Submitted by Natalia de Souza Gonçalves (natalia.goncalves@ufpe.br) on 2016-09-23T14:12:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE-Plinio correta.pdf: 2445148 bytes, checksum: af634e24f9f6564ebd9b8c37b5d9c2f8 (MD5) / Made available in DSpace on 2016-09-23T14:12:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE-Plinio correta.pdf: 2445148 bytes, checksum: af634e24f9f6564ebd9b8c37b5d9c2f8 (MD5) Previous issue date: 2016-03-10 / Serra Talhada, no sertão Pernambucano, registra grandes surtos de dengue. O armazenamento inadequado de água devido a estiagem prolongada, contribui para a perpetuação dos mosquitos. Além disso, Aedes albopictus também pode gerar maiores problemas para o município. Em nossas análises, o número de casos de dengue no período de 2012 a 2013, possui correlação com o número de ovos coletados no mês anterior. Apesar dos surtos intermitentes, o baixo número de casos de Dengue Hemorrágica, levanta questões a respeito de genes de suscetibilidade/resistência. Por isso, analisamos as frequências alélicas e genotípicas do marcador CCR5 e dos SNPs IL-4, IL-10 e Ly-6. Em nossos resultados, a mutação CCR5Δ32, apresentou baixa frequência. Os SNPs IL-4 e IL-10 encontraram-se em desequilíbrio de Hardy-Weinberg. Em relação às frequências alélicas, quando comparadas à população global, Ibérica, Peruana e Africana, observamos diferenças significativas para IL-4, mas não para IL-10. Já Ly-6 apresentou frequência alélica significativa em relação a Ibéricos, Peruanos e Africanos. A frequência genotípica dos genótipos CT e TT de IL-4, apresentaram diferenças significativas em relação a todas as populações. Enquanto que o genótipo TT de IL-10 foi significativamente diferente em relação aos Ibéricos. Para Ly-6, o genótipo GG foi significativamente diferente em relação à população africana, enquanto AG e AA diferiram das populações Ibérica, Peruana e Africana. Porém, só após o acréssimo de um grupo controle ou de outra categoria comparativa, teremos resultados conclusivos. / Serra Talhada, in semiarid of Pernambuco State, register every two years, dengue outbreaks. Inadequate water storage due prolonged drought, perpetuate the mosquito. Moreover, Aedes albopictus, may cause major problems for the municipality. Our analysis detected that the number of dengue cases in the period 2012-2013, has correlation with the number of eggs collected in the previous month. Despite facing intermittent outbreaks, the low number of cases of Hemorhagic Dengue, raises questions about susceptibility / resistance genes. Therefore, we analysed allelic and genotypic frequencies of the marker CCR5 and SNPs IL-4, IL-10 and Ly-6. About our results, the CCR5Δ32 mutation, showed a low frequency. About SNPs IL-4 and IL-10, Hardy-Weinberg disequilibrium were found. Regarding to allele frequency significant differences were observed for IL-4 but not IL-10 when compared to the overall population, Iberian, Peruvian and African. Ly-6 showed significant allele frequency compared to Iberian, African and Peruvian. Concerning to genotypic frequency, significant differences in relation to all populations were found to CT and TT genotype of IL-4. While the TT genotype of IL-10 was significantly different in relation to the Iberian. For Ly-6, the GG genotype was significantly different in relation to the African population, while AG and AA differ from the Iberian populations, Peruvian and African. However, to conclusive results regarding these markers, it becomes necessary to add a control group or another comparative category

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

Although the etiology surrounding many autoimmune diseases remains unknown, the underlying characteristic of many of these diseases is a disruption in the balance of pro- and anti- inflammatory cytokines It is well established that the dysregulation of the IL-12 family of cytokines, an increase in IL-12/IL-23 and a decrease in IL-27 production has been implicated in these conditions. We used ELISA, RT-PCR, Immunofluorescence and Western immunoblotting in conjunction with pharmalogical inhibitors and siRNA to demonstrate the role of SHP-1/Src in the regulation of IL-12, IL-23, IL-27 and IL-10 in LPS-stimulated human THP-1 cells, monocytes and MDMs. My results show for the first time that Src kinase activity relies on SHP-1 activity, and together this complex functions in TLR4-mediated MyD88 and TRIF pathways. Furthermore Src exhibits a dual role as a positive regulator for anti-inflammatory IL-10/IL-27 and as a negative regulator of pro-inflammatory IL-12/IL-23 downstream of TLR4. Moreover, the involvement of PI3K and JNK MAPK, dependent on SHP-1/Src complex, in the regulation of IL-12 family and IL-10 downstream of TLR4 was shown.

Formation of lipid-laden macrophage foam cells and inflammation are the central components in the initiation and progression of atherosclerosis. ABCA1 is well established as an anti-atherogenic factor that facilitates cellular cholesterol and phospholipid efflux, promotes reverse cholesterol transport, and suppresses pro-inflammatory cytokine secretion. Through these functions, ABCA1 is capable of reducing the lipid burden in atherosclerotic plaque. PKA signaling is an integral factor in promoting many anti-atherogenic functions of ABCA1; however, mechanistic aspects of PKA signaling associated with ABCA1 remain poorly defined. Thus, the first part of this study investigates the involvement of spatially regulated PKA signaling in ABCA1 activities through the use of st-Ht31, a PKA de-anchoring peptide. It appears that de-anchoring PKA robustly increases ABCA1-mediated microparticle release, one of the cholesterol efflux pathways of ABCA1, and reverses macrophage foam cell formation. These results highlight the significance of subcellular compartmentalization of PKA signaling in ABCA1 functions and present PKA de-anchoring as a potential therapeutic strategy for atherosclerotic lesion regression. The second part of this study provides evidence that ABCA1 activates PKA and promotes the secretion of anti-inflammatory IL-10, a cytokine crucial for inflammation resolution. Furthermore, we provide evidence that this elevated PKA activity is the underlying mechanism in which macrophage ABCA1 promotes M2-like inflammatory response. Our results also suggest that ABCA1 activates PKA by regulating cholesterol, which poises macrophages towards an anti-inflammatory or M2-activated phenotype. Collectively, we demonstrate that PKA signaling plays a crucial multifactorial role in anti-atherogenic functions of ABCA1.

Les lymphocytes B régulent la réponse immunitaire par la sécrétion de facteurs proinflammatoires ou immunosuppresseurs. Dans la Leucémie Lymphoïde Chronique (LLC),une sous-population de lymphocytes B CD5⁺ présente des propriétés immunosuppressives qui l’apparente aux lymphocytes B régulateurs notamment par la production d’IL-10. La survie des cellules leucémiques est, elle, associée à la réponse antigénique et à la production de cytokines dont l’IL-6. L’objectif de ce travail a été de caractériser dans la pathologie les populations produisant les cytokines pro-survie ou immunorégulatrices et d’analyser la relevance fonctionnelle de leur sécrétion. Nous avons identifié des sous-populations de cellules B leucémiques exprimant trois facteurs immunorégulateurs l’IL-10, le TGFβ1 et pour la première fois le facteur de transcription FOXP3, La proportion augmentée de cellules exprimant l’IL10 est associée à une diminution des cellules exprimant l’IL6. De manière importante, ce travail a identifié une boucle autocrine de stimulation de l’activité métabolique des cellules par l’IL10. La cytokine en se fixant à son récepteur permet l’activation des facteurs STAT3 et induit l’expression à la fois de protéines anti- apoptotiques de la famille Bcl2 mais surtout sa propre expression. Un blocage de cette boucle au niveau du récepteur à l’IL10 suspend l’avantage de survie des cellules tumorales. L’IL-6 ne déclenche pas ces mécanismes de maintien des cellules de LLC. Ce travail montre qu’en plus de son rôle sur les cellules du microenvironnement tumoral, l’IL-10 participe au maintien autocrine de la sous-population immunorégulatrice dans la LLC. / B cells produce pro-inflammatory or immunosuppressive factors to modulate the immuneresponse. In Chronic lymphocytic leukemia (CLL), a subset of the tumor lymphocytes produces IL10 and share immunoregulatory functions with regulatory B cells. CLL cell ssurvival is driven by antigenic response and pro-survival cytokines such as IL6. This project aimed at deciphering the cytokines profile of CLL subsets and analyzing their functional relevance. We identified immunoregulatory subsets producing IL-10, TGFβ1 and for the firsttime FOXP3. In patients, the increased proportion of cells expressing IL10 was correlated with decrease in IL6⁺ cells. Importantly we described an autocrine survival loop driven by IL10 in these cells. IL10 triggering led to STAT3 activation, induction of active pro-survival factors altogether with IL10 self-induction. Interrupting this loop with a blocking ab against IL10R prevented survival of the cells. IL6 did not manage such mechanisms. In conclusion,this work demonstrates that IL10 is an important mediator in CLL; the cytokine alters immune recognition of the tumor cells and sustains leukemic cells survival via the autocrine loop.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22122号 / 医科博第107号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 濵﨑 洋子, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The aim of the present study was to find out whether adaption to chronic hypoxia affects the expresion of TNF-α and IL-10 in rat myocardium. TNF-α is a proinflammatory cytokine, which amplifies inflammatory reaction, while IL-10 has opposite antiinflammatory effect. We also measured concentration of nitrotyrosine as a marker of nitrosative stress. We used male Wistar rats divided into four groups: 1) normoxic controls; 2) exposed to continous normobaric hypoxia (10% O2) for three days or 3) for three weeks and 4) exposed to intermittent normobaric hypoxia (10% O2) for three weeks with one hour daily reoxygenation. Cytosolic and membrane proteins (cytosolic and particulate fractions) were obtained from the left ventricle, right ventricle and interventricular septum. Concentrations of TNF-α and IL-10 in both fractions were measured by ELISA. Continous hypoxia increased TNF-α production in particulate fractions from all ventricular parts and decreased the ratio of IL-10/TNF-α in particulate and cytosolic fractions. Intermittent hypoxia redistributed TNF-α from cytosol into the particulate fraction and prevented the drop of IL-10/TNF-α ratio in the cytosolic fraction. The highest concentration of nitrotyrosine was found in the particulate fraction from the right ventricle after three days of hypoxia....

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21017号 / 医博第4363号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 濵﨑 洋子, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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