are members of the highly conserved family that are increased in NASH patients. Furthermore, these proteins have a well-studied role in muscle health, regeneration, and mass that has been hypothesized to be conserved between liver and muscle tissues. Surprisingly, novel expression of the myokine and negative regulator of muscle mass Gdf8 (myostatin) was increased in our in vivo model as well. Our studies focused on the molecular interactions of these TGFβ superfamily members and their role on liver disease progression. Through specific inhibition of these proteins (Activin A and Gdf8), we demonstrated that they appear to play key individual roles in the progression of the concomitant muscle atrophy observed in NASH patients. Interestingly, superior efficacy was gained with the treatment of a pan inhibitor of these proteins (Activin A, B, Gdf8 etc.) via a soluble decoy receptor (ActRIIB-Fc), suggesting an additional unaccounted for ligand. Activin B, was found to be increased in two separate in vivo models of liver fibrosis (two-hit model and BDL), has been implicated in regulating muscle mass. Our data suggest a pivotal role for several members of the TGFβ superfamily in NASH associated muscle atrophy. Therapies designed to treat liver fibrosis and the resultant decrements in muscle mass and force must account for these agents which will require pan inhibition of TGFβ superfamily ligands that signal through the ActRIIB receptor.