Phagozyten entfernen apoptotische Zellen während der Entwicklung und beseitigen Pathogene im Immunsystem. Die zugrundeliegenden molekularen und zellulären Mechanismen, insbesondere die Unterschiede zwischen Makrophagen und nicht-professionellen Phagozyten wie Gliazellen, sind weitestgehend unklar. Wir haben neuartige Zellkultur-basierte Assays entwickelt, um 86 Kandidatengene zu testen, die wir aus der Literatur sowie unserem Expressions-Profiling in embryonalen Gliazellen von Drosophila melanogaster zusammengestellt haben. Die Genfunktion wurde durch RNAi herabgesenkt und die Phagozytoseeffizienz wurde mittels FACS untersucht; um die funktionelle Spezifität der Gene zu messen, haben wir nicht nur apoptotische Zellen, sondern auch Bakterien und Beads als „Essen“ angeboten. Mit Hilfe von Null-Mutanten und transgenem RNAi wurden die Ergebnis in vivo validiert. Um die Phagozytose apoptotischer Zellen testen, haben wir untersucht, wie Makrophagen und Gliazellen tote Zellen während der Embryonalentwicklung entfernen, während zur Untersuchung der bakteriellen Phagozytoze adulte Fliegen mit Bakterien infiziert wurden. Unser Screen liefert einen Querschnitt durch die verschiedenen Schritte der Phagozytose. In Bezug auf die Erkennung apoptotischer Zellen finden wir sowohl bekannte als auch neue Akteure für Makrophagen und Gliazellen. Außerdem zeigen wir, dass Vesikeltransport für die Phagozytose apoptotischer Zellen erforderlich ist. Überraschenderweise werden Rezeptoren zur Bakterienerkennung auch für apoptotische Zellen benötigt. Umgekehrt sind Apoptose- Rezeptoren auch für bakterielle Phagozytose notwendig, wodurch eine grundlegende Kreuz-Spezifität zutage tritt. Unsere Arbeit liefert die erste systematische und vergleichende Analyse der verschiedenen Phagozytosearten. Durch die Identifizierung vieler neuer Faktoren legt diese Arbeit den Grundstein für ein mechanistisches Verständnis der Phagozytose von apoptotischen Zellen und Bakterien durch Makrophagen und Gliazellen. / Phagocytes remove apoptotic cells during development and eliminate pathogens in the immune system. The underlying molecular and cellular mechanisms, particularly the differences between macrophages and non-professional phagocytes like glia, are not well understood. We used novel cell-based assays to screen phagocytic function of candidate genes assembled from literature and our genome-wide transcription profiling of Drosophila melanogaster embryonic glia. Gene function was knocked-down by RNAi and phagocytic efficiency assessed by flow cytometry; to explore functional specificity, we offered not only bacteria, but also apoptotic cells and beads as ''food''. To validate results in vivo, we analysed glial clearance of apoptotic neurons in embryonic development and immune clearance of bacteria in adult flies using both genetic mutants and transgenic RNAi. Our screen provides a cross section of the different steps of phagocytosis from recognition to engulfment and phagosomal degradation. For the recognition of apoptotic cells, we confirm the involvement of known factors, such as the chaperone Calreticulin and PS-binding Annexin, and identify new players, such as NIMA for macrophage and Megalin for glial corpse clearance. We find components associated with vesicular trafficking including the v-SNARE Synaptobrevin and the cytochrome Cyp4g15 to be required for corpse clearance. Unexpectedly, receptors known for bacterial recognition, such as PGRP-LC and TEP2, are also strongly required for apoptotic clearance. Conversely, receptors previously implicated in apoptotic cell recognition are also required in bacterial clearance (SIMU, Draper), revealing cross-specificity of the system. Our work represents the first systematic and comparative assessment of the molecular repertoire of different types of phagocytosis, and, with the identification of many new players, lays the groundwork for a mechanistic dissection of bacterial and corpse clearance by glia and macrophages.

Au cours de ma thèse je me suis intéressée aux relations hôte-pathogène entre Drosophila melanogaster et Pseudomonas aeruginosa PA14. RhlR, un facteur de transcription bactérien permet à la bactérie d’échapper à la phagocytose. Mon projet de thèse consistait à identifier comment RhlR exerce cette fonction. Mes résultats suggèrent que RhlR exercerait également une fonction indépendante du quorum sensing. Un crible de mutants PA14 nous a permis d’isoler trois gènes importants pour la virulence de la bactérie et possiblement reliés à RhlR: xcpR, vfR et sltB1. L’utilisation de mutants de drosophile tep4, m’a permis de montrer que le rôle d’échappement à la phagocytose se ferait au niveau de la détection de la bactérie. Par ailleurs, mes résultats suggèrent aussi l’intervention d’un composé volatil qui permettrait de synchroniser la virulence de la bactérie. Dans une dernière partie, j’ai étudié les effets d’une co-infection entre un virus entérique et PA14. / During my PhD, I studied the host-pathogen interactions between Drosophila melanogaster and Pseudomonas aeruginosa PA14. We previously identified RhlR as a bacterial transcription factor that allows the bacteria to circumvent phagocytosis. My main PhD project was to study and identify how RhlR exerts this function. My first results suggested that RhlR plays also a role independently its the quorum sensing. A screen of PA14 mutants allowed me to identify three genes involved in PA14 virulence and possibility in RhlR function: xcpR, vfR and sltB1. By using tep4 fly mutants, I have shown that RhlR’s role against phagocytosis is most likely required at the level of PA14 detection. Beside this, my results indicated that possibly a volatile compound is involved to synchronize PA14 virulence. In the last part, I studied the effects of a co-infection between an enteric virus and PA14.

Malariapigment Hemozoin wird üblicherweise als nicht-toxische, hochmolekulare, parasitäre Speicherform des nicht abgebauten, toxischen Häms aus dem Wirtszell-Hämoglobins betrachtet. Unaufgereinigtes Pigment, wie wir es im infizierten Erythroyzten finden und wie es nach Schizontenruptur freigesetzt wird, kann man als die "natürliche Diät" bezeichnen, die Makrophagen in Malaria-infizierten Wirten aufnehmen. Nach Aufnahme in den Makrophagen persistiert Hemozoin in den Lysosomen und wird nicht abgebaut. Das Häm-abbauende Enzym, die Häm-Oxygenase wird nicht induziert. Hemozoin ist eine potente Quelle für Radikale, woraus Lipoperoxide und davon abgeleitete Hydroxyaldehyde,wie 4-Hydroxynonenal resultieren . 4- Hydroxynonenal in Konzentrationen, wie sie in Hemozoin-beladenen Monozyten nachgewiesen wurden, hemmen die Proteinkinase C. In immunopräzipitierter Proteinkinase C aus Hemozoin- haltigen Makrophagen wurden ProteinkinaseC-Hydroxynonenal-Komplexe nachgewiesen. Die Hydroxynonenal-bedingte Hemmung der Proteinkinase C (und anderer bisher nicht untersuchter Enzyme und Prozesse) könnte die Hemozoineffekte auf den oxydativen burst und die Phagozytose erklären. Der Phorbolester-induzierte oxydative burst ist irreversibel gehemmt in Monozyten, die entweder Hemozoin oder aber Hemozoin-haltige infizierte Erythrozyten phagozytiert haben. Die Hemmung der NADPH-Oxydase, das für den oxydativen burst verantwortliche Enzym, durch intrazelluläres Hemozoin, sollte beträchtlich zur burst -Hemmung beitragen. Monozyten phagozytieren Hemozoin-haltige, infizierte Erythrozyten oder isoliertes Hemozoin , sind danach jedoch unfähig, erneut zu phagozytieren, wie es Monozyten nach Phagozytose und Verdau von nicht-infizierten Erythrozyten physiologischerweise tun. Schließlich ist die Expression von Membranantigenen, die für die Immunantwort von Bedeutung sind, in Hemozoin-haltigen Monozyten vermindert. Die Induktion des für die Präsentation externer Antigene verantwortlichen Histokompatibilitätskoplexes (MHC) Klasse II durch Interferon-gamma ist in Hemozoin-beladenen Monozyten aufgehoben. Sowohl das Interzelluläre Adhäsionsprotein 1 (CD54) als auch p150,95 Integrin (CD11c) sind in Hemozoin-haltigen Monozyten vermindert Oberflächen-exprimiert. Trotz der verschiedenen funktionellen Einschränkungen sind Hemozoin-beladene Phagozyten vital. Bei Plasmodium-falciparum-Malaria enthält ein hoher Anteil von Gewebsmakrophagen und zirkulierender Monozyten und Leukozyten große Mengen an Hemozoin. Wichtige Funktionen wie oxydativer burst , Phagozytose und die Expression von MHC Klasse II sind in Hemozoin- beladenen Phagozyten gestört. Es scheint deshalb gerechtfertigt, die Hemozoin-Beladung als wichtigen Faktor in der gestörten Immunantwort bei der P.falciparum-Malaria zu betrachten. / Malaria pigment hemozoin is generally considered to be a non-toxic, high-molecular-weight, parasitic storage form of undigested,toxic, host-hemoglobin-heme.Crude pigment, as present in infected erythrocytes and shed after schizont rupture, may be considered the 'natural diet' ingested by macrophages in malaria-infected hosts. After ingestion by macrophages hemozoin persists in the lysosomes without being degraded. The heme-degrading enzyme, the heme-oxygenase, is not induced. Hemozoin is a powerfull source of radicals that generates lipoperoxides and derived, toxic hydroxyaldehydes such as 4-hydroxynonenal. High concentrations of 4-hydroxynonenal, which have been detected in hemozoin-fed macrophages, inhibit protein kinase C. Complexes between hydroxynonenal and protein kinase C have been detected in immunoprecipitated protein kinase C from hemozoin-fed macrophages. Hydroxynonenal-mediated inhibition of protein kinase C (and of other as yet unidentified enzymes and processes) may explain hemozoin-mediated effects on oxidative burst and phagocytosis. The phorbol ester-eliceted oxidative burst is irreversibly suppressed in monocytes fed with hemozoin or hemozoin-containing, infected erythrocytes. The inhibition of NADPH-oxidase, the enzyme responsible for oxidative burst, by ingested hemozoin should considerably contribute to burst inhibition. Monocytes avidly ingest infected hemozoin-containing erythrocytes or isolated hemozoin but are unable to repeat the phagocytic cycle as monocytes do after phagocytosis and digestion of non-infected erythrocytes. Finally , the expression of membrane antigens involved in the immune response is decreased in hemozoin-loaded monocytes. The induction of the major histocompatibility complex (MHC) class II by interferon-gamma, that is responsible for presentation of external antigens, is abrogated in hemozoin-loaded monocyte. The intercellular adhesion molecule 1 (CD54) as well as the p150,95 integrin (CD11c) are decreased on the surface of monocytes containing hemozoin. Despite multiple functional impairments, hemozoin-loaded phagocytes remain alive. In Plasmodium-falciparum malaria large portions of resident macrophages and circulating monocytes and leukocytes contain massive amounts of hemozoin. Important functions like oxidative burst, phagocytosis and the expression of MHC class II are severely impaired in hemozoin-fed phagocytes. It seems therefore likly that hemozoin loading may play an important role in the impairment of the immune response seen in P.falciparum malaria.

A hidroquinona (HQ) é um composto fenólico de origem natural ou antropogênica, encontrada em grandes concentrações no cigarro, além de ser produto da biotransformação do benzeno. Nosso grupo de pesquisa tem demonstrado que a exposição à HQ compromete a resposta inflamatória in vivo. Dando continuidade a estas investigações, este trabalho visou investigar os efeitos da exposição in vivo à HQ sobre funções do tecido traqueal e atividades de macrófagos alveolares. Para tanto, camundongos Swiss machos foram expostos à HQ 25ppm (1,5mg/60mL/1h; 5 dias) ou veículo (solução salina etanol 1:20) por via sistêmica (nebulização). Concentrações de mediadores inflamatórios (interleucina (IL) 1β, IL-6, IL-10, IL-4, IL-12 fator de necrose tumoral-α (TNF-α) ou proteína quimiotáxica de monócitos (MCP-1); ensaio imunoenzimático (ELISA) e óxido nítrico (NO; reação de Griess) foram quantificados no lavado bronco-alveolar (LBA) em condições basais ou 3 horas após estímulo inflamatório (LPS in vivo; 100µL/mL; 10min) e no sobrenadante de macrófagos (MΦs) alveolares ou de cultura da traquéia obtidos dos animais e posteriormente estimulados in vitro (MΦs: 5µg/mL de LPS + 10ng/mL de IFN-γ; traquéia: 1µg/mL de LPS; 24h). Atividades fagocítica e fungicida (microscopia óptica) e a expressão de receptores envolvidos na fagocitose toll-like receptor (TLR, TLR-2, TLR4 e dectina-1; citometria de fluxo) foram determinadas após incubação in vitro de MΦs alveolares com o fungo Candida albicans e a expressão protéica de MyD88 foi realizada em MΦs alveolares (western blot). Quantificação do RNAm para MCP-1 (reação da transcriptase reversa em cadeia de polimerase, RT-PCR) foi realizada em tecido traqueal e células de linhagem monocítica humana THP-1 foram empregadas em ensaios de quimiotaxia in vitro (Câmara de Boyden) frente a diferentes concentrações de MCP-1. Reatividade do tecido traqueal foi quantificada frente à metacolina. Os resultados obtidos mostraram que a exposição in vivo à HQ reduziu a concentração de MCP-1 (54,98% vs. controle LPS) e IL-12 (51,45% vs. controle LPS) no LBA após inflamação; reduziu a secreção de MCP-1 por MΦs (basal: 87,96%; LPS+INF-γ: 61,20%) e tecido traqueal em cultura (79,77% vs. controle LPS). Neste último tecido, a diminuição foi dependente da menor expressão gênica. Concentrações de MCP-1 semelhantes à detectada no sobrenadante de cultura de traquéia de animais expostos à HQ induziram migração de células THP-1 menor (38,2%) que à provocada pela concentração de MCP-1 no sobrenadante da cultura traquéia de animais controles. Traquéias de animais expostos à HQ apresentaram hiperreatividade (193,48%), a qual foi revertida pela remoção do epitélio traqueal. Adicionalmente, cultura de MΦs obtidos de animais expostos à HQ apresentaram maior atividade fagocítica (porcentagem de fagocitose: 36,30%; índice de fagocitose: 83,97%) e fungicida (68,47%), que não foram dependentes de alterações nos receptores TLR2, TLR4 e dectina-1, mas podem ser decorrentes da menor expressão protéica de MyD88. Em conjunto, os dados obtidos apontam para alterações importantes resultantes da exposição in vivo à HQ sobre funções de MΦs alveolares e do tecido traqueal que, em conjunto, podem ser determinantes para a toxicidade observada nestes animais que culmina com prejuízo na defesa do organismo. / Hydroquinone (HQ) is a phenolic compound of natural or anthropogenic source, also found in high concentrations in cigarette, as well as benzene´s metabolite. Our research group has demonstrated that exposure to HQ impairs in vivo inflammatory response. Following these investigations, this work aimed to study the effects of in vivo exposure to HQ on tracheal tissue and alveolar macrophages (MΦs) activities. For this purpose, male Swiss mice were systemically (aerolised) exposed to 25ppm HQ (1.5 mg/60mL/1h; 5 days) or vehicle (saline ethanol solution, 1:20). Concentrations of inflammatory mediators (interleukin (IL) IL-1β, IL- 6, IL-10, IL-4, IL-12 tumor necrosis factor-α (TNF-α ) or monocyte chemoattractant protein (MCP-1); (enzyme immune assay, ELISA)) and nitric oxide (NO; Griess reaction) were quantified in bronchoalveolar lavage (BAL) at baseline or 3 hours after inflammatory stimulus (in vivo LPS, 100µL/mL; 10min); in the supernatant of cultured alveolar macrophages (MΦs) or trachea obtained from animals and subsequently in vitro stimulated (MΦs: 5µg/mL of LPS plus 10ng/mL IFN-γ; trachea: 1µg/mL LPS; 24 hours). Phagocytic and fungicidal activities (light microscopy) and expression of receptors involved in phagocytosis (toll-like receptor (TLR, TLR2, TLR4 and dectin-1, flow cytometry) were determined after in vitro incubation of alveolar MΦs with Candida albicans fungus and expression of MyD88 pathway was held in alveolar MΦs (western blot). Quantification of mRNA for MCP-1 (reaction of reverse transcriptase polymerase chain reaction, RT-PCR) was performed in tracheal tissue and cells human monocytic THP-1 were used in in vitro chemotaxis assays (Boyden chamber) using different concentrations of MCP-1. Tracheal reactivity was measured in response to methacholine. The results showed that in vivo HQ exposure reduced the concentration of MCP-1 (54.98% vs. control) and IL-12 (51.45% vs. control) in the BAL after inflammation; decreased secretion of MCP-1 by MΦs (basal: 87.96%, LPS+INF-γ: 61.20%) and in tracheal culture after LPS stimulation (79.77% vs. control). In the latter tissue, the MCP-1 protein content was dependent on impaired gene expression. Concentrations of MCP-1 similar to those detected in the supernatant of tracheal from HQ exposed rats induced smaller migration of THP-1 cells (38.2%) than that evoked by the MCP-1 concentration obtained in trachea supernatants collected from control animals. Tracheas from HQ exposed rats showed hyper reactivity (193.48%), which was reversed by removal of the tracheal epithelium. Additionally, culture of MΦs obtained from HQ exposed rats showed increased phagocytic (percentage of phagocytosis: 36.30%; phagocytosis index: 83.97%) and fungicide activity (68.47%), which were not dependent on changes in the receptors TLR2, TLR4 and dectin-1, but could be due to reduced MyD88 expression. Together, these data point out important alterations on MΦs and trachea after in vivo HQ exposure, which may be crucial for the toxicity observed in these animals that culminates with impaired host defense.

Orientador: Luciane Alarcão Dias-Melicio / Resumo: Recentemente, a deficiência de vitamina D vem se tornando um problema de abrangência mundial em virtude de hábitos rotineiros da população, como o trabalho por períodos prolongados em ambientes fechados e diminuição da exposição solar. Trabalhos recentes demonstram que a vitamina D age não somente na homeostase do cálcio, mas também na regulação do sistema imune. Diante da multiplicidade de funções dessa vitamina, sua deficiência tem sido associada ao risco de desenvolvimento de uma série de doenças, entre elas doenças infecciosas como as causadas por S. aureus. As infecções por essa bactéria têm trazido expressiva preocupação para a população humana em decorrência do aumento da prevalência de cepas resistentes aos fármacos antibacterianos, dificultando, dessa maneira, o tratamento e contribuindo para a busca de métodos alternativos para combater esse tipo de infecção. Além disso, o S. aureus conta com um potente arsenal de fatores de virulência que contribuem para a evasão da resposta imune do hospedeiro. Nesse contexto, torna-se importante avaliar se a vitamina D pode modular os efeitos bactericidas de neutrófilos humanos através de mecanismos intra e extracelulares, favorecendo, portanto, o combate a infecções, especialmente aquelas causadas por microrganismos resistentes aos principais tratamentos. Dessa maneira, nós demonstramos que neutrófilos tratados com vitamina D e desafiados com duas cepas de S. aureus tiveram um aumento nas taxas de fagocitose e atividade bacteric... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the last years, vitamin D deficiency has become a worldwide problem due to routine population habits, such as prolonged work indoors and increased use of sunscreen/decreased sun exposure in attempt to avoid high rates of skin cancer. Recent studies demonstrated that vitamin D acts not only on calcium homeostasis, but also on the regulation and function of the immune system. Facing the countless functions of vitamin D, its deficiency has been associated with the risk of development of many diseases, including infectious diseases such as those caused by S. aureus. Infections caused by these bacteria have brought significant concern to the human population due to the increased prevalence of strains resistant to antibiotics, thus making it difficult to treat and contributing to the search for alternative methods to combat this type of infection. In addition, S. aureus have a variety of virulence factors, which confer the ability to evade host immune responses. In this context, it is important to evaluate whether vitamin D can modulate the bactericidal effects of human neutrophils through intra- and extracellular mechanisms, such as phagocytosis, bacterial killing and release of Neutrophil Extracellular Traps (NETs), thus contributing to the response against infections, especially those caused by microorganisms resistant to the main treatments. Thus, we demonstrated that neutrophils treated with Vitamin D and challenged with two strains of S. aureus had an increase in phagocyti... (Complete abstract click electronic access below) / Doutor

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-07-27T15:19:25Z No. of bitstreams: 1 JoãoBartholomeuNetoTese2018.pdf: 1992204 bytes, checksum: c127f6e78b3371ee05b0b87f14163392 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-07-27T15:19:43Z (GMT) No. of bitstreams: 1 JoãoBartholomeuNetoTese2018.pdf: 1992204 bytes, checksum: c127f6e78b3371ee05b0b87f14163392 (MD5) / Made available in DSpace on 2018-07-27T15:19:43Z (GMT). No. of bitstreams: 1 JoãoBartholomeuNetoTese2018.pdf: 1992204 bytes, checksum: c127f6e78b3371ee05b0b87f14163392 (MD5) Previous issue date: 2018-06-14 / Projections of the increase in absolute and relative numbers of elderly represent new challenges for public health. Aging is accompanied by gradual decline of physiological functions, immune function and muscle strength. In this way, the elderly is more susceptible to sarcopenia, osteoporosis, cancer and cardiovascular, metabolic and neurodegenerative diseases. Resistance Training (RT) has been shown to be an important intervention for increased muscle strength and decreased chronic inflammation in the elderly. However, little is known about the influence of RT on the functional response of cells that constitute the first line of defense during immunosenescence. In this sense, the present study aimed to investigate an association between RT adaptations induced by a program and a functional response of circulating phagocytes in elderly. The present cross-sectional study was composed of 54 elderly women, 71.3 ± 6.3 years, apparently healthy, non-institutionalized and divided into two distinct groups: Trained (n = 28) and Sedentary (n = 26). The elderly of the Trained group were participants of a RT program for 8.6 ± 0.3 months and performed a training consisting of 12 exercises, 3 sessions weekly, with intensity of 70% of a maximum repetition, while the Sedentary group did not participate of any physical activity program during the same period. Body mass, height, body mass index, waisthip ratio, fat free mass and fat mass were evaluated. The total energy intake and macronutrients in the usual diet were assessed. TNF-a, IL-6 and IGF-1 analyzes, total and differential leukocyte count, monocyte or neutrophil phagocytosis test, phagocytosis index (IF) and microbicidal activity of phagocytes were performed. Data analysis was performed using the Statistical Package for the Social Sciences (SPSS) program and considered statistically significant P <0.05 for two-tailed tests. The results show that the IF of neutrophils (but not of monocytes) in the Trained group was significantly higher (! <0.001, effect size, (") = 0.90, 95% CI: [0.75-1.04]) compared to that in Sedentary group. In contrast, the microbicidal activity of phagocytic cells was not significantly influenced by RT. Also, total energy and carbohydrate intake, as well as serum levels of IL-6, had a significant influence on the phagocytic activity of neutrophils (! = 0.04), being considered in the model. Multivariate regression identified the physical condition of the subject (β = 0.425; ! = 0.01) as a significant predictor of the IF. In conclusion, the circulating neutrophils of elderly women who had TR showed higher phagocytic activity, whereas microbicidal activity did not present a statistically significant difference between groups, demonstrating a positive effect of RT in the first line of defense of the organism. / As projeções do aumento nos números absoluto e relativo de idosos representa novos desafios para a saúde pública. O envelhecimento vem acompanhado declínio gradual das funções fisiológicas, da função imunológica e da força muscular. Dessa forma, idosos são mais susceptíveis a sarcopenia, osteoporose, câncer e doenças cardiovasculares, metabólicas e neurodegenerativas. O Treinamento Resistido (TR) tem se mostrado importante intervenção para aumento da força muscular e diminuição da inflamação crônica em idosos. No entanto, pouco se conhece a respeito da influência do TR na resposta funcional de células que constituem a primeira linha de defesa durante a imunossenescência. Nesse sentido, o presente estudo buscou investigar a associação entre adaptações induzidas por um programa de TR e a resposta funcional de fagócitos circulantes em idosas. O presente estudo de caráter transversal, foi composto por 54 idosas com idade 71.3 ± 6.3 anos, aparentemente saudáveis, não institucionalizadas e divididas em dois grupos distintos: Treinadas (n=28) e Sedentárias (n=26). As idosas do grupo Treinadas eram participantes de um programa de TR há 8,6 ± 0,3 meses e realizaram um treino composto por 12 exercícios, 3 sessões semanais, com intensidade de 70% de uma repetição máxima, enquanto o grupo Sedentárias não participou de nenhum programa de atividades físicas durante o mesmo período. Avaliou-se a massa corporal, estatura, índice de massa corporal, relação cintura-quadril, massa livre de gordura e massa gorda. Foi avaliado o consumo de energia total e de macronutrientes na dieta habitual. Foram realizadas análises de fator de necrose tumoral alfa (TNF-α), interleucina 6 (IL-6) e Fator de crescimento semelhante à insulina (IGF-1), contagem total e diferencial de leucócitos, teste de fagocitose por monócitos ou neutrófilos, índice de fagocitose (IF) e atividade microbicida de fagócitos. A análise dos dados foi realizada por meio do programa Statistical Package for the Social Sciences (SPSS) e considerado P < 0.05 estatisticamente significante para testes bicaudais. Os resultados demonstram que o IF de neutrófilos, mas não de monócitos, no grupo Treinadas foi significativamente maior (! <0,001; tamanho do efeito, (") = 0,90, IC 95%: [0,75–1,04]) em comparação com o grupo Sedentárias. Em contraste, a atividade microbicida das células fagocítica não foi influenciada significativamente pelo TR. Além disso, a ingestão total de energia e carboidratos, bem como os níveis séricos de IL-6, tiveram influência significativa na atividade fagocítica dos neutrófilos (! = 0,04), sendo considerados no modelo. Em conclusão, os neutrófilos circulantes de mulheres idosas que praticaram TR expressaram maior atividade fagocitária, enquanto atividade microbicida não apresentou diferença estatisticamente significante entre grupos, demonstrando efeito positivo do TR na primeira linha de defesa do organismo.

A aquicultura sofre diferentes pressões por um lado o impacto que as doenças causam na produção, e por outro a pressão por parte de grupos ambientalistas e do governo devido ao impacto ambiental. Diferentes soluções foram propostas para esses problemas como probióticos e imunoestimulantes para prevenir e controlar doenças com nenhum ou algum sucesso. Outra opção seria o biofloco para minimizar o impacto ambiental, com excelentes resultados zootécnicos. Porém não sabemos se o sistema de biofloco estimula ou inibe o sistema imune de camarões (Litopenaeus vannamei). Esse é o objetivo desse estudo, saber se o sistema de biofloco altera a contagem total e diferencial de hemócitos, os índices fagocíticos, utilizando ensaio de fagocitose in vitro com Sacaromises cerevisae e a produção de ânion superóxido pelo ensaio de Nitroblue tetrazolium (NBT). Na contagem total de hemócitos nos 2 primeiros meses os animais criados em água clara possuem uma contagem maior que os criados em biofloco enquanto que no 4° mês os animais de biofloco possuem uma contagem maior. Na contagem diferencial no 4°mês as células hialinas estão em maior porcentagem nos animais criados em biofloco enquanto que as granulares e simi-granulares estão em menor porcentagem nesses animais, quando comparamos com os animais criados em água clara. Os índices fagocíticos e a produção de ânion superóxido não apresentam diferença entre os animais criados em água clara e os criados em biofloco. / The aquaculture production suffers different pressure in one hand disease, and in the other its environment impact. Different solutions are offered to solve this problems such as probiotics and immunostimulant to prevent and control the diseases with some or no success. Another option is the biofloc to minimize its environmental impact, with excellent results to zootechincal characteristics. But it is not known whether biofloc stimulate or inhibits the shrimp (Litopenaeus vannamei) immune system. That is what our study aim to answer if the biofloc system affects or not the total and differential hemocytes count, the phagocityc indexes of in vitro phagocytosis with Sacaromises cerevisae and the production of superoxide anion with the Nitroblue Tetrazolium (NBT) assay. At the first 2 month the animal rare in clear water had a higher total hemocyte count, while it was higher in the animals rare in the biofloc at the 4th month. At the 4th month the hyalines cells had a higher concentration in animals rare at biofloc system, while the granular and semi-granular cells had a lower concentration in this animals when compared with the ones rare at the clear water system. The phagocityc indexes and the production of superoxide anion did not have any difference between animals rare at clear water system and biofloc system.

A citometria de fluxo é um método preciso, rápido e eficaz na avaliação de múltiplos parâmetros celulares, tanto estruturais como funcionais, propiciando a separação e o estudo de diferentes populações e sub-populações de células. No presente estudo, foram empregados métodos citométricos para caracterização dos diferentes tipos celulares sangüíneos de Centropomus parallelus, assim como para verificação da viabilidade celular, avaliação da atividade fagocítica e ativação do burst respiratório. Foram identificadas três sub-populações representativas de leucócitos: linfócitos, monócitos e trombócitos. Estas células foram estimuladas, in vitro, com Staphylococcus aureus, marcado com iodeto de propídeo (SAPI), lipopolissacarídeo de Escherichia coli (LPS) e zymosan, partículas de Saccharomyces cerevisiae. As respostas frente aos estímulos foram distintas de acordo com o tipo celular e o estímulo apresentado. Os monócitos apresentaram maiores percentuais de fagocitose frente ao estímulo provocado pela SAPI e pelo Zymosan; já a população que continha trombócitos entre outros tipos celulares (por exemplo: linfócitos), apresentou \"fagocitose\" significativa apenas para SAPI. O burst oxidativo detectado pela fluorescência emitida pelo diacetato 2´7´diclorofluoresceína (DCFH) foi significativo apenas quando estimulado com PMA (miristato-acetato de forbol), não apresentando resposta estatisticamente significante para os estímulos SAPI e LPS. Após a exposição ao naftaleno nas concentrações 10-3, 10-6, 10-9 M durante quatro horas, in vitro, houve um aumento na fagocitose realizado pelos monócitos e trombócitos, porém uma diminuição no burst oxidativo apresentado nas concentrações 10-6 e 10-9 M de naftaleno. Este resultado reflete, in vitro, uma resposta ao contaminante com significado imunológico desfavorável para o peixe, já que as células estão aumentando a atividade fagocítica sem conseguirem, teoricamente, destruir o agente invasor. A partir desses resultados preliminares, podemos avaliar melhor, algumas características da resposta imune inata desta espécie de peixe, presente na costa litorânea brasileira. Estes parâmetros imunofisiológicos podem servir como base para futuros estudos ecotoxicológicos tanto em laboratório mas também a campo. Estudos que utilizam a imunidade inata como indicador biológico de alterações ambientais causadas por poluentes diversos, podem evidenciar o grau de impacto toxicológico sobre esta espécie e agregar valor a sua importância econômica e ecológica. / Flow cytometry is a precise, fast, and effective method for the evaluation of several cellular parameters, both structural and functional, allowing the sorting and analysis of particular populations and sub-populations of cells. In this study, we employed cytometric methods on the caractherization of different blood cell types from Centropomus parallelus, and also verified cell viability, phagocytic activity and oxidative burst in these cells. Three sub-populations were identified: lymphocytes, monocytes, and trombocytes. These cells were stimulated in vitro with propidium iodide-conjugated Staphylococcus aureus (SAPI), Escherichia coli lipopolysaccharide (LPS), or zymosan (Saccharomyces cerevisae particles). Responses to each individual stimulus differed according to cell type. Monocytes displayed the higher percentages of phagocytosis in presence of SAPI or zymosan; on the other hand, the population that included trombocytes, among other cell types (such as lymphocytes) only performed phagocytosis in a relevant level in the presence of SAPI. The oxidative burst, detected by fluorescence emitted by 2´7´dichlorofluorescein diacetate (DCFH) was significant only after stimulation with PMA (phorbol myristate-acetate), but not when the stimulus was SAPI or LPS. Based on these preliminary results, the innate immune response in these animals (ubiquitous coastal waters of the Brazilian shore) can be further evaluated. Immunophysiological parameters in these species can build a solid ground for future ecotoxicological studies. Approaching innate immunity as a biological indicator of environmental changes induced by pollutants may support the degree of toxicological impact over these animals, and aggregate value to its current ecological and economical importance.

Membrane rafts are highly dynamic heterogeneous sterol- and sphingolipid-rich micro-domains on cell surfaces. They are generally believed to provide residency for cell surface molecules (e.g., adhesion and signaling molecules) and scaffolding to facilitate the functions of these molecules such as membrane trafficking, receptor transport, cell signaling, and endocytosis. Using laser scanning confocal microscopy and reflection interference microscopy (RIM), we studied the spatial and temporal distributions of membrane rafts and surface receptors, signaling molecules, and cell organelles during the formation of phagocytic contact areas. K562 cells, which naturally express CD32A, a cell surface receptor for the Fc portion of Immuno-globulin g (IgG), was chosen as a model for neutrophils. An opsonized target was modeled using a glass supported lipid bilayer reconstituted with IgG. CD32A was found to cluster and co-localize with membrane rafts. Placing the K562 cells on the lipid bilayer triggered a process of contact area formation that includes binding between receptors and ligands, their recruitment to the contact area, a concurrent membrane raft movement to and concentration in the contact area, and transport of CD32A, IgG, and membrane rafts to the Golgi complex. Characterization of these processes was performed using agents known to disrupt detergent resistant membranes (DRMs), dissolve actin microfilaments, and inhibit myosin motor activity, which abolished the CD32A clusters and prevented the contact area formation. The relevance to phagocytosis of contact area formation between K562 cells and lipid bilayers was demonstrated using micro-beads coated with a lipid bilayer reconstituted with IgG as the opsonized target instead of the glass supported planar lipid bilayer. Disruption of membrane rafts, salvation of the actin cytoskeleton, and inhibition of myosin II activity were found to inhibit phagocytosis. Here we have provided evidence that membrane rafts serve as platforms that are used to pre-cluster CD32A and transport CD32A along the actin cytoskeleton to the site of phagocytic synapse formation, followed by internalization to the Golgi complex.

Σα αρνητικά και θετικά κατά Gram βακτήρια E. coli και S. αureus αντίστοιχα αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων της C. capitata. Η πρόσδεση των βακτηρίων ενεργοποιεί τόσο τις β1 ιντεγκρίνες, όσο και σηματοδοτικά μονοπάτια που περιλαμβάνουν τα μόρια μεταγωγής σήματος Ras, Raf, MEK, ERK, FAK, Src, και GRB2. Οι παραπάνω ενεργοποιήσεις, σε συνδυασμό με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης, καταλήγουν στην επαγωγή της έκκρισης μορίων απαραίτητων για την κυτταροφαγία των βακτηρίων που είναι και το τελικό αποτέλεσμα των παραπάνω διαδικασιών. Σα συνθετικά πολυμερή σφαιρίδια, αλλά και πιθανόν και άλλοι αβιοτικοί παράγοντες, παρότι δεν έχουν καμία προηγούμενη εξελικτική σχέση με τα αιμοκύτταρα, ως σύγχρονο προϊόν της ανθρώπινης γνώσης, αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων από άγνωστους μέχρις στιγμής υποδοχείς. Η κυτταροφαγία τους προωθείται μέσω ενεργοποίησης σηματοδοτικών μονοπατιών που περιλαμβάνουν την ενεργοποίηση των μορίων FAK, Src και MAP κινασών καθώς και με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης. Ο LPS αναγνωρίζεται και δεσμεύεται στην επιφάνεια των αιμοκυττάρων, ενεργοποιεί άγνωστους μέχρις στιγμής υποδοχείς και διαμέσου σηματοδοτικών μονοπατιών που περιλαμβάνουν τις Ras, ενεργοποιεί τις MAP κινάσες και το σύστημα της έκκρισης. Αν και ενεργοποιεί και τις τρεις MAP κινάσες, μόνο η ERK και η p38 απαιτούνται τόσο στη διαδικασία της έκκρισης, όσο και στη διαδικασία της ενδοκυττάρωσής του. Η FAK, αν και ενεργοποιείται από τον LPS, δεν εμπλέκεται στην διαδικασία της ενδοκυττάρωσής του. Σα παραπάνω δείχνουν ότι τα αιμοκύτταρα έχουν αναπτύξει διακριτούς μηχανισμούς για την κυτταροφαγία των παθογόνων, των μικρομορίων και των αβιοτικών παραγόντων, γεγονός που δείχνει την ικανότητα εξέλιξης των εντόμων έτσι ώστε να καλύπτουν τις ανάγκες της επιβίωσή τους. / Gram negative and Gram positive bacteria, E. coli and S. αureus respectively, are recognized and they are bound on the C. capitata hemocyte surface. After binding, they activate β1 integrins and intracellular signalling pathways, involving the kinases Ras, Raf, MEK, ERK, FAK, Src, and GRB2. This signal transduction, with the participation of the cytoskeleton of actin and tuboulin filaments, leads to a regulated secretion, that is a prerequisite for phagocytosis. Latex beads and probably other abiotic factors, despite having no previous evolutionary relation to the hemocytes, being a new product of human knowledge, are recognized and they are bound on the hemocyte surface, by hitherto unknown receptors. They activate intracellular signalling pathways that involves FAK, Src and MAP kinases and they promote, with the participation of actin and tuboulin cytoskeleton, their phagocytosis. LPS is recognized and bound on the hemocyte surface and activates so far unknown receptors and through unknown intracellular signalling pathways involving Ras, activates the MAP kinases and the regulated secretion. Although it activates all three MAPKs, only the ΕRΚ and p38 are required not only for the secretion, but also for its internalization. Although FAK is activated by LPS, it does not get involved in the process of its internalization. All of the above mentioned results indicate that the hemocytes have developed distinct mechanisms for phagocytosis of pathogens, micromolecules and abiotic factors, a fact that underlines insects evolutionary adaptations, so that they can survive.