Biblioteca Digital

AIMS: The relationship between the activity of eosinophils and platelets has been observed in recent decades by many scientists. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. MAIN METHODS: Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. KEY FINDINGS: Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with l-Nitro-Arginine-Methyl-Ester (l-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP...

Introdução: Estudos iniciais sugeriram que o risco para tromboembolismo venoso (TV) era atribuído ao componente estrogênico dos contraceptivos de forma dose-dependente. Estudos epidemiológicos têm sugerido que o risco para TV é maior com contraceptivos combinados que contêm progestagênios de terceira geração (gestodeno, desogestrel) comparados com aqueles com progestagênios de segunda geração (levonorgestrel). Esses achados inesperados têm sido alvo de muitos debates sem uma explicação definitiva. Assim, a questão das diferenças nas propriedades de cada progestagênio sobre a hemostasia tem sido levantada. Apesar dos progestagênios não serem associados a alterações marcantes nos parâmetros hemostáticos, existem poucos estudos sobre os efeitos dessas drogas, especialmente os progestagênios de terceira geração, no sistema hemostático. Objetivo: Avaliar o efeito do implante subdérmico de etonogestrel sobre a agregação plaquetária de mulheres hígidas, em seis meses de tratamento. Casuística e Métodos: Vinte e quatro mulheres saudáveis e voluntárias foram selecionadas neste estudo longitudinal e prospectivo, para usar um implante contraceptivo subdérmico de etonogestrel (metabólito biologicamente ativo do desogestrel). A agregação plaquetária foi avaliada em todas as mulheres...

Esse trabalho apresenta um estudo experimental de agregação da Acridina Laranja (AL) em solução aquosa homogênea e na sua interação com o surfactante Sodium Dodecyl Sulfate (SDS). Este estudo foi realizado com o objetivo de definir estruturas e características temporais de formação de agregados de compostos orgânicos com sistema desenvolvido de conjugação-, usados como fotossensibilizadores e marcadores de estruturas biológicas, em sua interação com nanoestruturas heterogêneas. A informação sobre as características de agregação de fotossensibilizadores é importante devido à influência deste processo na eficácia de suas aplicações práticas, em particular na biologia e medicina. A AL foi escolhida como modelo devido aos seguintes motivos: AL é um composto bem conhecido e várias de suas características já estão bem estabelecidas na literatura; Possui alto rendimento quântico de fluorescência, alta fotoatividade, alta afinidade com estruturas biológicas (membrana celular, DNA e RNA, em particular), por isso a AL é amplamente usada como marcador fluorescente e fotossensibilizador em fototerapia; AL possui a tendência de formar agregados em solução aquosa; A agregação da AL modifica seus espectros de absorção e de fluorescência...

This study investigates the thrombocyte aggregation process in the South American fresh water turtle (Phrynopys hilarii) using electron microscopy. Blood was taken from surgically exposed lateral neck vessels often turtles Phrynopys hilarii during the spring and summer seasons, when the mean temperature is 37°C. Blood samples were fixed with Karnovsky solution for processing by transmission electron microscopy. The turtle thrombocytes were spindle-shaped with lobulated nuclei. Prominent vesicles and canaliculi were found throughout the cytoplasm. The cytoplasm organelles showed an agranular endoplasmatic reticulum, Golgi complex near the centrioles and scattered free ribosomes. These cells are similar to bird thrombocytes but distinct from fish and frog thrombocytes. Blood clotting time was 5 min ± 30 sec measured by the Lee and White method. Structural alterations resulting from the aggregation process occurred after activation. Thrombocytes developed numerous filopodial projections, an increased number of vacuoles and changed from spindle to spherical shape. P. hilarii thrombocytes have different morphologic characteristics compared to other non-mammalian vertebrate cells. These cells can participate in the aggregation process...

A sepse e ainda causa de muitos óbitos em hospitais do mundo todo. A gravidade da sepse esta relacionada ao estado de ativação de plaquetas. Trabalho prévio do nosso grupo mostrou que o tratamento de ratos com lipopolissacarideo (LPS) leva a inibição da agregação plaquetaria e aumento da formação de espécies reativas de oxigênio (EROs) via NADPH oxidase. Entretanto, o efeito inibitório do LPS na agregação não e dependente da liberação de EROs. Portanto, o objetivo do presente trabalho foi investigar as vias de sinalização envolvidas na inibição da agregação e na ativação da NADPH oxidase em plaquetas de ratos tratados com LPS. Para tanto, ratos Wistar machos foram injetados com LPS (1 mg/kg, i.p.) e apos 6h ou 48h o sangue foi coletado. A agregação plaquetaria foi induzida por ADP (10 μM) na ausência e na presença de diferentes inibidores enzimáticos. A formação de EROs em plaquetas foi determinada por citometria de fluxo utilizando a sonda fluorescente DCFH-DA e a concentração intraplaquetaria de GMPc por imunoensaio. Também foram realizados ensaios de western blotting para a analise da ativação das enzimas c-Src, AKT e NADPH oxidase, bem como para a detecção de proteínas contendo resíduos de nitrotirosina. A analise do western blotting mostrou que a fosforização da c-Src quinase no resíduo Tyr 416...

Os eosinófilos participam de processos inflamatórios e alérgicos. Estando relacionados com o sistema de imunidade inata do organismo, eles representam uma linha fundamental de defesa contra invasão microbiana e, quando ativados, produzem uma série de mediadores solúveis que atuam nas respostas inflamatórias e alérgicas. A relação entre a atividade dos eosinófilos e plaquetas foi observada nas últimas décadas por muitos cientistas. Estas observações incluem o aumento do número de eosinófilos associados a desordens plaquetárias, incluindo alterações na cascata de coagulação e agregação plaquetária. Com base nessas observações, a interação entre os eosinófilos e plaquetas foram analisadas na agregação plaquetária. Plaquetas humanas foram incubadas com a fração citosólica de eosinófilos, linhagem celular promielocítica humana HL-60 clone 15 e proteína catiônica do eosinófilo (ECP). A agregação em plasma rico em plaquetas (PRP) foi induzida por difosfato de adenosina, fator de ativação plaquetária, ácido araquidônico e colágeno, e as plaquetas lavadas (PL) foram ativadas por trombina. A agregação induzida por todos os agonistas foi inibida de maneira concentração de células dependente pela fração citosólica de eosinófilos. Esta inibição foi apenas parcialmente revertida pela prévia incubação dos eosinófilos com L-Nitro-Arginina-metil-éster (L-NAME). A prévia incubação com indometacina não impediu a inibição induzida pela fração citosólica. A separação da fração citosólica de eosinófilos por gel filtração em Sephadex G-75 mostrou que a atividade inibitória foi concentrada na fração de peso molecular mais baixo. As células HL -60 clone 15 diferenciadas em eosinófilos por 5 e 7 dias foram capazes de inibir a agregação plaquetária. A proteína de ECP inibiu a agregação plaquetária em PRP e PL. Esta inibição foi mais evidente em PL...

As plaquetas são importantes células na inflamação, entretanto, os trabalhos que estudam as citocinas na reatividade plaquetária são raros. O objetivo do presente trabalho foi estudar os efeitos do fator de necrose tumoral-alfa (TNF-α) em plaquetas. Ensaios de agregação foram realizados incubando-se plaquetas com crescentes concentrações de TNF-α (1 – 3000 pg/ml) por diferentes intervalos de tempo (5 – 60 min), na ausência ou presença do antagonista não seletivo dos receptores TNFR1 e TNFR2, o R7050. Também foi estudado o efeito do TNF-α na viabilidade plaquetária utilizando-se o MTT. O efeito do TNF-α na mobilização de Ca2+ em plaquetas foi investigado através de ensaios de fluorescência utilizando-se o fluo-3-AM; os ensaios de western blotting foram realizados para o estudo da ativação da enzima c-Src e do receptor de fibrinogênio. Finalmente, foram determinados os níveis intraplaquetários de AMPc e GMPc por ELISA. O TNF-α inibiu a agregação plaquetária induzida por ADP ou trombina de forma dependente da concentração da citocina e do tempo de incubação. O efeito inibitório máximo do TNF-α na agregação induzida por ADP (5 μM) foi obtido com a concentração de 300 pg/ml por um tempo de incubação de 30 min (90 ± 7% de inibição)...

A thorough understanding of how proteins induce nanoparticle (NP) aggregation is crucial when designing in vitro and in vivo assays and interpreting experimental results. This knowledge is also crucial when developing nano-applications and formulation for drug delivery systems. In this study, we found that extraction of immunoglobulin G (IgG) from cow serum results in lower polystyrene NPs aggregation. Moreover, addition of isolated IgG or fibrinogen to fetal cow serum enhanced this aggregation, thus demonstrating that these factors are major drivers of NP aggregation in serum. Counter-intuitively, NP aggregation was inversely dependent on protein concentration; i.e., low protein concentrations induced large aggregates, whereas high protein concentrations induced small aggregates. Protein-induced NP aggregation and aggregate size were monitored by absorbance at 400 nm and dynamic light scattering, respectively. Here, we propose a mechanism behind the protein concentration dependent aggregation; this mechanism involves the effects of multiple protein interactions on the NP surface, surface area limitations, aggregation kinetics, and the influence of other serum proteins.

Conformational disorders such as Alzheimer’s, Parkinson’s, familial amyloidotic polyneuropathy and spongiform encephalopaties are a consequence of protein misfolding and aggregation predominantly in the form of amyloid fibrils. These pathologies represent a major health problem, which most probably will overwhelm the health systems of developed countries in the near future. Significant progress has been made recently to understanding the underlying mechanism of protein misfolding and aggregation. The current picture of protein aggregation is a phenomenon resulting from protein conformational fluctuations leading to misfolded intermediates prone to form non-native interactions with other intermediates, resulting in amyloid fibril formation. Fortunately just a small group of proteins are associated with human conformational disorders. The primary causes that lead this group of proteins to misfolding and aggregation are point mutations, protein over-expression and failure of protein quality-control system. Beside amyloid formation, there are other types of aggregation available to a misfold-disease-related polypeptide chain in the proteinfree energy landscape. Among them, native-like aggregation is becoming a widely studied topic of research.; This aggregation type...

JEL Classification: C22, C15; More than four decades have passed and the Sharpe Ratio (SR) continues to be one of the most popular portfolio risk adjusted performance measures. We comment on Lo’s (2002) results for the time aggregation of SR considering a different approach to deal with the conditional heteroskedasticity of returns. Based on a theorem proposed by Diebold (1986, 1988) we verify, for the series of financial returns with no serial correlation, that the most common method for time aggregation, the product of the higher-frequency SR by the square root of the number of periods contained in the lower-frequency holding period, can still be used in the presence of heteroskedasticity, when higher-frequency returns have been generated by a GARCH process and aggregated returns converge to the normal distribution. In an empirical application based on 65 investment funds, the convergence to normality is illustrated, showing that in 70% of the cases the convergence is held at least when daily returns are aggregated into annual frequency. Moreover, we show that serial correlation tends to disappear when the number of periods in the aggregation process tends to infinity and the most common method of SR time aggregation should not be disregarded as a valid method. The results are in accordance with Lo (2002) who roughly states that when serial correlation is not significant...

Triatomine bugs show a temporal modulation of many activities. Here, we analyse the daily modulation of the aggregation behaviour of Triatoma infestans larvae and its chronobiological basis. In the laboratory, groups of six bugs were released over an experimental arena during six consecutive days, where their aggregation behaviour was quantified every hour. When submitted to a 12/12 h photoperiod (L/D), the larvae of T. infestans exhibited a cyclic pattern of aggregation with a 24 h period, evincing the existence of a daily rhythm of aggregation in this species. Bugs exhibited the maximum aggregation tendency at the end of the scotophase (7:00 h), moment in which they naturally search for refuges. The minimum aggregation (i.e. maximal dispersion) was observed during the last part of the photophase and beginning of the scotophase (15:00 to 1:00 h). This cyclic pattern disappeared when constant conditions of illumination (L/L) or darkness (D/D) were imposed to the bugs, suggesting the absence of an endogenous circadian control of this behaviour. Insects submitted to L/L and D/D photoperiods presented lower global levels of aggregation than those submitted to L/D conditions. The lack of an endogenous control and the relevance of light cycles as a synchronization signal are discussed as the temporal modulation of this behaviour might play an important role in the nocturnal habits of this species.

BACKGROUND: The study of platelet aggregation is essential to assess in vitro platelet function by different platelet activation pathways. OBJECTIVE: To assess aggregation and biochemical parameters of random platelet concentrates produced at the Fundação HEMOAM using the quality control tests defined by law. METHODS: Whole blood samples from 80 donors and the respective platelet concentrate units were tested. Platelet concentrates were tested (platelet count, aggregation and pH) on days 1, 3 and 5 of storage. Additionally a leukocyte count was done only on day 1 and microbiological tests on day 5 of storage. Collagen and adenosine diphosphate were used as inducing agonists for platelet aggregation testing. RESULTS: Donor whole blood had normal aggregation (aggregation with adenosine diphosphate = 67% and with collagen = 78%). The median aggregation in platelet concentrates with adenosine diphosphate was low throughout storage (18% on day 1, 7% on day 3 and 6% on day 5) and the median aggregation with collagen was normal only on day 1 and low thereafter (54.4% on day 1, 20.5% on day 3 and 9% on day 5). CONCLUSION: Although the results were within the norms required by law, platelet concentrates had low aggregation rates. We suggest the inclusion of a functional assessment test for the quality control of platelet concentrates for a more effective response to platelet replacement therapy.

Background: Spatial data on cases are available either in point form (e.g. longitude/latitude), or aggregated by an administrative region (e.g. zip code or census tract). Statistical methods for spatial data may accommodate either form of data, however the spatial aggregation can affect their performance. Previous work has studied the effect of spatial aggregation on cluster detection methods. Here we consider geographic health data at different levels of spatial resolution, to study the effect of spatial aggregation on disease mapping performance in locating subregions of increased disease risk. Methods: We implemented a non-parametric disease distance-based mapping (DBM) method to produce a smooth map from spatially aggregated childhood leukaemia data. We then simulated spatial data under controlled conditions to study the effect of spatial aggregation on its performance. We used an evaluation method based on ROC curves to compare performance of DBM across different geographic scales. Results: Application of DBM to the leukaemia data illustrates the method as a useful visualization tool. Spatial aggregation produced expected degradation of disease mapping performance. Characteristics of this degradation, however, varied depending on the interaction between the geographic extent of the higher risk area and the level of aggregation. For example...

Comparison between two time points of the same categorical variable for the same study extent can reveal changes among categories over time, such as transitions among land categories. If many categories exist, then analysis can be difficult to interpret. Category aggregation is the procedure that combines two or more categories to create a single broader category. Aggregation can simplify interpretation, and can also influence the sizes and types of changes. Some classifications have an a priori hierarchy to facilitate aggregation, but an a priori aggregation might make researchers blind to important category dynamics. We created an algorithm to aggregate categories in a sequence of steps based on the categories’ behaviors in terms of gross losses and gross gains. The behavior-based algorithm aggregates net gaining categories with net gaining categories and aggregates net losing categories with net losing categories, but never aggregates a net gaining category with a net losing category. The behavior-based algorithm at each step in the sequence maintains net change and maximizes swap change. We present a case study where data from 2001 and 2006 for 64 land categories indicate change on 17% of the study extent. The behavior-based algorithm produces a set of 10 categories that maintains nearly the original amount of change. In contrast...

The effects of salt with different valences (NaCl, CaCl2 and CrCl3) on the aggregation of O-carboxylmethylchitosan (OCMCS) in dilute aqueous solution were investigated using viscometry, dynamic laser light scattering (DLS) and atomic force microscopy (AFM). With increasing OCMCS concentration beyond a critical aggregation concentration (cac) of ∼0.045 g/l, the aggregation of OCMCS appears in solution. The driving forces of the OCMCS aggregation are intermolecular hydrogen bond, hydrophobic interaction and electrostatic repulsion. The OCMCS aggregation behavior strongly depends on the valence of salt. When NaCl is added, the aggregate size increases with NaCl concentration. When CaCl2 or CrCl3 is added to a given OCMCS concentration, there exists a critical concentration each of Ca2+ and Cr3+. Before the critical concentration, the aggregates decrease in size with increasing salt concentration due to the intra-aggregate complexation; while after the critical concentration, the size of the aggregates increases with salt concentration due to the inter-aggregate complexation. Moreover, the effect of Cr3+ on the OCMCS aggregation is greater than that of Ca2+. The formation of the intra-aggregate complexation is found to be a kinetic process and the aggregate size decreases with time; the formation of the inter-aggregate complexation is also kinetic where the aggregate size increases with time. The aggregates dominated by the intra-aggregate complexation are small...

Aggregation of disease-specific proteins occurs in a variety of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and the prionoses. Hence, the term “proteopathies” has been used to refer to these disorders. Among them, the most prevalent is Alzheimer’s diseases with an increasing number of people affected due to the aging of our society. The aggregation of amyloid beta (Aβ) in the brain of patients with Alzheimer’s disease is considered an initial and early event in disease pathogenesis, followed by neurofibrillary tangle formation, neuronal loss, and progressive cognitive decline. Therefore, it is important to understand the underlying mechanisms and impact of early Aβ aggregation that in turn, is a prime target for therapy. The aim of this thesis was to study mechanistic similarities between the induction of Aβ-aggregation and prions, the infectious protein aggregates in prion diseases. In the first project, the transmissibility of β-amyloidosis in an amyloid precursor protein (APP) transgenic (tg) mouse model was studied. The aim was to investigate the possible induction of cerebral Aβ deposition in young APP tg mice after peripheral inoculations with brain extract containing aggregated Aβ (“Aβ seeds”). Previously...

Protein aggregation can proceed via disordered or ordered mechanisms, with the latter being associated with amyloid fibril formation, which has been linked to a number of debilitating conditions including Alzheimer's, Parkinson's and Creutzfeldt-Jakob diseases. Small heat-shock proteins (sHsps), such as αB-crystallin, act as chaperones to prevent protein aggregation and are thought to play a key role in the prevention of protein-misfolding diseases. In this study, we have explored the potential for small molecules such as arginine and guanidine to affect the chaperone activity of αB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation. The effect of these additives is highly dependent upon the target protein undergoing aggregation. Importantly, our results show that the chaperone action of αB-crystallin against aggregation of the disease-related amyloid fibril forming protein α-synucleinA53T is enhanced in the presence of arginine and similar positively charged compounds (such as lysine and guanidine). Thus, our results suggest that target protein identity plays a critical role in governing the effect of small molecules on the chaperone action of sHsps. Significantly, small molecules that regulate the activity of sHsps may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some protein-misfolding diseases.; Heath Ecroyd and John A. Carver

La Enfermedad de Parkinson (EP) es el desorden neurodegenerativo motor más frecuente, caracterizado por la presencia de agregados proteicos amiloideos intra y extraneuronales conocidos como Cuerpos de Lewy (CL). El principal componente de estas estructuras es alfa-sinucleína (AS), una proteína presináptica de 140 aminoácidos abundante en células dopaminérgicas del cerebro aunque su función fisiológica permanece aún desconocida. En su forma libre nativa AS carece de una estructura secundaria definida pudiendo adoptar múltiples conformaciones en solución y en asociación con otras proteínas y membranas. Se cree que los CL y las fibras amiloides maduras (mafs) son el resultado de un proceso de mal plegamiento, auto-ensamblado y asociación de AS y que posiblemente sirven como mecanismos de protección para la célula, a través de la redistribución en la población de las especies tóxicas intermediarias solubles asociadas a etapas tempranas. Estudio in vitro de las especies y procesos involucrados en la agregación de AS brindan la posibilidad de entender los mecanismos subyacentes en EP. El diseño de nuevas formas de detección de las especies intervinientes en el proceso de agregación y el estudio de los mecanismos de formación de dichos intermediarios tempranos poseen una alta importancia. En esta tesis...

En els darrers 15 anys, l'estudi de l'agregació de proteïnes ha evolucionat de ser una part de la química de proteïnes que tradicionalment generava poc interès, a convertir-se en una àrea d'investigació dinàmica que ha ampliat el seu abast a diferents camps de recerca incloenthi la bioquímica, la biotecnologia, la nanotecnologia y la biomedicina. Dins d'aquests camps, l'anàlisi de l'agregació de proteïnes un interès particularment rellevant en les àrees biomèdica i biotecnològica. Això es degut, per una banda, a que la aparició de depòsits proteics insolubles está relacionada amb un nombre creixent de malalties humanes, moltes d'elles amb conseqüències fatals per als malalts. Per l'altre costat, l'agregació proteica es una complicació habitual en la expresió recombinant de proteïnes a nivell industrial, com ara a la producció d'agents terapèutics de naturalessa proteica, com els anticossos. Conseqüentment, l'exploració de mecanismes que permetin prevenir l'agregació proteica es subjecte actualment d'una intensa tasca d'investigació adreçada a desenvolupar métodes per a la prevenció i l'intervenció terapèutiques d'aquestes malalties greus; i també amb l'objectiu d'incrementar els rendiments en la producció biotechnològica de proteïnes. La gran capacitat de les eines computacionals desenvolupades per tal de predir l'agregació proteica ha donat un gran impuls als esforços destinats a identificar els determinants de l'agregació de polipèptids...

Roberts, Christopher; Global aggregation behavior of a model IgG1 protein, anti-streptavidin (antiSA), was characterized as a function of pH and [NaCl] in acidic conditions. The relative rate of aggregation as illustrated by T2h (temperature at which isothermal incubation resulted in an aggregation half-life of two hours), and mechanisms of aggregation at the different solution conditions were monitored. It was found that relative rate of aggregation is strongly influenced by conformational stability of the protein but generally independent of colloidal interactions. A state diagram of aggregation mechanisms suggested that the mechanism of aggregate growth, and thus the resulting aggregate type, is strongly dependent on electrostatic, inter-molecular interactions. When this state diagram was overlayed with a phase diagram from reversible phase transition studies, it was found that there was a strong correlation between the two, suggesting that the formation of insoluble aggregates from kinetic and thermodynamic means are by the same or similar mechanisms. Several structural assays (far-UV CD, and fluorescence (intrinsic and ThT binding)) were used to analyze the changes in secondary and tertiary structure due to aggregation by the various mechanisms. It was found that ThT bound equally to all types of soluble aggregates...