In dieser Arbeit wurde die Myeloperoxidase in neutrophilen Granulozyten untersucht. Bei Entzündungsprozessen wandern Neutrophile ins Gewebe ein haben großen Einfluss auf den Entzündungsverlauf. Dabei spielt das Enzym Myeloperoxidase (MPO) eine wichtige Rolle. Die MPO produziert starke Oxidationsmittel, insbesondere auch Hypochlorsäure (HOCl), was als chlorierende Aktivität bezeichnet wird. HOCl trägt zur Pathogenabwehr bei und ist zudem an der Regulation und auch der Terminierung des Entzündungsprozesses beteiligt. Im Rahmen dieser Arbeit wurden neutrophile Granulozyten aus dem Vollblut gesunder Spender isoliert. Die chlorierende MPO-Aktivität konnte mithilfe des Fluoreszenzfarbstoffs Aminophenylfluorescein (APF) durchflusszytometrisch gemessen werden. Diese Methode wurde systematisch optimiert. Außerdem wurde erstmals die chlorierende Aktivität der MPO am Zellmodell (ex vivo) moduliert. Unter verschiedenen Bedingungen wurde dabei der Einfluss des Flavonoids (–)-Epicatechin auf die chlorierende MPO-Aktivität untersucht. Man nimmt an, dass unter Entzündungsbedingungen Komplex II akkumuliert, ein Intermediat der MPO, das keine chlorierende Aktivität aufweist. Einige Substanzen, wie (–)-Epicatechin, sind in der Lage, in vitro die chlorierende MPO-Aktivität zu steigern, indem sie Komplex II wieder in aktive Formen überführen. Dieser Sachverhalt wurde in dieser Arbeit erstmals am Zellmodell (ex vivo) untersucht. Unter Bedingungen, die zur Akkumulation von Komplex II führen, zeigte sich eine Steigerung der chlorierende MPO-Aktivität durch (–)-Epicatechin. Darüber hinaus konnten Grundlagen für eine Untersuchung der MPO am Vollblut gelegt werden: mithilfe von APF gelang eine durchflusszytometrische Differenzierung der Zellfraktionen anhand ihrer chlorierenden MPO-Aktivität und Granularität.

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In dieser Arbeit wurde die Myeloperoxidase in neutrophilen Granulozyten untersucht. Bei Entzündungsprozessen wandern Neutrophile ins Gewebe ein haben großen Einfluss auf den Entzündungsverlauf. Dabei spielt das Enzym Myeloperoxidase (MPO) eine wichtige Rolle. Die MPO produziert starke Oxidationsmittel, insbesondere auch Hypochlorsäure (HOCl), was als chlorierende Aktivität bezeichnet wird. HOCl trägt zur Pathogenabwehr bei und ist zudem an der Regulation und auch der Terminierung des Entzündungsprozesses beteiligt. Im Rahmen dieser Arbeit wurden neutrophile Granulozyten aus dem Vollblut gesunder Spender isoliert. Die chlorierende MPO-Aktivität konnte mithilfe des Fluoreszenzfarbstoffs Aminophenylfluorescein (APF) durchflusszytometrisch gemessen werden. Diese Methode wurde systematisch optimiert. Außerdem wurde erstmals die chlorierende Aktivität der MPO am Zellmodell (ex vivo) moduliert. Unter verschiedenen Bedingungen wurde dabei der Einfluss des Flavonoids (–)-Epicatechin auf die chlorierende MPO-Aktivität untersucht. Man nimmt an, dass unter Entzündungsbedingungen Komplex II akkumuliert, ein Intermediat der MPO, das keine chlorierende Aktivität aufweist. Einige Substanzen, wie (–)-Epicatechin, sind in der Lage, in vitro die chlorierende MPO-Aktivität zu steigern, indem sie Komplex II wieder in aktive Formen überführen. Dieser Sachverhalt wurde in dieser Arbeit erstmals am Zellmodell (ex vivo) untersucht. Unter Bedingungen, die zur Akkumulation von Komplex II führen, zeigte sich eine Steigerung der chlorierende MPO-Aktivität durch (–)-Epicatechin. Darüber hinaus konnten Grundlagen für eine Untersuchung der MPO am Vollblut gelegt werden: mithilfe von APF gelang eine durchflusszytometrische Differenzierung der Zellfraktionen anhand ihrer chlorierenden MPO-Aktivität und Granularität.

This thesis describes the design and synthesis of small molecule derivatives and their polypeptide conjugates as high affinity binders for proteins: the D-dimer protein (D-dimer), a biomarker for diagnosis of thromboembolic diseases; human myeloperoxidase (MPO), a biomarker for cardiovascular diseases; and chitinases, potential targets for asthma therapy. The interactions between the synthetic binder molecules and those proteins were evaluated by surface plasmon resonance (SPR) biosensor analysis and fluorescence spectroscopy. Competition SPR experiments or other methods proved that the small molecule components of the binder molecules were critical for binding and specifically bound to the original binding site of small molecules. The binder molecules consisted of a 42-residue helix-loop-helix polypeptide conjugated to a small molecule via aliphatic spacers of suitable length. The small molecules could be any type of moderately binding structure. In the binder development for the D-dimer, the tetrapeptide GPRP with a dissociation constant Kd of 25 μM was used and the affinity of 4C15L8GPRP obtained was estimated to be approximately 3 nM. In the binder development for MPO, salicylhydroxamic acid (SHA) with Kd of 2 μM was used and the affinity of 4C37L34C11SHA obtained was estimated to be approximately 0.4 nM. In the binder development for chitinases, a theobromine derivative (pentoxifylline) with a Kd of 43±10 μM was used and the affinity of 4C37L34-P obtained was estimated to be considerably higher than that of pentoxifylline. The binder molecules were identified from a 16-membered pool of candidates obtained by conjugating the small molecules to each member of a set of 16 designed polypeptides. The affinities were greatly enhanced by 2-3 orders of magnitude, compared to the small molecule. The polypeptides did not bind to the proteins with measurable affinities. The discovery of these new synthetic binders for protein targets can pave the way to diagnostic tests in vivo or in vitro, independent of antibodies.

To elucidate the correlation between host susceptible genes and the carcinogenesis of gastric cancer and duodenal ulcer, myeloperoxidase (MPO) -463 G/A polymorphism was detected by PCR-RFLP and nucleotide autosequencing, respectively. On the other hand, E-cadherin (CDH1) -160 C/A polymorphism was analyzed by nucleotide autosequencing. No positive correlation among MPO genotype distributions, gastric cancer (p=0.26,

Made available in DSpace on 2014-06-11T19:26:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-29Bitstream added on 2014-06-13T18:29:59Z : No. of bitstreams: 1 martins_ab_me_arafcf.pdf: 992180 bytes, checksum: 3281d39ebadb109f527844b299d91644 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Níveis séricos elevados de mieloperoxidase (MPO) estão associados com disfunção endotelial e risco aumentado de acidente cárdio-vascular. Há muito tempo se sabe que o hábito do tabagismo é um fator de risco para doenças cardiovasculares.Uma vez que o infarto do miocárdio está associado também com leucocitose neutrofílica, e fumantes apresentam neutrofilia, nós hipotetizamos que o nível sérico de MPO em fumantes também poderia estar elevado. O estudo incluiu quarenta adultos voluntários e saudáveis. O grupo controle foi composto por vinte indivíduos não tabagistas e o grupo de estudo por vinte indivíduos tabagistas. Hemograma, Interleucina 8 (CXIL8) sérica e MPO sérica foram determinados. Encontramos contagens de neutrófilos e monócitos aumentados (p<0,05) e o nível sérico de interleucina-8 (CXIL8) foi cerca de quatro vezes maior em fumantes quando comparados a não fumantes (p<0,05). O nível sérico de MPO foi também significativamente superior (p<0,05) nos indivíduos fumantes. Este último resultado se correlaciona perfeitamente com a neutrofilia e o aumento da CXIL8 (potente quimioatrator e ativador de neutrófilos), que caracterizam indivíduos fumantes. Propõe-se que esta alta concentração sérica de MPO possa estar diretamente envolvida com a alta prevalência de doenças coronarianas arteriais entre tabagistas / Elevated myeloperoxidase (MPO) serum levels are associated with endothelial dysfunction and cigarrete smoking is a risk factor for cardiovascular diseases. Since myocardial infarctation is associated with leucocytosis and smokers present increased levels of neutrophils, here we hypothesized that levels of serum MPO in smokers subjects could be also elevated. The study included forty adult healthy volunteers. The control group consisted of twenty non-smokers and the study group was twenty smoker subjects. The hemogram, interleukin-8 (CXIL8) and MPO serum levels were mensured. We found that neutrophil and monocytes counts are increased (p<0,05) and the serum level of interleukin-8 (CXIL8) was about 4-fold higher in smokers compared to non-smokers (p<0,05).The level of MPO was also higher among the group of tabagists (p<0,05). This result correlated perfectly with the increased in neutrophil counts and CXIL-8 serum level (a potent neutrophil chemo-attractant and activator), which characterize smoking subjects. We propose that the high level of serum MPO could be directly involved in the higher prevalence of coronary artery diseases among smokers

Resumo: Níveis séricos elevados de mieloperoxidase (MPO) estão associados com disfunção endotelial e risco aumentado de acidente cárdio-vascular. Há muito tempo se sabe que o hábito do tabagismo é um fator de risco para doenças cardiovasculares.Uma vez que o infarto do miocárdio está associado também com leucocitose neutrofílica, e fumantes apresentam neutrofilia, nós hipotetizamos que o nível sérico de MPO em fumantes também poderia estar elevado. O estudo incluiu quarenta adultos voluntários e saudáveis. O grupo controle foi composto por vinte indivíduos não tabagistas e o grupo de estudo por vinte indivíduos tabagistas. Hemograma, Interleucina 8 (CXIL8) sérica e MPO sérica foram determinados. Encontramos contagens de neutrófilos e monócitos aumentados (p<0,05) e o nível sérico de interleucina-8 (CXIL8) foi cerca de quatro vezes maior em fumantes quando comparados a não fumantes (p<0,05). O nível sérico de MPO foi também significativamente superior (p<0,05) nos indivíduos fumantes. Este último resultado se correlaciona perfeitamente com a neutrofilia e o aumento da CXIL8 (potente quimioatrator e ativador de neutrófilos), que caracterizam indivíduos fumantes. Propõe-se que esta alta concentração sérica de MPO possa estar diretamente envolvida com a alta prevalência de doenças coronarianas arteriais entre tabagistas / Abstract: Elevated myeloperoxidase (MPO) serum levels are associated with endothelial dysfunction and cigarrete smoking is a risk factor for cardiovascular diseases. Since myocardial infarctation is associated with leucocytosis and smokers present increased levels of neutrophils, here we hypothesized that levels of serum MPO in smokers subjects could be also elevated. The study included forty adult healthy volunteers. The control group consisted of twenty non-smokers and the study group was twenty smoker subjects. The hemogram, interleukin-8 (CXIL8) and MPO serum levels were mensured. We found that neutrophil and monocytes counts are increased (p<0,05) and the serum level of interleukin-8 (CXIL8) was about 4-fold higher in smokers compared to non-smokers (p<0,05).The level of MPO was also higher among the group of tabagists (p<0,05). This result correlated perfectly with the increased in neutrophil counts and CXIL-8 serum level (a potent neutrophil chemo-attractant and activator), which characterize smoking subjects. We propose that the high level of serum MPO could be directly involved in the higher prevalence of coronary artery diseases among smokers / Orientador: Luiz Marcos da Fonseca / Coorientador: Valdecir Ximenes Farias / Banca: Christiane Pienna Soares / Banca: Marcelo Nalin / Mestre

Cardiovascular disease and the associated endothelial dysfunction are characterized by leukocyte activation, decrease endothelial nitric oxide synthase (eNOS) activity, and increased endothelial cell adhesion molecules expression. This leads to the release of myeloperoxidase (MPO) by activated neutrophils and monocytes. MPO is a peroxidase enzyme that plays an important role in innate immune host defense, however it has been shown to play a pathogenic role in cardiovascular disease, mainly by causing endothelial dysfunction. The molecular mechanisms through which MPO induces endothelial damage are not fully understood. Calpains are a family of calcium-dependent proteases. Two calpain isoforms, µ- and m-calpain, are expressed in the vascular wall, including endothelial cells. Activation of calpains has been recently implicated in inflammatory disorders of the vasculature. The goal of this study was to test the hypothesis of a role for calpains in the molecular mechanism(s) through which MPO causes endothelial dysfunction and vascular inflammation. To explore if MPO activates calpain and to identify the calpain isoform(s) involved, we first studied the effects of MPO treatment on calpain activity in mouse lung microvascular endothelial cells (MMVEC). MMVECs were stimulated with 10 nmol/L MPO for 60, 120, 180, and 240 min. Using a fluorescent calpain activity assay, we found that MPO time dependently activates calpain in endothelial cells, with peak activity reached within 180 min. Using immunoblot analysis techniques we demonstrated that the calpain isoform targeted by MPO is µ-calpain. Interestingly, using a biotin switch assay,10 nmol/L MPO appears to activate the µ-calpain isoform via denitrosylation of its C-terminus domain. Using MMVECs, we studied the effects of the MPO/µ-calpain signaling on endothelial dysfunction. MMVECs were stimulated with 10 nmol/L MPO for 180 min. Expression levels of Protein Phosphatase 2 (PP2A), total 5' AMP-activated protein kinase (AMPK), Thr172 phospho-AMPK, total endothelial nitric oxide synthase (eNOS),Ser1177 phospho-eNOS, total protein kinase B (AKT), Ser473 phospho AKT, Adiponectin receptor 1 (AdipoR1), and Adiponectin receptor 2 (AdipoR2), were measured by immunoblot analysis. Interestingly, MPO impaired Thr172AMPK, Ser1177eNOS, but not Ser 473 AKT phosphorylation in a calpain dependent manner. On the other hand, MPO significantly increased the expression levels of PP2A. Inhibition of PP2A with okadiak acid decreased the phosphorylation levels of AMPK, and eNOS, indicating that PP2A is a downstream target of the MPO/calpain system. MPO treatment significantly increased the expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Pharmacological inhibition of calpain activity attenuated expression of VCAM-1. MPO also decreased protein levels of AdipoR1, and AdipoR2 in a calpain dependent manner. The treatment of MMVEC with adiponectin in the presence of MPO was not able to restore AdipoR2 expression levels. Using genetically modified mice, we found evidence of reduced leukocyte adhesion to the aortic endothelium in response to MPO in µ-calpain deficient mice, compared to wild-type mice . These effects appears to be attributed to the endothelial calpain, since incubating wild type aortas with calpain deficient leukocytes did not reduce leukocyte adhesion to the endothelium. The data in this study first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO, with MPO activating the µ-calpain isoform via denitrosylation. Our data also report that increased calpain activity downregulats the expression of a number of signaling molecules important for endothelial cell function. This study may provide the MPO/calpain/PP2A signaling pathway as a novel pharmacological targets for the treatment of inflammation-driven vascular disorders. / Biomedical Sciences

Made available in DSpace on 2017-07-21T14:13:02Z (GMT). No. of bitstreams: 1 Liliane Werle Lima.pdf: 3601191 bytes, checksum: 5783e9d4437866bebca28c4080abc069 (MD5) Previous issue date: 2014-11-10 / Pesticides are widely used around the world for pest control and guarantee increased productivity. As is harmful to insects and weeds, are also toxic to humans . Pesticides are related to alterations of the immune system, are neurotoxic, and involved with alterations em endocrine system, besides interfering with metabolism. Exposure to pesticides is monitored by measurement of biomarkers, especially cholinesterase , and through changes in laboratory tests. The most commonly used pesticides are those belonging to the class of organophosphates, whose mechanism of action is irreversible inhibition of cholinesterase. Pesticides being involved in alterations of the immune system, and cardiovascular disease, this study aimed to evaluate the activity of myeloperoxidase (present in phagocytic cells) in individuals chronically exposed to pesticides, in addition to other laboratory markers. This study included 79 volunteers , divided into 3 groups : 1 ) EDP : direct exposure to pesticides ; 2) EIP : indirect exposure to pesticides , and 3) C : control, volunteers not exposed, or low exposure to pesticides. Laboratory tests for glucose, total cholesterol and fractions , triglycerides , AST, ALT, urea, creatinine, myeloperoxidase (MPO) , alpha-1- acid glycoprotein (AGP), high sensitive C-reactive protein (Hs-CRP), total antioxidant capacity, cholinesterase were performed and CBC . There were significant increases in urea in the EDP group. High levels of MPO, AGP and glucose were found for the EDP and EIP groups. The Hs-CRP showed a direct correlation with the values of AGP, but was elevated only for the EIP group. Significant reduction in total antioxidant capacity of serum was found in groups EDP and EIP. The cholinesterase values showed significantly higher activity in the EDP group. With these results we concluded that chronic exposure to pesticides can lead to an adaptive increase in cholinesterase activity resulting in decreased anti-inflammatory action of acetylcholine, with increased levels of myeloperoxidase and other markers of inflammation. / Os praguicidas são largamente utilizados em todo o planeta para controle de pragas e garantia de aumento de produtividade. Assim como são nocivos para insetos e ervas daninhas, também são tóxicos para os seres humanos. Os pesticidas estão relacionados com alterações do sistema imunológico, nervoso, endócrino, alem de interferir no metabolismo. A exposição aos praguicidas é monitorada pela dosagem de biomarcadores, especialmente a colinesterase, e através de alterações em exames laboratoriais. Os pesticidas mais usados são aqueles pertencentes à classe dos organofosforados, que tem como mecanismo de ação a inibição irreversível das colinesterases. Estando os pesticidas envolvidos em alterações do sistema imunológico, e com a doença cardiovascular, este estudo teve por objetivo avaliar a atividade da enzima mieloperoxidase (presentes em células fagocíticas) em indivíduos expostos cronicamente a pesticidas, alem de outros marcadores laboratoriais. Este estudo incluiu 79 voluntários, divididos em 3 grupos: 1) EDP: exposição direta aos praguicidas; 2)EIP:exposição indireta aos praguicidas; e 3) C: controle, voluntários com pouca ou nenhuma exposição aos praguicidas. Foram realizados exames laboratoriais para dosagem de glicose, colesterol total e frações, triglicerídeos, TGO, TGP, ureia, creatinina, mieloperoxidase (MPO), alfa-1-glicoproteína ácida (AGP), proteína C reativa ultrassensível (PCR-us), colinesterase, capacidade antioxidante total e hemograma. Ocorreram aumentos significativos na ureia no grupo EDP. Níveis elevados de MPO, AGP e glicose foram encontrados para os grupos EDP e EIP. A PCR-us apresentou correlação direta com os valores de AGP, contudo mostrou-se elevada apenas para o grupo EIP. Significante redução na capacidade antioxidante total do soro foi encontrada nos grupos EDP e EIP. A colinesterase apresentou valores de atividade significativamente maiores no grupo EDP. Com estes resultados concluímos que a exposição crônica aos pesticidas pode levar a um aumento adaptativo da atividade da colinesterase resultando em diminuição da ação anti-inflamatória da acetilcolina, com aumento dos níveis de mieloperoxidase e outros marcadores da inflamação.

Made available in DSpace on 2014-06-11T19:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-09-29Bitstream added on 2014-06-13T20:41:01Z : No. of bitstreams: 1 vellosa_jcr_dr_arafcf.pdf: 1454785 bytes, checksum: 6e53ccd18d72b4b09811858784aa22d3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A pesquisa de produtos naturais é uma fonte importante de informações para o desenvolvimento de novos fármacos que tenham ação em diferentes processos oxidativos teciduais. Estudou-se o perfil bioquímico para o mecanismo de ação antioxidante dos flavonóides kaempferol, afzelina, lespedina, pteroginoside, quercetina e isoquercetrina isolados de Pterogyne nitens. Buscou-se avaliar desde os níveis mais simples de atividade até os níveis mais complexos de interação para as atividades observadas. Nenhuma das amostras foi eficiente como scavenger de H2O2, o que se mostrou interessante na comprovação dos resultados de inibição de peroxidases observados. Mostrou-se que a variação nas estruturas dos flavonóides pode levar a diferentes padrões de atividade de acordo com o sistema avaliado, revelando assim especificidade sobre a interação estudada. A quercetina mostrou-se, em geral, o agente mais eficiente, com alteração entre as posições dos demais flavonóides. Observou-se ainda, que os flavonóides apresentam potencial lesivo contra eritrócitos e neutrófilos quando em presença de radicais livres e outras espécies reativas. Na pesquisa da ação de flavonóides e outros produtos naturais em processos oxidativos modelo deve-se buscar possíveis efeitos protetores e agressores aos sistemas biológicos traçando-se um perfil bioquímico de ação que elucide os mecanismos pelos quais tais substâncias agem. / The research on natural products Field is na inportant source of informations to the development of new medicines wich act over tecidual oxidative processes. It was studied the biochemical profile of the antioxidant action from kaempferol, afzelin, lespedin, pterogynoside, quercetin and isoquercetrin isolated from Pterogyne nitens. It was intended to evaluate diferent interaction levels to the studied systems. None of the samples were able to act against hydrogen peroxide, what strengthens the observations on peroxidase inhibition. It was showed that chemical structural variations can lead to different activities patterns according to the studied system, revealing that exist specificity by the evaluated interaction. The flavonoid quercetin usually were the most efficient agent and the others agents had alternated position on different systems. It was observed too that the studied flavonoids were potentially lesive to the erytrocytes and neutrophils when they were put together free radicals and others reactive species. In the flavonoids and others natural products researches on model oxidative processes it must be made sought possible protective and lesive effects to biological systems by drawing the biochemical action profile of these sbstances.