Orientador: Camila Renata Corrêa / Coorientador: André Ferreira do Nascimento / Banca: Luciane Dias Alargão Dias Melicio / Banca: José César Rosa Neto / Resumo: A prevalência da obesidade tem aumentado durante as duas últimas décadas; esse aumento tem sido atribuído a mudanças no hábito dietético onde prevalece o aumento de gorduras e açúcares. É fator de risco para doenças cardiovasculares e o processo inflamatório favorece esse contexto. Porém ainda não está bem esclarecido o efeito do tempo de consumo de dieta desequilibrada sobre o processo inflamatório via receptor Toll-like 4 (TLR-4) e sua associação com alterações metabólicas. Sendo assim o objetivo do presente estudo foi testar a hipótese que um aumento da exposição a uma da sobrecarga nutricional acarreta em aumento da expressão de TLR-4 e esse inicia a resposta inflamatória com conseqüentes alterações metabólicas. Para isso foram utilizados ratos Wistar machos (n=32 animais), com 30 dias de idade, que foram casualmente divididos para receberem dieta controle (C) ou hiperlipídica + água com açúcar (HFD), durante 6 ou 24 semanas, perfazendo 4 grupos experimentais independentes (C6, C24, HFD6, HFD24; n=8 animais/grupo). Os ratos C receberam ração padrão para roedores e o grupo HFD uma ração hiperlipídica. Foi avaliado peso corporal, índice de adiposidade (IA), hipertensão arterial, perfil glicêmico e lipídico, avaliação hormonal, inflamatória no plasma. No tecido adiposo foram avaliados os indicadores de inflamação: fator de necrose tumoral-alfa (TNF-α), Interleucina -6 (IL-6), fator transcricional NFkB e receptor Toll like 4 . Ao final de 6 semanas, os animais apresentaram maior IA, triglicérides, glicemia, insulina, leptina e menor adiponectina em comparação a C6, caracterizando um quadro de síndrome metabólica. Ao final de 24 semanas, além das alterações metabólicas, os animais apresentaram aumento da expressão de TLR-4, NFkB e das citocinas inflamatórias TNF-α e IL-6 no tecido adiposo. Assim, concluímos que um tempo prolongado de consumo de dieta rica em açúcar e gordura agrava ... / Abstract: The prevalence of obesity has increased over the past two decades , and this increase has been attributed to changes in dietary habit where the increase of fats and sugars prevails . It is a risk factor for cardiovascular disease and inflammatory process favors this context. But is not yet well understood the effect of time -consuming unbalanced diet on inflammation via Toll-like receptor 4 ( TLR - 4 ) and its association with metabolic abnormalities . Therefore the aim of this study was to test the hypothesis that increased exposure to a nutritional overload leads to increased expression of TLR-4 and this initiates the inflammatory response with consequent metabolic changes. Male Wistar rats ( n = 32 animals ) were used , with 30 days of age, who were randomly divided to receive control diet ( C ) or high fat + sugar water ( HFD ) for 6 to 24 weeks , totaling 4 groups independent experimental ( C6 , C24 , HFD6 , HFD24 n = 8 animals / group ) . The C mice fed standard rodent chow diet and HFD group a high-fat diet . Body weight , adiposity index (AI ) , hypertension , glucose and lipid profile , hormonal evaluation , inflammatory plasma was evaluated . Tumor necrosis factor -alpha ( TNF - α ) , interleukin 6 ( IL - 6 ) , transcription factor NFkB and Toll like receptor 4 : the adipose tissue indicators of inflammation were evaluated . At the end of 6 weeks, the animals showed higher AI , triglycerides , glucose , insulin, leptin and lower adiponectin compared to C6 , featuring a picture of metabolic syndrome . At the end of 24 weeks , in addition to metabolic changes , the animals showed increased expression of TLR - 4 , NFkB and inflammatory cytokines TNF - α and IL - 6 in adipose tissue . Thus, we conclude that a prolonged time consumption of a diet rich in sugar and fat aggravates the situation of metabolic syndrome , and trigger an inflammation via TLR - 4 / Mestre

Os tecidos periodontais estão em confronto contínuo com microrganismos capazes de disparar mecanismos da resposta imune inata, dando origem ao infiltrado inflamatório neutrofílico. A participação dos receptores tipo Toll (TLRs) na resposta de neutrófilos frente à periodontopatógenos associados à doença periodontal precisam ser determinados. Nesse estudo procuramos caracterizar o infiltrado inflamatório presente no peritônio de animais deficientes de TLR2-/-, avaliar a atividade fagocítica, bem como a produção de óxido nítrico (NO) e a atividade de mieloperoxidase (MPO) no curso da infecção por Aggregatibacter actinomycetemcomitans (Aa). Os resultados revelaram um menor recrutamento de leucócitos para o peritônio de animais deficientes de TLR2 (TLR2-/-), com predomínio de macrófagos no exsudato peritoneal tanto de animais selvagens (WTTLR2-/-) como nos animais nocautes. A análise da atividade fagocítica revelou uma menor taxa de fagocitose pelas células do exsudato de animais TLR2-/- e, ainda, a deficiência do receptor TLR2 inibiu a produção de NO (óxido nítrico) e aatividade de MPO (mieloperoxidase). Adicionalmente, são apresentados os resultados referentes ao protocolo de doença periodontal experimentalmente induzida (DPEI) com Aggregatibacter actinomycetemcomitans (Aa) em camundongos deficientes de TLR2. Os resultados mostraram que 100% dos animais deficientes de TLR2 sobreviveram à infecção durante o período de observação. Em relação à análise de perda óssea os dados revelaram uma maior perda progressiva de osso alveolar na região dos molares de animais deficientes de TLR2. A ausência do receptor interferiu com a disseminação da bactéria, uma vez que se observou um grande número de bacilos no linfonodo e baço dos animais que não expressaram TLR2, diferente do observado para os animais selvagens (WTTLR2-/-). Os resultados indicam a importância da sinalização via TLR2 durante a resposta imune contra Aggregatibacter actinomycetemcomitans. / Polymorphonuclear leukocytes (PMNs) are the first line of defense against bacterial infections. PMNs express a numerous pattern-recognition receptors (PRR) that facilitate identification of invading microorganisms. Toll-like receptors (TLRs) represent the main class of PRR involved to a recognize pathogenic microorganism. However, the role played by TLR-2 in the recognition and killing of Aggregatibacter actinomycentemcomitans by PMNs is unknown. Thus, we investigated the ability of TLR-2 to mediate neutrophil phagocytosis and bactericidal activity. To determine the role of A. actinomycentemcomitans in triggering neutrophil infiltration, TLR2-/- mice were infected intraperitoneally (2x109 bacteria) and sacrificed after 24 hours. Peritoneal inflammatory cells were isolated and analyzed by optical microscopy. Examination of local inflammatory infiltrates revealed that neutrophil influx into peritoneal cavity of TLR2-/- mice was similar than that observed into their littermate controls wild type C57BL/6 mice (WT). A. actinomycentemcomitans was detected in the spleen of the TLR2-/- mice but not in WT. In the phagocytic assays, TLR2-/- presented minor number of ingested inflammatory cells than WT. Peritoneal cells were stimulated with A. actinomycetemcomitans antigens, and NO and myeloperoxidase was measured after 48 hours; results showed that TLR2-/- cells produce less NO and MPO than WT. In addition, TLR2-/- deficient mice presented higher bone loss following oral infection with A. actinomycetemcomitans when compared with WT and higher tissue destruction.

Orientador : Alexandre Leite Rodrigues de Oliveira / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:27:25Z (GMT). No. of bitstreams: 1 Freria_CamilaMarques_D.pdf: 9799936 bytes, checksum: 9a9057c6ab5ca87ea1bc6767c4f71490 (MD5) Previous issue date: 2013 / Resumo: Lesões nervosas periféricas e centrais levam à inflamação local e retrógrada, resultando em alterações axonais, perdas neuronais e sinápticas significativas. Juntamente a tais alterações, as células gliais tornam-se reativas, influenciando na remodelação do SNC após lesão. Os mecanismos que desencadeiam tais mudanças não são completamente compreendidos, mas é evidente que as moléculas classicamente relacionadas com o sistema imune estão envolvidas em tais eventos diretamente ou através da modulação da reatividade glial. Assim, nossa hipótese é que o controle da sinalização inflamatória após a lesão central ou periférica possa afetar indiretamente nos mecanismos endógenos de reparação no SNC, resultando em maior preservação das conexões neurais e melhor recuperação funcional. Para isso, realizamos lesões periféricas e centrais expondo os animais a diferentes microambientes de lesão a fim de investigar o papel das células gliais na sobrevivência, capacidade regenerativa axonal e estabilidade sináptica de motoneurônios medulares. Os resultados mostraram que, após lesão, a modulação da sinalização inflamatória através da administração de citocinas ou deleção de moléculas expressas na superfície das células gliais podem influenciar direta ou indiretamente na estabilidade dos circuitos neuronais, na regeneração axonal e sobrevivência neuronal. Desse modo, conclui-se que o controle da inflamação e da reatividade glial são, provavelmente, críticos para a plasticidade no Sistema Nervoso viabilizando, assim, novas estratégicas de tratamentos / Abstract: Central or peripheral lesions result in local and retrograde inflammation, leading to axonal degeneration, synaptic and/or neuronal loss. Additionally, after injury, reactive glial cells are recruited to the lesion site, influencing the plasticity of the nervous system. The mechanisms which trigger such changes are not completely understood, but evidences have shown that molecules classically related to the immune system are involved in such events directly or indirectly by glial modulation. Based on this, our hypotheses is that the control of inflammatory signaling after central or peripheral injury may indirectly affect the endogenous repair mechanisms, resulting in a greater synaptic preservation and better functional recovery. In this sense, animals were submitted to both central and peripheral lesions in order to investigate the effects of glial cells on neuron survival, axonal regeneration and synaptic plasticity. The results showed that, after lesion, the modulation of inflammatory signaling by cytokines or knocking down molecules on glial surface, directly or indirectly influence the stability of neural circuits, neuronal survival and axonal regeneration. Thus, we believe that this is important findings that may be critical to the development of new therapeutic strategies following nervous system injury / Doutorado / Clinica Medica / Doutora em Ciências

INTRODUÇÃO: O Sarcoma de Kaposi (SK) é a neoplasia mais frequente dos doentes com Aids. É causada pelo herpes-vírus 8 (HHV-8). As células dendríticas plasmocitóides (CDp) são especializadas na produção de interferon tipo 1 e participam da resposta imune aos vírus. Os receptores \"toll-like\" são os principais receptores de reconhecimento de padrão, sendo que os receptores toll-like (TLR) 3 e 9 têm função no reconhecimento de vírus. O D2-40 é o anticorpo que reconhece a podoplanina, uma proteína transmembrana, presente no endotélio linfático e que tem função na imunidade. OBJETIVO: Demonstrar e comparar os componentes da imunidade inata: CDp e TLR 3 e 9, nas lesões cutâneas de SK associado a Aids e esporádico. Identificar a presença do HHV-8 nas CDp. Verificar o componente endotelial linfático na progressão das lesões de SK e comparar a expressão dos elementos da imunidade inata estudados, nas lesões com menor e maior componente endotelial linfático. MÉTODOS: Estudo retrospectivo de 50 biopsias de pacientes com diagnóstico de SK, todos com comprovação pelo exame histopatológico e demonstração do antígeno nuclear associado à latência (LANA) do HHV-8. Foram avaliados 11 biopsias de SK da forma clássica (SKc), 22 lesões de doentes com Aids (SK-Aids) e de 17 de doentes com Aids submetidos a tratamento com terapia antirretroviral altamente eficaz (SK-Aids/HAART). Os espécimes foram submetidos a exame por técnica imuno-histoquímica para evidenciar a presença de CDp (anticorpo CD303/BDCA-2), a expressão de TLR 3 e 9, bem como de podoplanina (anticorpo D2-40). Foi realizada também técnica de dupla marcação com CD303 e LANA, objetivando a identificação de CDp infectadas pelo HHV-8.Vinte e três espécimes de granuloma piogênico constituíram o grupo controle. A população de CDp e expressão de TLR 3 e TLR 9 também foi comparada nas lesões cutâneas de SK de doentes com e sem comprometimento visceral pela neoplasia; lesões não tumorais (máculo-papulares/placas) foram comparadas às lesões tumorais (nodulares) e de acordo com níveis sanguíneos de linfócitos T CD4+ (menor e igual ou maior que 350 células/mm3). RESULTADOS: As CDp foram mais numerosas nos espécimes de SK-Aids quando comparado com o granuloma piogênico. Foram identificadas CDp infectadas pelo HHV-8. A expressão de TLR 3 foi menor nas lesões de SK, independente da forma epidemiológica, do que no granuloma piogênico. Para todas as outras comparações da densidade de CDp e expressão de TLR 3 e de TLR 9 não houve diferença entre os grupos. Não houve diferença no componente endotelial linfático das lesões máculo-papulares/placas e tumorais do SK, assim como na expressão dos elementos da imunidade inata estudados entre as lesões com maior e menor componente endotelial linfático. CONCLUSÕES: Demonstrou-se pela primeira vez a presença de CDp e a expressão de TLR 3 e 9 em lesões cutâneas do Sarcoma de Kaposi, bem como a infecção de CDp pelo HHV-8 \"in situ\" nos tumores. Os resultados obtidos sugerem a participação das células CDp e do TLR 3 na patogênese das lesões cutâneas do Sarcoma de Kaposi, independente da presença do vírus da imunodeficiência humana. A imunomarcação de SK com o anticorpo D2-40, tanto nas fases precoce como tardia das lesões, confirma a natureza endotelial linfática das células neoplásicas. Esta parece não ter relação com a expressão dos elementos da imunidade inata estudados / Introduction: Kaposi\'s sarcoma (KS) is the most common Aids-associated malignancy. It is caused by human herpesvirus-8. Plasmacytoid dendritic cells (pDC) are professional interferon producing cells, and participate in the immune response against viruses. Toll-like receptors (TLR) are the main pattern recognition receptors, and TLR 3 and TLR 9 participate in the recognition of viruses. Podoplanin, recognized by antibody D2-40, is a transmembrane protein identified on lymphatic endothelial cells with functions inimmunity. Objective: Demonstrate and compare some innate immunity components: pDC, TLR 3 and TLR 9, in cutaneous lesions of Aids-associated Kaposi\'s sarcoma and classic Kaposi\'s sarcoma. Identify the infection of pDC by HHV-8. Compare the lymphatic endothelial component in the course of tumor progression and compare the expression of innate immunity elements in lesions with a predominance of lymphatic endothelial components or not. Methods: Retrospective study of 50 biopsies diagnosed as Kaposi\'s sarcoma withpositive staining for latency-associated nuclear antigen (LANA) of HHV-8. Eleven classic KS, 22 Aids-associated KS and 17 Aids-associated KS from patients undergoing highly active antiretroviral therapy (HAART) were assessed. Paraffinembedded tissue was submitted to immunohistochemistry technique in order to demonstrate pDC (CD303/BDCA-2 antibody), expression of TLR 3, TLR 9 and podoplanin (D2-40 antibody). We performed double staining with CD303 and LANA in order to identify pDC infection with HHV-8. Twenty-three pyogenic granuloma(PG) specimens were analyzed as a control group. Plasmacytoid dendritic cells population, TLR 3 and TLR 9 expressions were compared between patients with and without visceral disease, nodular stageandpatch/plaque stage and according to bloodlymphocytes T CD4 count(=350 cells/mm3). Results: Plasmacytoid dendritic cells density in Aids-associated SK was higher than in PG. We could identify pDC infection by HHV-8. The expression of TLR 3 in all forms of KS was less extensive than PG. All others comparisons about pDC density, TLR 3 and 9expressions were similar. We found no difference in D2-40 expression between nodular and patch/plaque stages. When comparing tumors with extensive expression of D2-40 (>= 50% of cells) and tumors with less expression (<50% of cells), we found no differences in density of pDC and expression of TLR 3 and TLR 9. Conclusion: This is the first time that pDC, TLR 3 and TLR 9 have been demonstrated in skin lesions of KS, as well as the infection of pDC in the lesions. Our results suggest that pDC and TLR 3 participate in the pathogenesis of KS, independently of HIV presence. The positive staining with D2-40 antibody, in all the stages of KS, confirmsthe lymphatic nature of neoplastic cells. It seems that podoplanin is not related to the innate immunity elements studied here

Memoria para optar al título de Bioquímico / Durante la inflamación de las mucosas, las células epiteliales y los macrófagos expresan el receptor de tipo Toll 4 (TLR4) y secretan citoquinas, las que potencian el cuadro. Sin embargo, hasta ahora existe escasa información acerca de la regulación de la expresión de TLR4 por la combinación de moléculas pro-inflamatorias y antiinflamatorias. Utilizando la línea celular de epitelio pulmonar, las células A549, se evaluó la expresión del TLR4 a nivel de transcrito, proteína y actividad transcripcional de mutantes con deleciones en la región promotora del receptor, posterior a la exposición al factor de necrosis tumoral á (TNFα) en presencia o ausencia del glucocorticoide sintético Dexametasona (Dex), mediante PCR en tiempo real, inmunoblot o citometría de flujo, y ensayos del gen reportero luciferasa, respectivamente. El aumento de la expresión de TLR4 inducido por TNFα, tanto a nivel del mRNA como de la proteína, es revertido por la adición de Dex. La regulación de la actividad transcripcional del promotor de TLR4 por estas moléculas fue similar a la observada a nivel de proteína, y el efecto inhibitorio de Dex estaría comandado por al menos un elemento de respuesta del receptor de glucocorticoides, GR (GRE). En conjunto, estos resultados demuestran un efecto antagónico de Dexametasona sobre la expresión de TLR4 inducida por TNFα, aparentemente por un mecanismo que involucra al menos la regulación de GR a nivel del promotor de TLR4. Se evidenció claramente en estas células los fuertes efectos antagónicos que ejercen sobre la expresión de TLR4, moléculas pro-inflamatorias como es TNFα y antiinflamatorias como los glucocorticoides

Background: In the last two decades the discovery of the Toll-Like Receptor (TLR) family in humans revealed the importance of innate immunity in providing host defence against invading pathogens. Moreover, TLRs have a vital role in mediating the interactions between the reproductive and immune systems. Similar to TLRs, (NOD)-like receptors (NLRs); retinoic acid-inducible gene-1, RIG-1-like receptors (RLRs) are also important pattern recognition receptors in the female reproductive system. Successful implantation requires effective reciprocal interactions between receptive endometrium and competent embryo. The endometrial innate immunity during implantation has been intensively investigated. However, little is known about the expression of innate immunity during the early stages of human embryo development. Objective: To investigate the expression of innate immunity molecules during the early stages of human embryo development and to determine the functions of TLRs in blastocysts. Material and methods: The expression of TLRs, a panel of downstream signalling molecules, NLRs, RLRs, inflammatory cytokines, chemokines and the hyaluronic acid system were investigated in the following developmental stages: oocyte, 4- cell, blastomeres, 8- cell, blastocyst, inner cell mass and trophectoderm using Affymetrix Human Genome U133 Plus 2.0 arrays. Microarray data were validated by Q-PCR. TLR function in human blastocysts was investigated by treating day five blastocysts with TLR3 or TLR5 specific ligands; Poly (I:C) and flagellin respectively, for 24 hours. The culture media was analysed for elevated cytokine and chemokine levels using cytometric bead array. Results: TLRs, NLRs, RLRs, TLR downstream signalling molecules, cytokines and chemokines involved during implantation event and the hyaluronic system were all found to be positively expressed in the early stages of human embryo development. The expression levels of the above molecules were generally moderate to low (CT 24-34) and varied across the embryonic developmental stages. Stimulation of TLR3 and TLR5 in day 5 blastocysts produced cytokines and chemokines. In addition, there were alterations in gene expression patterns in the Poly (I:C) and flagellin treated blastocysts in comparison to the untreated blastocysts. Conclusion: The varied expression levels of the investigated molecules involved in early embryonic developmental suggests a potential role for these molecules in early pregnancy loss and implantation failure. Specifically, the relationship between the level of TLR expression, function and embryo quality is worth investigating in the future as a potential marker for embryo competence.

This thesis examines the impact of the real exchange rate on the amount of revenues from a system of tolled and time tolled roads in the Czech Republic in the years 1999-2014. This effect is investigated on monthly data using regression analysis on two systems of tolled roads separately, system of tolled roads and system of time-tolled roads. Based on available dataset was shown a statistically significant negative effect of the real exchange rate on toll revenues in the system of tolled roads. In the case of time-tolled roads system was demonstrated negative effect of real exchange rate on time-toll revenues, but this effect is questionable, since the influence has been demonstrated only in a model with limited explanatory power.

The Master's Thesis deals with the toll system in the Federal Republic of Germany. The theoretical part of the thesis describes technologies that are used in electronic fee collection systems, satellite navigation systems and provides a snapshot of German road infrastructure. The main part of the work deals with German toll system that is provided by Toll Collect GmbH. Various aspects of toll payment via fuel cards are discussed in the practical part that also includes an alternative payment option. The study case assesses whether hauliers should or should not try to avoid using toll roads.

taxes on vehicles in Czech republic. criterions of efficiency are - technology of toll collect system, analysis of avoiding toll system, income and cost analysis

Electronic Fee Collection (EFC) is spread almost all over the Europe. Its main duty is to collect fees by on board units. The system of electronic toll does not need to be inevitably only a tool for fee collection. The combination of DSRC and DNSS systems might bring new horizons in terms of telematic data usable for wide variety of services. This diploma thesis in its theoretical part deals with electronic toll systems in general and shows how the system works in chosen countries of Europe. Special focus is on Czech toll system, its implementation and evaluation of its economic contributions year after year. From this point of view, the Czech system works fully. In practical part, the focus is on widening of the system functionality. At this moment, the on board units works only as a tool for collecting fees. However, thanks to combination of microwave and satellite system, the on board unit could become also a very intelligent tool for collecting of telematic data.