Cardiovascular disease is a leading cause of death worldwide. Changes in the cardiac extracellular matrix (ECM) are associated with cardiac pathologies such as cardiomyopathy and cardiac hypertrophy. The ECM is a dynamic scaffold of proteoglycans, fibrous proteins, and glycoproteins that sheathes and protects many organs and tissues, including the heart, by attenuating mechanical stress. Misregulation of ECM proteins triggers changes in matrix stiffness, which can lead to age-associated and congenital heart defects. ECM rigidity is also important to the migration of cells, such as hemocytes, the invertebrate blood cells. In the embryo, hemocytes also perform fibroblast functions, through the deposition of the ECM proteins Collagen and Laminin. Hemocytes are hypothesized to be critical for ECM assembly, and by extension, for heart development. The consequences of impaired hemocyte function in the embryo and during larval growth are unknown and are the focus of this research. Using Drosophila melanogaster as a model, I used genetic tools to manipulate hemocyte survival and motility to assess their role in ECM organization and structure around the heart. Concerted gene knockdown and confocal microscopy techniques were employed to evaluate the effects of altered hemocyte abundance and motility on hemocyte behaviour and resulting changes to the ECM. Here I provide evidence to support a role for hemocytes in the turnover of a vital ECM protein, the Type IV Collagen Viking. I also developed a novel protocol to photobleach and observe fluorescence recovery in intact, living larvae using confocal microscopy. Recovery of fluorescence of GFP tagged ECM is a measure of the rate of ECM protein turnover during development or growth. This novel technique has allowed for assessment of recovery of Viking-GFP after photobleaching in vivo, as a measue of Viking protein turnover at the cardiac ECM. This new technique can be employed to determine the turnover of other major ECM proteins. Combining hemocyte impairment with photobleaching provides the opportunity to observe innate protein turnover at the ECM in real time, both in normal and hemocyte-deprived matrices. Recovery of Viking-GFP fluorescence was also observed in hemocyte-deprived conditions. My findings reveal gradual recovery of Viking-GFP at the cardiac ECM in controls, and potentially slower recovery in hemocyte-impaired conditions. These observations suggest a role of hemocytes in ECM protein turnover. This work will help reveal the role of hemocytes in organizing the cardiac ECM and provides a novel technique for the in vivo assessment of ECM protein turnover. Ultimately, this research sheds light on how hemocyte function affects overall structure of the cardiac ECM and contributes to an enhanced understanding of how changes in this ECM influence predisposition to and progress of cardiac disease. / Thesis / Master of Science (MSc)

Cell migration is essential for embryonic development, it occurs in adult organisms during processes like wound healing and its misregulation contributes to pathological conditions such as metastasis. Despite this, most studies of cell migration have been undertaken in vitro. Ena/VASP proteins, believed to be actin anti-capping proteins, have been studied extensively in fibroblasts in vitro, and using Drosophila macrophages (hemocytes) within the developing embryo, the role of the Drosophila homologue of Mena, Ena, is investigated in vivo. Consistent with data from fibroblasts in vitro, Ena localised to regions of actin dynamics within migratory hemocytes, where this protein stimulated lamellipodial dynamics and positively regulated filopodial number and length. However, whilst overexpression of Ena/VASP proteins in fibroblasts reduced migration speeds, Ena overexpression in hemocytes dramatically increased migration speeds in three different assays. This positive regulation of migration speed closely resembled the increased motility of breast cancer cells that overexpress Mena and evidence presented here, suggests that this key difference may be explained by spatial constraints that are imposed upon cells within three dimensional environments. Indeed, such constraints prevented ruffling, a more detrimental form of retraction, in hemocytes in vivo. Furthermore, fibroblasts overexpressing Mena in vitro form membrane ruffles more frequently. Therefore Ena/VASP proteins drive migration by enhancing lamellipodial protrusion, but in certain environments these protrusions are lost as ruffles slowing migration. The method by which Ena regulates lamellipodial protrusion and migration speeds was then investigated: Ena increased Fascin-mediated actin bundling and the number of Fascin rich-actin bundles that coalesced. Analysis of individual actin bundles revealed that coalescence increased protrusion rate and that both protrusion rate and coalescence, increased cell migration speeds. This suggests that Ena facilitates an increase in cell migration by promoting the coalescence of Fascin bundles, and positions Ena as a key regulator of migration speeds in vivo.

An increase in attachment of SRBCs to Procambarus clarkii hemocytes has been shown after the crayfish were injected with a live or killed Pseudomonas strain RS2b. The increase in attachment occurred at 8 hours post injection and peaked at 24 hours for both experimental groups. The population of hemocytes with receptors for LPS and mannose also increased at 8 hours post injection and peaked at 24 hours for both experimental groups. At 96 hours post injection the number of receptor bearing hemocytes and hemocytes bound to SRBCs began to decrease to the level of the control for both groups. The protein concentration of hemolymph from the experimental groups remained stable at 8 and 24 hours post injection and increased at 96 hours. The correlation of the protein concentration increase at 96 hours with the decrease of receptor bearing hemocytes may be due to the degranulation of the receptor bearing hemocytes.

Made available in DSpace on 2016-06-21T13:43:16Z (GMT). No. of bitstreams: 2 985.pdf: 1940501 bytes, checksum: 018fcc4969f375effb75a13c7d6c7974 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Made available in DSpace on 2016-07-05T22:16:55Z (GMT). No. of bitstreams: 3 985.pdf.txt: 141267 bytes, checksum: a925e4eaf511d9977eb6efbe2cc45af4 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 985.pdf: 1940501 bytes, checksum: 018fcc4969f375effb75a13c7d6c7974 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / O sucesso dos insetos em explorar diversos ambientes é devido, em grande parte, à habilidade em se defender contra patógenos e parasitas. Nos insetos, os principais mecanismos de defesa são desempenhados pelos hemócitos. A classificação dos tipos de hemócitos presentes na hemolinfa ainda é bastante controversa. A biodiversidade desses organismos tem proporcionado modelos importantes para o estudo de suas estratégias antimicrobianas, as quais podem fornecer informações relevantes para o combate a diversas doenças, bem como para o estudo da imunologia geral. O objetivo do presente estudo foi caracterizar a resposta imune celular de Aedes aegypti sob o ponto de vista morfológico, funcional e ultra-estrutural através da microscopia óptica e eletrônica de transmissão. Em nossos estudos identificamos seis tipos morfológicos de hemócitos circulantes na hemolinfa de Ae. aegypti através da microscopia de luz, eletrônica de transmissão e contraste de interferência diferencial, são eles: prohemócitos, adipohemócitos, granulócitos, plasmatócitos, oenocitóides e trombocitóides. Os prohemócitos foram as menores células encontradas na hemolinfa. Sua principal característica é a presença de um citoplasma ocupando uma pequena área em torno do núcleo. Os adipohemócitos foram os mais abundantes tipos celulares presentes e exibiam grandes inclusões lipídicas preenchendo praticamente todo o citoplasma. Os granulócitos possuem um citoplasma contendo diversos grânulos elétron-densos. Os plasmatócitos exibiram morfologia bastante polimórfica e diversos filopódios e pseudópodes. Os oenocitóides possuem citoplasma homogêneo com poucas organelas. Os trombocitóides são raros e possuem características similares aos oenocitóides com organelas pouco desenvolvidas. Os hemócitos responsáveis pela resposta imune contra partículas de látex conjugadas a FITC foram identificados através da microscopia laser confocal, assim como as lectinas que marcam os hemócitos. Os granulócitos foram as únicas células envolvidas na fagocitose de alvos abióticos in vitro e in vivo. As lectinas BSI, ConA, HPA, LCA, PNA, UEA e WGA marcaram os hemócitos com variações na intensidade. A WFA e LPL não marcaram hemócitos de Ae. aegypti (AU).

Procambarus clarkii hemocytes have been shown to bind to both LPS and a mannose containing neoglycoprotein conjugated to fluorescent probes. A decrease in the observed number of fluorescent hemocytes indirectly indicated that the binding of FITC-LPS to these cells is impaired by initial incubation with LPS. Prior treatment of hemocytes with horseradish peroxidase, a mannose rich glycoprotein, decreased their capacity to bind mannose. Mannan had no inhibitory effect on the binding of either ligand. Mixed hemocytes and hemocyte subpopulations separated by buoyant density were incubated with both ligands and revealed that a subpopulation of granular hemocytes bound both LPS and mannose. These results suggest the possible presence of two different pattern recognition cell surface receptors on this subpopulation of granular hemocytes from the red swamp crayfish, Procambarus clarkii, which may specifically recognize and bind to both LPS from gram-negative bacteria and to mannose containing glycoproteins found on microbial surfaces.

Crayfish exhibit innate immune responses via hemocytes and their products. There are 3 hemocyte populations: hyaline cells, granular cells, and semigranular cells. Hemocytes from laboratory housed, untreated crayfish (normal crayfish) have been quantified on the basis of cell type, cell size, and cell granularity using Flow Cytometry. These data present the first overall picture of normal hemocytes from Red Swamp Crayfish with regard to cell type, cell size, and cell granularity and will serve as a baseline for all future studies in our lab. Experiments using crayfish injected with Pseudomonas aeruginosa, Staphylococcus aureus, or crayfish saline alone showed significant and consistent changes in cell type in cells from crayfish injected with bacteria with a decrease in hyaline cells and an increase in granular cells. This effect was greater in crayfish injected with Gram - bacteria. In addition, crayfish injected with Pseudomonas aeruginosa showed a significant difference in Granular cell size with a shift to larger cells and a significant decrease in granularity in the Granular cell population. Cells from crayfish treated with Staphylococcus aureus did not show these changes.

Os invertebrados impedem o estabelecimento de uma infecção através de uma resposta imune eficiente. Esta resposta envolve reações celulares e humorais. Existem poucos trabalhos sobre a imunidade das aranhas. O principal objetivo deste estudo foi verificar o papel dos hemócitos e dos peptídeos antimicrobianos na resposta imune da aranha Acanthoscurria gomesiana. Inicialmente, a localização relativa da gomesina e da acanthoscurrina foi determinada, mostrando que 58% dos hemócitos armazenam os dois peptídeos antimicrobianos. Além disso, foi verificado que a gomesina é direcionada aos grânulos dos hemócitos da forma de pró-peptídeo. Observou-se ainda, que após um desafio, os hemócitos migram para o sítio de infecção, onde deve secretar componentes da cascata de coagulação e peptídeos antimicrobianos. Além disso, os resultados mostraram que a fagocitose não é o principal mecanismo ativado após uma infecção. Assim sendo, as principais respostas imunes da aranha são através da coagulação e secreção dos peptídeos antimicrobianos. / Invertebrates avoid the infection establishment through an efficient immune response. This response consists in cellular and humoral reactions. Few data are available concerning the spider\'s immunity. The main aim of this study was to determine the role of hemocytes and antimicrobial peptides on the immunity of the spider Acanthoscurria gomesiana. Initially, the localization of gomesin and acanthoscurrin was determined, showing that 58% of hemocytes store both antimicrobial peptides. Moreover, our results show that gomesin is addressed to the hemocyte granules as a pro-peptide. We also demonstrate, by in vivo and in vitro experiments, that hemocytes migrate to the site of microbial infection. Once at the site of infection, hemocytes might secrete components of coagulation cascade and antimicrobial peptides. Besides, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore the main reactions involved in the spider defense might be through the coagulation and antimicrobial peptides secretion.

Neste estudo avaliamos a ocorrência de fagocitose por parte de alguns tipos celulares presentes na hemolinfa do carrapato bovino B. microplus e a produção de espécies reativas de oxigênio durante a resposta imune. As técnicas empregadas para avaliação da produção de espécies reativas de oxigênio foram luminescência amplificada por luminol, oxidação de fenol vermelho, microscopia de fluorescência e fluorimetria com o corante dihidrorrodamina 123 (DHR). Observamos um aumento da luminescência amplificada por luminol quando hemócitos foram incubados na presença de bactérias Micrococcus luteus ou zimosam ou PMA. Esta luminescência foi inibida por superóxido dismutase (SOD) e por catalase (CAT), enzimas antioxidantes que removem superóxido e peróxido de hidrogênio, respectivamente. LPS não elicitou aumento da luminescência dos hemócitos em relação ao controle. Através da oxidação de fenol vermelho em reação inibida por CAT, verificamos aumento nos níveis de H2O2 produzido pelos hemócitos quando estimulados com PMA e Micrococcus luteus, enquanto não houve aumento quando o estímulo foi LPS, corroborando os resultados da luminescência. Usando microscopia de fluorescência para avaliar a produção de ERO pelos hemócitos, encontramos que cerca de 25% dos hemócitos fluorescem com maior intensidade quando estimulados com zimosam, sendo esta fluorescência inibida por CAT. Através de fluorimetria usando DHR observamos um aumento na intensidade de fluorescência dos hemócitos estimulados com PMA em reação inibida por cisteína, substância redutora que remove peróxido de hidrogênio e peroxinitrito. Nosso conjunto de resultados permitem concluir que os hemócitos do carrapato bovino produzem espécies reativas de oxigênio durante a resposta imune, semelhantemente ao que ocorre em vertebrados e em invertebrados como moluscos, crustáceos e insetos. Este é o primeiro trabalho mostrando produção de ERO pelos hemócitos de aracnídeos. / The phagocytic activity and the reactive oxygen species (ROS) production during immune response of some hemocytes of the cattle tick Boophilus microplus were evaluated in this study. The ROS production was evaluated by luminol amplified luminescence, phenol red oxidation, dyhydrorhodamine (DHR) fluorescence microscopy and fluorimetry. The luminol-amplified luminescence increased when hemocytes were incubated with bacteria (Micrococcus luteus) or zymosam or phorbol 12-miristate 13 acetate (PMA). The superoxide dismutase (SOD) and catalase (CAT), antioxidant enzymes that removes superoxide and hydrogen peroxide, respectively, inhibited this luminescence. Lipopolysaccharide (LPS) did not elicit luminescence of hemocytes in relation to controls. The catalase-inhibittable phenol red oxidation assay also showed an increase in the level of hydrogen peroxide produced by hemocytes stimulated with PMA or Micrococcus luteus. LPS did not stimulate the hemocytes, similarly to the observed by luminescence assay's. We also evaluated ROS production by fluorescence microscopy and we found approximately 25% more fluorescent hemocytes when zymosam was used. This fluorescence was inhibited by catalase. In DHR fluorimetry assay we observed an increase in the intensity of fluorescence in PMA stimulated hemocytes. This fluorescence was inhibited by cystein, a reducing agent that removes hydrogen peroxide and peroxinitrite. We conclude that hemocytes of the tick, like other invertebrate such as mollusks, crustaceans and insects and vertebrate, produce reactive oxygen species during the immune response. This is the first report of reactive oxygen species production by arachnid hemocytes.

As formigas-cortadeiras do gênero Atta são apontadas como os principais herbívoros da região Neotropical, sendo capazes de causar grandes danos à agricultura, às pastagens e à silvicultura em especial. No controle químico dessa praga, a tática mais usual e efetiva tem sido o uso de iscas granuladas tóxicas. Todavia, têm-se procurado métodos alternativos para o controle de cortadeiras, sobretudo por pressão de agências certificadoras de manejo florestal como o FSC (Forest Standarship Council). Recentemente, isolados de Trichoderma spp. começaram a ser testados no controle de formigas-cortadeiras em razão das suas propriedades antagonísticas ao fungo simbionte por elas cultivados. Além disso, destacam-se substâncias que têm o potencial de inibir a resposta imune inata dos insetos. Por exemplo, alguns elementos químicos como, cádmio e zinco. O presente trabalho objetivou o preparo de uma formulação com iscas granuladas e encapsuladas do fungo Trichoderma harzianum. Para encapsulação do fungo, utilizou-se uma mistura de alginato de sódio, farelo de trigo, suco concentrado de laranja e micélio triturado do antagonista. Esta mistura foi gotejada em solução (0,25 M) de CaCl2, o que permitiu a formação de grânulos esféricos de diâmetro regular. Paralelamente testou-se iscas contendo sulfato de zinco ZnSO4 (0,25 g/L), produzidas a partir da mistura de alginato, farelo de trigo e suco concentrado de laranja. Após o fornecimento das iscas fez-se a contagem total de hemócitos das operárias a fim de verificar alterações da sua resposta imune. Não ocorreu declínio na quantidade de hemócitos. Apesar das iscas não terem promovido a morte das colônias, elas apresentaram boa aceitação pelas operárias e promoveram a redução do volume do fungo simbionte. O cloreto e o sulfato de zinco foram empregados nas concentrações de 0,15; 0,25; 0,5; 1,5; 2;5 e 5;0 g/L em placas de Petri em meio BDA para o teste de desenvolvimento dos fungos simbionte e antagonista, e os resultados mostraram inibição no crescimento nas doses máximas tanto em Leucoagaricus gongylophorus como em Trichoderma harzianum. As operárias foram imersas em soluções de sulfato de zinco com as mesmas concentrações daquelas empregadas no teste de inibição dos fungos. Após o tempo de 24 e 48 horas fez-se a contagem total de hemócitos e verificou-se um decréscimo dos mesmos em altas concentrações. Conclui-se que as iscas contendo T. harzianum e sulfato de zinco apresentaram boa aceitação por parte das colônias, elas não promoveram a morte das colônias, no entanto, reduziram o volume do fungo simbionte. Altas doses de cloreto e sulfato de zinco inibem o desenvolvimento do fungo antagonista e do fungo simbionte e elevadas concentrações zinco e o maior tempo de exposição das operárias ao mesmo afetam o seu sistema imune. / The leaf-cutting ants of the genus Atta are cited as the main herbivores of the Neotropical region, being capable of causing major damage to agriculture, pasture and forestry in particular. In the chemical control of this plague, the most common and effective tactic has been the use of toxic granular baits. However, there have been alternative methods for control of cutting, especially by pressure certifying agencies forest management as the FSC (Forest Standarship Council). Recently, Trichoderma spp. They began to be tested in the control of leaf-cutting ants because of their antagonistic properties to the symbiotic fungus cultivated by them. Furthermore, they highlight substances that have the potential of inhibiting the innate immune response of the insects. For example, some chemical elements such as cadmium and zinc. This study aimed to the preparation of a formulation with granulated baits and encapsulated fungus Trichoderma harzianum. For encapsulation fungus, a mixture of sodium alginate was used wheat bran, concentrated orange juice and triturated antagonist mycelium. This mixture was dripped into solution (0,25 M) CaCl2, which allowed the formation of spherical granules of regular diameter. Parallel tested for baits containing zinc sulfate ZnSO4 (0,25 g/L) produced from the mixture of alginate, wheat bran and concentrated orange juice. After the supply of baits made up the total count of hemocytes of the workers in order to verify changes in their immune response. There was no decline in the amount of hemocytes. Despite the baits have not promoted the death of the colonies, they had good acceptance by workers and promoted the reduction of the symbiont fungus volume. Chloride and zinc sulfate were used in concentrations of 0.15; 0.25; 0.5; 1.5; 2, 5 and 5; 0 g /L in Petri dishes on PDA medium for the development and test of antagonist symbiont fungi, and the results showed growth inhibition in both maximal doses Leucoagaricus gongylophorus as Trichoderma harzianum. The ants were dipped in zinc sulfate solutions with the same concentrations of those employed in the fungal inhibition assay. After time 24 and 48 hours we made the total hemocytes count and there was a decrease in high concentrations thereof. We conclude that the baits containing T. harzianum and zinc sulfate showed good acceptance by the colonies, they did not promote the death of the colonies, however, reduced the volume of the symbiont fungus. High doses of zinc chloride and sulfate inhibit the development of the antagonist fungus and symbiont fungus and high concentrations of zinc and the longer exposure time of workers at the same affect your immune system.

Neste estudo avaliamos a ocorrência de fagocitose por parte de alguns tipos celulares presentes na hemolinfa do carrapato bovino B. microplus e a produção de espécies reativas de oxigênio durante a resposta imune. As técnicas empregadas para avaliação da produção de espécies reativas de oxigênio foram luminescência amplificada por luminol, oxidação de fenol vermelho, microscopia de fluorescência e fluorimetria com o corante dihidrorrodamina 123 (DHR). Observamos um aumento da luminescência amplificada por luminol quando hemócitos foram incubados na presença de bactérias Micrococcus luteus ou zimosam ou PMA. Esta luminescência foi inibida por superóxido dismutase (SOD) e por catalase (CAT), enzimas antioxidantes que removem superóxido e peróxido de hidrogênio, respectivamente. LPS não elicitou aumento da luminescência dos hemócitos em relação ao controle. Através da oxidação de fenol vermelho em reação inibida por CAT, verificamos aumento nos níveis de H2O2 produzido pelos hemócitos quando estimulados com PMA e Micrococcus luteus, enquanto não houve aumento quando o estímulo foi LPS, corroborando os resultados da luminescência. Usando microscopia de fluorescência para avaliar a produção de ERO pelos hemócitos, encontramos que cerca de 25% dos hemócitos fluorescem com maior intensidade quando estimulados com zimosam, sendo esta fluorescência inibida por CAT. Através de fluorimetria usando DHR observamos um aumento na intensidade de fluorescência dos hemócitos estimulados com PMA em reação inibida por cisteína, substância redutora que remove peróxido de hidrogênio e peroxinitrito. Nosso conjunto de resultados permitem concluir que os hemócitos do carrapato bovino produzem espécies reativas de oxigênio durante a resposta imune, semelhantemente ao que ocorre em vertebrados e em invertebrados como moluscos, crustáceos e insetos. Este é o primeiro trabalho mostrando produção de ERO pelos hemócitos de aracnídeos. / The phagocytic activity and the reactive oxygen species (ROS) production during immune response of some hemocytes of the cattle tick Boophilus microplus were evaluated in this study. The ROS production was evaluated by luminol amplified luminescence, phenol red oxidation, dyhydrorhodamine (DHR) fluorescence microscopy and fluorimetry. The luminol-amplified luminescence increased when hemocytes were incubated with bacteria (Micrococcus luteus) or zymosam or phorbol 12-miristate 13 acetate (PMA). The superoxide dismutase (SOD) and catalase (CAT), antioxidant enzymes that removes superoxide and hydrogen peroxide, respectively, inhibited this luminescence. Lipopolysaccharide (LPS) did not elicit luminescence of hemocytes in relation to controls. The catalase-inhibittable phenol red oxidation assay also showed an increase in the level of hydrogen peroxide produced by hemocytes stimulated with PMA or Micrococcus luteus. LPS did not stimulate the hemocytes, similarly to the observed by luminescence assay's. We also evaluated ROS production by fluorescence microscopy and we found approximately 25% more fluorescent hemocytes when zymosam was used. This fluorescence was inhibited by catalase. In DHR fluorimetry assay we observed an increase in the intensity of fluorescence in PMA stimulated hemocytes. This fluorescence was inhibited by cystein, a reducing agent that removes hydrogen peroxide and peroxinitrite. We conclude that hemocytes of the tick, like other invertebrate such as mollusks, crustaceans and insects and vertebrate, produce reactive oxygen species during the immune response. This is the first report of reactive oxygen species production by arachnid hemocytes.