Orientador: Wilson Nadruz Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T13:58:57Z (GMT). No. of bitstreams: 1 Paim_LaydeRosane_M.pdf: 776318 bytes, checksum: f8e1046411d4b45041c7d37ca207a89e (MD5) Previous issue date: 2014 / Resumo: Estudos prévios mostram que indivíduos com lesão crônica na medula espinhal (LM) apresentam maior risco cardiovascular em comparação com indivíduos fisicamente normais. O presente estudo investigou a relação entre os níveis plasmáticos da lipoproteína de baixa densidade oxidada (LDLox), as metaloproteinases de matriz (MMP) e seus inibidores teciduais (TIMPs) e o remodelamento vascular em pacientes com LM, e o papel da atividade física nesta relação. Foram estudados 42 homens com LM (? 2 anos), [18 sedentários (S-LM) e 24 fisicamente ativos (A-LM)] e 16 homens fisicamente saudáveis por meio de análise clínica, antropométrica, laboratorial e de espessura íntima-média da carótida (EIM). Todos os participantes estudados eram normotensos, não diabéticos, não fumantes e normolipêmicos. As concentrações plasmáticas de LDLox, MMP-2, MMP-8, MMP-9, TIMP-1 e TIMP-2 foram determinados por ensaio imunoenzimático (ELISA). Os resultados mostraram que a EIM da carótida, razão EIM/diâmetro e as concentrações de LDLox dos A-LM e dos indivíduos fisicamente normais não foram diferentes estatisticamente. Por outro lado, indivíduos com S-LM apresentaram maior EIM, razão EIM/diâmetro e concentrações aumentados de LDLox em comparação com A-LM (p<0,01, p<0,001 e p=0,01, respectivamente) e indivíduos controles (p<0,001 para todos). Os resultados da análise de correlação bivariada, incluindo todos os indivíduos com LM, demonstrou que o EIM de carótida e a razão EIM/diâmetro se correlacionaram apenas com LDLox, MMP-8 e com a relação MMP-8/TIMP-1. Além disso, a análise de regressão ajustada para a presença ou não de atividade física e idade mostrou que a LDLox foi associada à EIM carotídea e com a relação EIM/diâmetro, enquanto que MMP-8 foi associado com o índice EIM/diâmetro em indivíduos com LM. Em conclusão, as concentrações plasmáticas de LDLox e MMP-8 estão associados com aterosclerose carotídea e há interação entre a inatividade física, aterosclerose e LDLox em indivíduos com LM / Abstract: Previous reports have indicated that subjects with chronic spinal cord injury (SCI) exhibit increased cardiovascular risk compared to able-bodied individuals. This study investigated the relationship between plasmatic oxidized low-density lipoprotein (OxLDL), matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) levels and vascular remodeling in SCI subjects and the role of physical activity in this regard. We studied 42 men with chronic (?2 years) SCI [18 sedentary (S-SCI) and 24 physically active (PA-SCI)] and 16 able-bodied men by clinical, anthropometric, laboratory, and carotid intima-media thickness (IMT) analysis. All enrolled subjects were normotensive, non-diabetics, non-smokers and normolipemic. Plasmatic OxLDL, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay. The results showed that carotid IMT, IMT/diameter ratio and OxLDL levels of PA-SCI and able-bodied subjects were statistically similar. Conversely, S-SCI subjects exhibited higher IMT, IMT/diameter ratio and OxLDL levels compared to PA-SCI (p<0.01, p<0.001 and p=0.01, respectively) and able-bodied (p<0.001 for all) individuals. Results of bivariate correlation analysis including all injured subjects showed that carotid IMT and IMT/diameter ratio only correlated with OxLDL, MMP-8 and MMP-8/TIMP-1 ratio. Further stepwise regression analysis adjusted for the presence or not of physical activity and age showed that OxLDL was associated with carotid IMT and IMT/diameter ratio, while MMP-8 was associated with IMT/diameter ratio in SCI individuals. In conclusion, plasmatic OxLDL and MMP-8 levels are associated with carotid atherosclerosis and there is an interaction among physical inactivity, atherosclerosis and OxLDL in SCI individuals / Mestrado / Clinica Medica / Mestra em Clínica Médica

La souche de P. aeruginosa PA14 est un isolat humain hautement virulent. PA14 possède deux îlots de pathogénicité. L'îlot de pathogénicité PAPI-1 de 108 kb est un élément intégratif et conjugatif (ICE), capable de s'auto-transférer à des souches de Pseudomonas par un mécanisme de conjugaison. Le mécanisme de transfert fait intervenir un pilus de Type IVb, encodé dans l'îlot PAPI-1. Mon travail de doctorat a eu pour but de caractériser à un niveau moléculaire le pilus de Type IVb (Pil-PAPI-1). J'ai d'abord, inséré un promoteur constitutif en place du promoteur endogène pour activer l'expression du locus pil2. J'ai démontré que 9 des 10 gènes sont requis pour le transfert d'ADN. J'ai ensuite initié la caractérisation de composants formant la machine de conjugaison. J'ai caractérisé le produit des gènes pilL2 et pilN2. J'ai démontré que PilL2 est une protéine de membrane externe (ME) et exposée dans le périplasme. Cette protéine est essentielle pour la fonctionnalité (transfert d'ADN) de la machinerie de conjugaison. Malgré ses caractéristiques prédites de lipoprotéine, aucune des mutations réalisées n'a pu modifier la localisation de ME de PilL2. J'ai aussi démontré que PilL2 forme un sous complexe de ME avec PilN2, la sécrétine du système. PilN2 forme des multimères stable, et présente les caractéristiques d'une liposécretine, capable d'auto-insertion et d'auto-multimérisation dans la ME. J'ai démontré que le N-ter de PilN2 mature est critique pour la formation d'un pore fonctionnel, mais n'est pas impliqué dans l'interaction avec PilL2. Ces résultats suggèrent que PilL2 et PilN2 forment un nouveau type de sous complexe de ME dans la famille des TFPb. / The P. aeruginosa PA14 strain is a highly virulent human isolate. PA14 possesses two pathogenicity islands. The 108-kb pathogenicity island PAPI-1 is an integrative and conjugative element (ICE), capable of self-transferring to any recipient Pseudomonas strain, by a conjugative mechanism. The transfer mechanism is mediated by a Type IVb pilus, encoded within the PAPI-1 Island. My PhD work aimed to characterize this Type IVb pilus (Pil-PAPI-1) at a molecular level. The pil2 locus is poorly expressed under laboratory condition. I, first, introduced a constitutive promoter to turn on expression of the pil2 locus. I demonstrated that 9 of the 10 genes are required for DNA transfer. Then, I initiated the characterization of components forming the conjugation machinery. I characterized the products of pilL2 and pilN2 genes. I demonstrated that PilL2 is an OM protein and protruding in the periplasm. This protein is essential for the functionality (DNA transfer) of the conjugative TFPb machinery. Despite its predicted lipoprotein-hallmarks, none of the mutations engineered were able to abrogate the OM-localization of PilL2. We also demonstrated that PilL2 forms an OM sub-complex with PilN2, the secretin of the system. We provide evidence that PilN2 forms stable multimers, which presents the features of a liposecretin, capable of self-insertion and self-multimerization in the OM. We demonstrated that while the N-terminus of the mature PilN2 is required for the formation of a functional pore, it is not involved in interaction with PilL2. These results suggest that PilL2 and PilN2 could form new type of OM sub-complex in the TFPb family.

Heat shock protein (HSP)27 is traditionally described as an intracellular chaperone and signaling molecule, but growing evidence suggests it is released from immune cells where it plays an anti-inflammatory role during atherogenesis. Previously, the O’Brien lab found that overexpression of HSP27 led to augmented HSP27 serum levels in female apolipoprotein E knockout (ApoE-/-) mice, attenuated atherogenesis, and inhibited macrophage foam cell formation via physical binding with scavenger receptor (SR)-A. However, the precise mechanism of atheroprotection remained elusive. This thesis sought to ascertain the mechanism(s) by which HSP27 prevents foam cell formation, and determine if SR-A, a key receptor involved in the uptake of lipid into macrophages, plays an important role in HSP27-mediated atheroprotection. Pre-treatment of human macrophages with recombinant HSP27 (rHSP27) inhibited acytelated low density lipoprotein (acLDL) binding and uptake independent from receptor competition effect. Reduction in uptake was associated with attenuation of expression of SR-A mRNA, total protein, and cell surface expression. To explore the signaling mechanism by which HSP27 modulated SR-A expression it was hypothesized that nuclear factor-kappa B (NF-kB), a major regulator of many atherosclerosis gene programs, is altered by extracellular HSP27. Indeed, rHSP27 markedly activated NF-kB signaling in macrophages. Using an inhibitor of NF-kBsignaling there was an attenuation of rHSP27-induced inhibition of SR-A gene and protein expression, as well as lipid uptake, suggesting that SR-A expression is regulated by NF-kB activation. Lastly, to investigate if SR-A is required for HSP27-mediated atheroprotection in vivo, ApoE-/- and ApoE-/-SR-A-/- mice fed a high fat diet were treated with rHSP25, the mouse orthologue of HSP27, or PBS for 3 weeks. While rHSP25 therapy equally reduced serum cholesterol levels in the mouse cohorts, aortic atherogenesis, assessed using en face and sinus cross-sectional analyses, was attenuated in ApoE-/- mice but not ApoE-/-SR-A-/- mice. In conclusion, rHSP27 inhibits foam cell formation by downregulating SR-A expression. This effect may be associated with NF-kB activation. Reductions in atherosclerotic burden by rHSP27 require SR-A, and are independent of changes in serum cholesterol levels, highlighting the importance of macrophage lipid uptake in atherogenesis. Results presented in this thesis demonstrate that SR-A is a major target for HSP27 atheroprotection in the vessel wall, and provide an impetus for further studies that investigate the potential therapeutic value of HSP27.

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

A associação de uma série de influências ambientais como dietas com excesso de gordura ou falta de atividade física regular (sedentarismo) são importantes fatores que podem levar ao desenvolvimento da obesidade e dislipidemias. Portanto, a prática de atividade física regular, caracterizada pelo treinamento, mostra-se atualmente como parte de estratégias para combater problemas como dislipidemias. Sabe-se que o aumento do gasto calórico e a melhora no desempenho podem ser atingidos com treinamentos de endurance ou intermitentes, uma vez que ambos levam à alterações fisiológicas e metabólicas semelhantes. O treinamento intermitente é caracterizado pela execução de repetidas sessões de curtos ou longos períodos, preferencialmente de alta intensidade (aproximadamente 100% do VO2máx.), intercaladas por pausas ou períodos de menor intensidade, visando a recuperação do indivíduo. Dada a importância do fígado no metabolismo lipídico em repouso e no exercício foi nosso interesse avaliarmos o comportamento do fígado frente a oito semanas de treinamento intermitente de alta intensidade e comparar tais alterações às promovidas pelo treinamento de endurance em ratos, com especial atenção a síntese e secreção de VLDL. Os animais foram divididos em três grupos: sedentário (SD), treinamento contínuo (TC) e treinamento intermitente de alta intensidade (TI). Os dois protocolos de treinamento resultaram em menor ganho de peso em comparação com o grupo SD. Ainda o grupo TI apresentou maior secreção de VLDL em comparação aos grupos TC e SD. Além disso, a expressão gênica da MTP, proteína chave na montagem da VLDL e da LPL muscular responsável por catalisar a liberação de TAG da VLDL para captação pelo músculo esquelético demonstrou aumento no grupo TI em comparação ao SD. Tais resultados inferem que o treinamento intermitente modulou o transporte de TAG para a periferia e contribui para um efeito hipotrigliceridêmico após o exercício de alta intensidade / The combination of environmental influence, including high fat diets and lack of regular physical activity (sedentary lifestyle) are important factors leading to the development of obesity and dyslipidemias. Regular practice of physical activity, characterized by training, appears as important strategy to reduce such problems. Increased caloric expenditure and improvement of physical performance can be reached with endurance or intermittent training since both lead to similar physiological and metabolic adaptations. Intermittent training is characterized by the execution of repeated bouts of physical effort (high intensity, approximately 100% of VO2max.) Due to the importance of the liver in lipid metabolism during rest and exercise, we examined the adaptations of organ to 8 weeks of high intensity intermittent training, compared with the effect of endurance exercise with special attention to VLDL synthesis and secretion. The animals were randomized into three groups: sedentary (SD), continuous training (TC) and intermittent training (TI). Both training protocols resulted in reduced weight body gain compared with SD, although IT presents higher VLDL secretion, compared with TC and SD. In addition, gene expression of MTP, a key protein in the assembly of VLDL and LPL muscle responsible for catalyzing the release of TAG for VLDL uptake by skeletal muscle, showed an increase in TI compared to SD. These results suggest that intermittent training modulated the transport of TAG to the periphery and contributed to an hypotriglyceridemic effect caused by high intensity exercise

This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.

Background: The 5 subunits of native pentameric C-reactive protein (CRP) are dissociated to generate the monomeric form of CRP (mCRP) in some in vitro conditions, both physiological and non-physiological, and also in vivo. Many bioactivities of mCRP generated by urea-treatment of CRP and of mCRP generated by mutating the primary structure of CRP have been reported. The bioactivities of mCRP generated by spontaneous dissociation of CRP are largely unexplored. Methods: We purified mCRP generated by spontaneous dissociation of CRP and investigated the binding of mCRP to enzymatically-modified low-density lipoprotein (E-LDL). Results: mCRP was approximately 60 times more potent than CRP in binding to E-LDL. In the presence of the small-molecule compound phosphoethanolamine (PEt), at 37 °C, the binding of mCRP to E-LDL was enhanced <2-fold, while the binding of CRP to E-LDL was enhanced >10-fold. In contrast, PEt inhibited the binding of both CRP and mCRP to pneumococcal C-polysaccharide, another phosphocholine-containing ligand to which CRP and mCRP were found to bind. We have not investigated yet whether PEt alters the structure of CRP at 37 °C. Conclusions: Combined data suggest that the targeting of CRP with the aim to monomerize CRP in vivo may be an effective approach to capture modified forms of LDL.

The connection between C-reactive protein (CRP) and atherosclerosis lies on three grounds. First, the concentration of CRP in the serum, which is measured by using highly sensitive (a.k.a. 'hs') techniques, correlates with the occurrence of cardiovascular disease. Second, although CRP binds only to Fcγ receptor-bearing cells and, in general, to apoptotic and damaged cells, almost every type of cultured mammalian cells has been shown to respond to CRP treatment. Many of these responses indicate proatherogenic functions of CRP but are being reinvestigated using CRP preparations that are free of endotoxins, sodium azide, and biologically active peptides derived from the protein itself. Third, CRP binds to modified forms of low-density lipoprotein (LDL), and, when aggregated, CRP can bind to native LDL as well. Accordingly, CRP is seen with LDL and damaged cells at the atherosclerotic lesions and myocardial infarcts. In experimental rats, human CRP was found to increase the infarct size, an effect that could be abrogated by blocking CRP-mediated complement activation. In the Apob 100/100 Ldlr -/- murine model of atherosclerosis, human CRP was shown to be atheroprotective, and the importance of CRP-LDL interactions in this protection was noted. Despite all this, at the end, the question whether CRP can protect humans from developing atherosclerosis remains unanswered.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20810号 / 医博第4310号 / 新制||医||1025(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 清水 章, 教授 萩原 正敏, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

It has been found in previous studies that there is a correlation between the placenta regulatory genes and the weight gain of the mother, Body Mass Index (BMI) as well as the birthweight of the fetus. When the mother gains weight / is overweight, this will affect the gene expression in the placenta, and in turn this triggers the weight gain of the fetus. The aim of the study was to investigate the correlation between the lipoprotein lipase gene and the mother's BMI, weight gain and the child's birth weight by extracting RNA from the placentas and analysing its quality and concentration. cDNA was generated from RNA using reverse transcription and gene expression was amplified using real-time PCR. The data from real-time PCR was used in the comparative Ct-method to calculate a 2˄(-ΔΔCt)-value which represents the RNA-level of the LPL-gene. Lastly this value was analysed by using the two-statistic methods, Pearson's rank correlation and Spearman's correlation, which showed that the value of the correlation coefficient for all the variables was close to the value of zero. The closer the value is to zero, the weaker the association becomes between the different variables. The correlation was 0.045, 0.112 and 0.044 for the child's birth weight, mother's BMI respective weight gain. The results from this study shows that there is no correlation between LPL and the mother's weight gain, BMI, or the child's birth weight.