A galectina-1 (Gal-1) é uma lectina homodimérica multifuncional capaz de reconhecer e se ligar a beta-galactosídeos por meio de um domínio denominado carbohydrate recognition domain (CRD). A Gal-1 humana (Gal-1h) e a Gal-1 de camundongo (Gal-1c) mantêm 88,15% de homologia e, apesar de não existirem mutações em aminoácidos-chave do CRD, há substituições próximas a esses resíduos. Considerando as implicações dessas diferenças em estrutura e função, e que é comum a utilização de modelos murinos para estudar a função Gal-1, o presente trabalho objetiva analisar comparativamente a Gal-1c e a Gal-1h por meio de ensaios de cristalização e determinação estrutural da Gal-1c, além da avaliação comparativa da atividade lectínica da Gal-1h e da Gal-1c por glycan array e hemaglutinação. Também foi avaliada a capacidade de ambas as Gal-1 em induzir a exposição de fosfatidilserina (FS) em neutrófilos ativados provenientes de medula de camundongos normais ou deficientes de ?-2 integrina (Mac-1), de modo a investigar se a interação Gal-1/Mac-1 estaria envolvida nesse processo. Preparações homogêneas e ativas de Gal-1c e Gal-1h foram utilizadas nos ensaios. Os cristais de Gal-1c foram obtidos em 20% de polietilenoglicol 3350 e 0,2 M de fluoreto de amônio. Os dados de difração de raios X foram coletados e processados, obtendo-se uma estrutura com resolução de 2,4 Å. Observou-se que substituições de aminoácidos entre a Gal-1c e a Gal-1h estão localizadas em regiões expostas ao solvente, próximas do CRD e distantes da interface de dimerização. A análise comparativa entre Gal-1c e Gal-1h mostrou que estas substituições conferem a Gal-1c um caráter mais polar, com consequente aumento da distribuição de volume molecular. Nos ensaios de hemaglutinação, pode-se observar que é necessária uma concentração 2 vezes maior de Gal-1c para aglutinar eritrócitos humanos, de carneiro e de coelho na mesma proporção que a Gal-1h. Por meio do glycan array, pode-se determinar o perfil de ligação a glicanas de ambas as Gal-1. As duas Gal-1 apresentam afinidade por glicanas ramificadas contendo galactose terminal, e a Gal-1h apresentou maior intensidade de ligação às glicanas quando comparada à Gal-1c. Preparações de Gal-1c e Gal-1h induzem níveis semelhantes de exposição de FS na superfície de neutrófilos deficientes ou não de Mac-1, sugerindo que a interação Gal-1/Mac-1 não esteja envolvida no processo de exposição de FS na superfície de neutrófilos ativados. Assim, a diferença sequencial entre a Gal-1c e a Gal-1h é capaz de gerar diferenças estruturais consideráveis que implicam no reconhecimento diferencial de glicanas, o que, entretanto, não se reflete na capacidade de indução de FS na superfície de neutrófilos ativados. Além disso, a interação Gal-1/Mac-1 parece não participar desse processo, o que pode indicar que o papel da Gal-1 no turnover de neutrófilos, via reconhecimento fagocítico, seja um processo complexo e independente dessa interação. / Galectin-1 (Gal-1) is a homodimeric and multifunctional lectin that recognizes and binds to beta-galactoside by a carbohydrate recognition domain (CRD). Human Gal-1 (hGal-1) and mouse Gal-1 (mGal-1) are 88.15% identical, and although there are no mutations in key amino acids within the CRD, there are differences in the amino acids sequence near the CRD. Given the potential of these differences to alter overall structure and function, and the common utilization of murine models to study Gal-1 function, we sought to directly compare key biochemical features of hGal and mGal-1. Thus, we performed crystallization and structure determination assays of mGal-1, and determined the carbohydrate binding specificy of mGal-1 and hGal-1 using a glycan array and using hemagglutination assay. We also evaluated the ability of both Gal-1 to induce exposure of phosphatidylserine (PS) in activated neutrophils from the bone marrow of normal or ?-2 integrin (Mac-1) deficient mice, in order to investigate the involvement of Gal-1/Mac-1 interaction in this process. To accomplish this, homogeneous and active preparations of hGal-1 and mGal-1 were used in the study. mGal-1 crystals were obtained in 20% polyethylene glycol 3350 and 0.2 M ammonium fluoride. Data from X-ray diffraction were collected and processed, yielding a structure with a final resolution of 2.4 Å. The amino acid substitutions found between mGal-1 and hGaI-1 are detected on the solvent-exposed surfaces where the CRDs are located and not on the proteins dimerization surfaces. A comparative structural analysis between mGal-1 and hGal-1 shows that these amino acid substitutions confer to mGal-1 a greater number of ionizable residues, polar character, appearance of the acid regions clustered, and a slight increase of volume distribution. In hemagglutination assays, twice the concentration of mGal-1 was required to cause equivalent agglutination of human, sheep or rabbit erythrocytes as hGal-1. Glycan array analysis demonstrated that both galectins have affinity for branched glycans containing terminal galactose residues. However, hGal-1 appeared to display higher levels of binding that mGal-1. Preparations of mGal-1 and hGal-1 induced similar levels of PS exposure on normal or Mac-1 deficient neutrophils, suggesting that the interaction Gal-1/Mac-1 is not involved in this process. Thus, hGal-1 and mGal-1 appear to possess considerable differences in glycan recognition that likely reflects subtle difference in amino acid sequence. Furthermore, the interaction Gal-1/Mac-1 do not appear to participate in this PS exposure process, which suggest that other Gal-1 receptors are likely important in this process.

O mecanismo responsável pela exposição da fosfatidilserina (PS) nas membranas celulares não está bem definido. Uma atividade dependente de ATP está envolvida, provavelmente uma ATPase tipo-P. ATPases tipo P são uma família de proteínas transmembranares envolvidas no transporte de metais, íons e fosfolipídios através da membrana plasmática. As P4 ATPases translocam aminofosfolipidios (APTLs) como a PS durante a apoptose. No entanto, o sentido do transporte de PS pela APLT não está claramente definido. Os macrófagos reconhecem a PS exposta na superfície das células apoptóticas, o que inibe sua capacidade microbicida. Formas promastigotas e amastigotas de Leishmania ssp. sofrem apoptose, porém a exposição de PS na superfície dos promastigotas sempre leva à morte, enquanto que nos amastigotas não está necessariamente associada à morte e permite a internalização desses protozoários e sua sobrevivência no macrófago. Esse trabalho teve como objetivo a caracterização molecular da APLT de L. (L.) amazonensis e a avaliação de seu papel na exposição de PS nesse parasita. / The mechanism responsible for phosphatidylserine (PS) exposure in biological membranes is still an open subject. An ATP-dependent activity is involved, probably a Type P- ATPase. Type P ATPases are a family of transmembrane proteins involved in the transport of metals, ions and phospholipids across plasma membrane. P4 ATPases mediate phospholipid transport (APLT) as PS during the process of cell death by apoptosis. However, the direction (inwards or outwards) of this translocation has not been defined. Macrophages recognize exposed PS on the surface of apoptotic cells, what inhibits their microbicidal capacity. Promastigotes and amastigotes of Leishmania ssp. die by apoptosis, but PS exposure on promastigotes always leads to apoptosis, whereas PS exposure by amastigotes is not necessarily associated to death and allows their internalization and survival in the macrophage. This work aimed to characterize APLT from L. (L.) amazonensis and to evaluate its role in PS exposure in this parasite.

Promastigotas de Leishmania são encontradas no trato digestivo do inseto vetor e podem ser cultivados em alguns meios de cultura, onde ocorre a diferenciação regulada geneticamente entre procíclicos e metacíclicos infectivos. Amastigotas são intracelulares e irão se estabelecer e se multiplicar dentro dos vacúolos parasitóforos dos macrófagos. Os metacíclicos e os amastigotas possuem capacidade de evasão do sistema de defesa do hospedeiro vertebrado e conseguem estabelecer uma relação parasita/hospedeiro bem-sucedida. A apoptose, em organismos multicelulares, é uma forma controlada geneticamente de suicídio celular. A eliminação das células apoptóticas é feita pelos fagócitos sem que seja induzida uma resposta inflamatória. Em Leishmania, os eventos da apoptose parecem seguir os mesmos dos encontrados em metazoários, tais como: queda do potencial da mitocôndria, ativação de caspases, clivagem de substratos naturais de caspases, além da externalização de fosfatidilserina (PS) e seu papel na modulação da fagocitose. Nossos dados demonstraram que Leishmania é capaz de mimetizar e utilizar um dos fenômenos observados na apoptose em metazoários, a exposição de PS, como um mecanismo adaptativo para se estabelecer como um parasita de mamíferos. / Promastigotes of Leishmania are found in the midgut environment of the vector insect and can be grown in some culture medium. In culture the genetically regulated differentiation from procyclic to infective metacyclic can be observed. Amastigotes are intracellular parasites and which multiply inside phagolysosomes of macrophages. Metacyclics and amastigotes are endowed with the capacity to evade from the innate and adaptive immune system of the vertebrate host and to establish successful infections. Apoptosis, in multicellular organisms, is a form genetically controlled cellular suicide. The elimination of apoptotic cells is made by phagocytes without initiating an inflammatory event. In Leishmania, the events of apoptosis are similar to those observed in multicellular organisms such as: decrease in mitochondrial transmembrane potential, caspase activation, cleavage of caspase substrates besides exposure of phosphatidylserine (PS) and its role in the modulation of phagocytosis. Our data showed that Leishmania is capable of mimicking one of the features of apoptosis, mainly PS exposure, and to use as an adaptive mechanism for survival in mammalian hosts.

No description available.

<p>  </p> <p>Ebola virus (EBOV) infection remains to be a challenge to human health by its high mortality rate. Though it has been discovered for almost 50 years, there are only two antibody-based therapies approved today, and the mortality rate is still greater than 30% with the treatment. Authentic EBOV studies are strictly limited to biosafety level-4 (BSL-4) labs, which slows the development of treatment. While more simple and safer systems have been developed to understand different stages of EBOV infection, such as the matrix protein (VP40) virus-like particle (VLP) and minigenome systems, we still lack a systematical view of EBOV infection. On the other hand, mathematical modeling has been used to assist biological and medical studies for many years, as it has the advantage of integrating data and providing quantitative insight to a biosystem. In our study, we took advantage of mathematical modeling and build the primary ordinary differential equation-based (ODE-based) model of EBOV at subcellular level step by step. We built the budding pathway of EBOV VP40 first, calibrated and validated our model with experimental data. We proposed that phosphatidylserine (PS) can directly influence the stability of VP40 filaments and the budding process of VLPs. Also, the oligomerization of VP40 filaments may follow the nucleation-elongation process. Next, we conducted in-silico simulation to evaluate the treatment efficiency of fendiline, a drug lowering cell membrane PS level, in treating EBOV. We found that while in general, fendiline can decrease VLP production, there can be fendiline-induced VLP production increases at certain time points due to slow filament growth or fast VLP budding rates. Also, we concluded that fendiline is relatively more effective when applied in the budding stage of EBOV life cycle. Moreover, fendiline efficacy may increase when applied with a VLP budding step targeted treatment. Finally, we integrated nucleoprotein (NP) into our model. We reproduced the two-stage interaction between NP and VP40 and predict that NP increases VLP production through influencing filament oligomerization and VLP budding steps. Also, the dual-effect of NP on VLP production may exist, as a too high NP/VP40 production ratio can decrease VLP production. From the aspect of protein expression time, we found that a bit earlier NP production than VP40 production is beneficial for both inclusion body-containing (IB-containing) VLP production and prevention in energy waste on production of VLPs without IBs. Overall, we have built a solid foundation towards a mathematical EBOV model and demonstrated the value of models in assisting experimental EBOV studies.</p>

No description available.

No description available.

In großen Teilen der Welt verursachen intrazelluläre Parasiten der Spezies Leishmania schwerwiegende Infektionen beim Menschen. Die Exposition eines Phospholipids (Phosphatidylserin, PS) steht unter Verdacht Fresszellen zur Aufnahme der Parasiten zu stimulieren. Bisher ist die Regulation der Phospholipidverteilung in der Plasmamembran dieser Parasiten kaum erforscht. In der vorliegenden Arbeit wurde ein lipidtransportierender Proteinkomplex identifiziert, der einen wesentlichen Beitrag zur asymmetrischen Lipidverteilung in der Plasmamembran von Leishmania donovani leistet. Die Zerstörung des Komplexes führte zum Verlust des einwärts gerichteten Lipidtransports und zur Anreicherung von Phosphatidylethanolamin (PE) auf der Zelloberfläche des Parasiten. Diese veränderte Lipidasymmetrie hatte jedoch keinen Einfluss auf die Phagozytose durch Makrophagen. Darüber hinaus brachte die Untersuchung des Insektenstadiums (Promastigote) verschiedener Leishmania Spezies zu Tage, dass die Menge an PS unterhalb des Detektionslimits modernster Nachweisverfahren liegt. Des Weiteren konnte gezeigt werden, dass der Parasit über einen Scramblase-Mechanismus verfügt, der durch intrazelluläres Kalzium stimulierbar ist. Die Scramblase-Aktivität ist, im Gegensatz zu dem zuvor beschriebenen einwärts gerichteten Lipidtransport, energieunabhängig und ermöglicht die bidirektionale Translokation von fluoreszenzmarkiertem Phosphatidylcholin (PC), PE, PS und Sphingomyelin (SM). Dementsprechend konnte nach Kalziumstimulierung endogenes PE auch in der äußeren Lipidschicht der Plasmamembran detektiert werden, wobei deren Barrierefunktion nicht beeinträchtigt wurde. Diese Ergebnisse geben neue Einblicke in die dynamische Regulation der Lipidverteilung über die Plasmamembran des Parasiten und verdeutlichen, dass die Exposition von PS und PE nicht essentiell für das Eindringen der Leishmanien in die Wirtszellen ist. / The protozoan parasite Leishmania causes severe infections in humans throughout the world. Following the transmission via sand flies to its mammalian host the extracellular parasite has to gain entry into phagocytic cells to initiate a successful infection. Specific surface exposed phospholipids have been implicated in Leishmania macrophage-interaction, but the mecha-nisms controlling and regulating the plasma membrane lipid distribution remains to be eluci-dated. In the present work a lipid transporting protein complex was identified in Leishmania dono-vani which plays an essential role in maintaining an asymmetric lipid distribution across the plasma membrane. Loss of the protein complex abolishes the inward-directed lipid transport and thus e.g. to an increased cell surface exposure of phosphatidylethanolamine (PE). In spite of this altered lipid asymmetry the uptake by macrophages is unaffected. Moreover, Leishma-nia promastigotes of different species lack detectable amounts of phosphatidylserine (PS) although being infective. Furthermore, a scramblase activity following a cytosolic calcium signal was demonstrated. This scramblase mechanism facilitated, in contrast to the previous described inward directed lipid transport, the bidirectional movement of fluorescent lipid analogues of PC, PE, PS and SM in an energy-independent manner. In accordance with these findings endogenous PE was exposed to the outer plasma membrane leaflet following the Ca2+-signal, while the plasma membrane itself remained intact. These results provide novel insight into the dynamic regulation of the transbilayer lipid distri-bution across the parasite plasma membrane and reveal that exposure of PS and PE is not cru-cial for invasion of the host cell by Leishmania donovani promastigotes.

Made available in DSpace on 2017-07-24T19:22:04Z (GMT). No. of bitstreams: 1 Leticia Antonelo Campos.pdf: 2246502 bytes, checksum: 0d9288acf39d871d8aedcd4d1fc4e5ca (MD5) Previous issue date: 2014-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The biofilm accumulation in the subgingival region produces an inflammatory process that can lead to alveolar bone resorption and periodontal attached loss through due inflammatory mediators that induce the production and activation of enzymes and periodontal degradation tissues. Phosphatidylserine liposomes can mimic apoptotic cells effects and may modulate the host inflammatory response. This present study evaluated the effect of phosphatidylserine liposomes on the inflammatory response and alveolar bone loss ligatureinduced in rats. We used a phosphatidylserine liposome solution preparation in order to evaluate the cytotoxicity in lineage osteoblastic cells. The antiinflammatory effect was evaluated by mouse ear edema (topical application) and rat paw edema (systemic application) methods. Then, we evaluated the phosphatidylserine liposome action on gingival inflammatory response and alveolar bone loss ligature-induced in rats. The results showed phosphatidylserine liposome 50 and 250 μM concentrations were safer considering cell viability and proliferation. The phosphatidylserine liposome gel (topical application) had no anti-inflammatory action on mouse ear edema model and alveolar bone loss ligature-induced in rats. The phosphatidylserine liposome solution (systemic application) has anti-inflammatory action on rat paw edema. Although, we did not observe an effect on alveolar bone loss ligatureinduced. We conclude phosphatidylserine liposome gel and solution are easy to prepare and they have low cost. Phosphatidylserine liposoma solution (systemic application) have antiinflammatory effect on rat paw edema model. On the other hand, it have no effect on alveolar bone loss ligature-induced in rat. / O acúmulo de biofilme na região subgengival produz um processo inflamatório que pode conduzir à reabsorção óssea e perda de inserção, devido mediadores inflamatórios que induzem a produção e ativação de enzimas e degradação dos tecidos periodontais. Lipossomas de fosfatidilserina mimetizam o efeito de células apoptóticas podendo modular a resposta inflamatória do hospedeiro. Este estudo avaliou o efeito do tratamento com lipossomas de fosfatidilserina sobre a reposta inflamatória e perda óssea alveolar induzida por ligadura em ratos. Utilizamos uma preparação de solução de lipossomas de fosfatidilserina avaliando a citotoxicidade em uma linhagem de células osteoblásticas. Avaliamos a ação anti-inflamatória por meio dos métodos de edema de orelha em camundongos (aplicação tópica) e edema de pata em ratos (aplicação sistêmica). Em seguida, avaliamos a ação de lipossomas de fosfatidilserinana na resposta inflamatória gengival e perda óssea alveolar induzida por ligadura em ratos. Os resultados mostraram que as concentrações de 50 e 250 μM dos lipossomas de fosfatidilserina foram seguras, considerando a viabilidade e proliferação celular. O gel lipossomal de fosfatidilserina (aplicação tópica) não teve ação anti-inflamatória no modelo de edema de orelha em camundongo nem na perda óssea induzida por ligadura em ratos. A solução de lipossomas de fosfatidilserina (aplicação sistêmica) teve ação anti-inflamatória no modelo de edema de pata em ratos. Porém, não observamos efeito na perda óssea induzida por ligadura. Pode-se concluir que o gel e a solução de lipossomas de fosfatidilserina são de fácil preparo e de baixo custo. Lipossomas de fosfatidilserina em solução (aplicação sistêmica) possui efeito anti-inflamatório no modelo edema de pata em ratos. Por outro lado, não interferiu na perda óssea alveolar induzida por ligadura em ratos.

Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates.