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Made available in DSpace on 2014-06-11T19:31:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2001-12-17Bitstream added on 2014-06-13T19:20:40Z : No. of bitstreams: 1 queiroz_ces_dr_arafo.pdf: 331073 bytes, checksum: 5b0cc47532d72c27e47c5ac40cc15e8e (MD5) / O autor avaliou a capacidade do hidróxido de cálcio em neutralizar o LPS de Pseudomonas aeruginosa, através de duas metodologias, a liberação de óxido nítrico e Fator de Necrose Tumoral Alfa (TNF-a) em cultura de macrófagos peritoneais de camundondos. O autor concluiu que o LPS bacteriano é um potente estimulador da produção de NO e TNF-a e que o tratamento deste LPS com hidróxido de cálcio causa sua inativação. / It was evaluated the calcium hydroxide in neutalysing Pseudomonas aeruginosa's LPS. Two methodologies were used; NO and TNF-a liberation in macrophage's rat culture. It was concluded that Ca(OH)2 inhibitted TNF-a and NO liberation.

Oxidative stress and inflammation are important factors involved in the progression of heart failure. An important cytokine produced during myocardial infarction (MI) is tumor necrosis factor alpha (TNF-α). TNF-α may induce oxidative stress, cell damage, apoptosis and cardiac dysfunction. Considering the anti-inflammatory and anti-oxidant properties of extra-virgin olive oil and its major component (80%) oleic acid (OA), and their benefits to the cardiovascular system, we hypothesized that the negative effects of TNF-α in the pathogenesis of heart failure will be mitigated by olive oil consumption. This hypothesis was tested by examining the effects of a special diet supplemented with 10% olive oil, in coronary artery ligated animal model of MI. Corn oil (10%) supplementation was used as a control for matching caloric intake. Animals in the sham and ligated groups fed regular chow, olive oil, and corn oil were studied at 4 and 16 weeks post myocardial infarction (PMI). Mortality, diet consumption, weight gain and conduction system abnormalities were comparable among all ligated groups. Echocardiography showed that MI deteriorated cardiac function, and olive oil restored the function. At 16 weeks PMI, only corn oil fed groups showed significant increase in both total cholesterol and HDL. Corn oil was not able to offer protection to the heart, suggesting that the beneficial effects of olive oil are not due to increased caloric intake or increased HDL. MI increased myocardial TNF-α, oxidative stress, lipid peroxidation, pro-apoptotic protein expression (Bax, cleaved Caspase 3, cleaved PARP, TGFβ, Bnip3), cytochrome C release, MAP kinase activation (p38, JNK) and decreased anti-apoptotic protein Bcl-xL expression at both 4 and 16 weeks PMI, and these changes were modulated by olive oil. In order to further test the central role of TNF-α PMI, we examined the possible miti-gation of TNF-α induced changes by OA in isolated adult rat cardiomyocytes. TNF-α in-creased oxidative stress, cell damage, cell death, and apoptosis, while OA treatment miti-gated these TNF-α induced effects. We concluded that TNF-α is implicated in the progression of heart failure subsequent to MI and that OA in olive oil may prevent this progression, through its anti-oxidant, anti-inflammatory, anti-hypertensive, and inotropic effects.

Resumo: O autor avaliou a capacidade do hidróxido de cálcio em neutralizar o LPS de Pseudomonas aeruginosa, através de duas metodologias, a liberação de óxido nítrico e Fator de Necrose Tumoral Alfa (TNF-a) em cultura de macrófagos peritoneais de camundondos. O autor concluiu que o LPS bacteriano é um potente estimulador da produção de NO e TNF-a e que o tratamento deste LPS com hidróxido de cálcio causa sua inativação. / Abstract: It was evaluated the calcium hydroxide in neutalysing "Pseudomonas aeruginosa"'s LPS. Two methodologies were used; NO and TNF-a liberation in macrophage's rat culture. It was concluded that Ca(OH)2 inhibitted TNF-a and NO liberation. / Orientador: Renato de Toledo Leonardo / Coorientador: Iracilda Zeponi Carlos / Banca: Fábio Luiz Camargo Villela Berbert / Banca: Caio César Randi Ferraz / Banca: Odilon Mattos Rasquin / Doutor

Transplantation of multipotent mesenchymal progenitor cells is a valuable option for treating tendon disease. Tenogenic differentiation leading to cell replacement and subsequent matrix modulation may contribute to the regenerative effects of these cells, but it is unclear whether this occurs in the inflammatory environment of acute tendon disease. Equine adipose-derived stromal cells (ASC) were cultured as monolayers or on decellularized tendon scaffolds in static or dynamic conditions, the latter represented by cyclic stretching. The impact of different inflammatory conditions, as represented by supplementation with interleukin-1β and/or tumor necrosis factor-α or by co-culture with allogeneic peripheral blood leukocytes, on ASC functional properties was investigated. High cytokine concentrations increased ASC proliferation and osteogenic differentiation, but decreased chondrogenic differentiation and ASC viability in scaffold culture, as well as tendon scaffold repopulation, and strongly influenced musculoskeletal gene expression. Effects regarding the latter differed between the monolayer and scaffold cultures. Leukocytes rather decreased ASC proliferation, but had similar effects on viability and musculoskeletal gene expression. This included decreased expression of the tenogenic transcription factor scleraxis by an inflammatory environment throughout culture conditions. The data demonstrate that ASC tenogenic properties are compromised in an inflammatory environment, with relevance to their possible mechanisms of action in acute tendon disease.

Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor (TNF) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNF-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNF to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNF-mediated signaling, supporting the functionality with an NF-B luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNF to its receptors and influencing TNF-mediated signaling via its interaction with progranulin.

<p><strong>Abstract</strong></p> <p>Sandhoff disease (SD) is a monogenic lysosomal storage disorder caused by a lack of a functional β-subunit of the beta-hexosaminidase A and B enzymes. The clinical phenotype of <em>Hexb</em>^-/-mouse model recapitulates the symptoms and signs of Tay-Sachs and Sandhoff diseases in human. To gain insight into the neuropathology of Sandhoff disease, we defined the role of TNFα in the development and progression of Sandhoff disease pathology in mice, by generating a <em>Hexb^-/-Tnf</em><em>a</em><em>^-/-</em> double knock-out mouse. Behavioural testing and immunostaining data revealed the neurodegenerative role of TNFα in disease pathology. Double knock-out mice showed ameliorated clinical course, with prolonged life span. TNFα-deficient Sandhoff mice also demonstrate decreased levels of astrogliosis, and reduced neuronal cell death. Deletion of <em>Tnfα</em> in Sandhoff mice inhibited JAK2/STAT3 pathway, implicating its role in glia cell activation. This result points to TNFa as a potential therapeutic target to attenuate neuro-pathogenesis.</p> <p>To investigate whether blood-derived or CNS-derived TNFα has the major impact on neurological function, we transplanted <em>Hexb^-/-Tnfα^+/+</em> with bone marrow from either <em>Hexb^-/-Tnfα^-/-</em>or <em>Hexb^-/-Tnf</em><em>a</em><em>^+/+</em> mice donors. Neurological tests shows a significant clinical improvement for Hexb<em>^-/-Tnfα^-/-</em> compared to <em>Hexb^-/-Tnf</em><em>a</em><em>^+/+</em> recipient, regardless the genotype of donor cells. These findings highlight the importance of resident-derived TNFα during the robust neurodegenerative consequences in Sandhoff disease. To understand of the role of microRNAs in Sandhoff pathology, we investigated the miRNA profile in Sandhoff brains. A pattern of dys-regulated microRNAs was evident in Sandhoff CNS. Microarray identified miR-210 and miR-96 dys-regulated pattern in the CNS of Sandhoff mice. Strikingly, neuronal pentraxin, a putative target gene for miR-210, was induced in Sandhoff brains.</p> <p>Taken together, this work establishes the proinflammatory role of TNFα in Sandhoff pathology, leading to massive neuro-apoptosis. Importantly, our studies propose that neuronal pentraxin as a novel target gene for microRNA-210 in Sandhoff brain samples, providing a potential modulator of neurodegeneration.</p> / Doctor of Philosophy (PhD)