Acidentes envolvendo abelhas africanizadas são freqüentemente relatados, particularmente na América do Sul. As picadas de abelhas causam reações localizadas e sistêmicas e os sintomas do envenenamento incluem náuseas, vômitos, hemólise, falência renal e coagulação intravascular disseminada. Durante muito tempo todas as reações tóxicas eram atribuídas à presença de uma fosfolipase A2, mesmo sendo o veneno uma mistura complexa de substâncias. A melitina, o componente mais abundante e tóxico do veneno de abelha, é um peptídeo de 26 aminoácidos com a habilidade de interagir e danificar membranas celulares. A melitina é também capaz de modular muitas proteínas, aumentando a diversidade de atividades biológicas do peptídeo. Até recentemente, acreditava-se que a estrutura tri-dimensional biologicamente ativa do peptídeo, que possui conformação em hélice, era um tetrâmero. Neste trabalho avaliamos a conformação da melitina e seus estados oligoméricos em solução por dinâmica molecular e a interferência da melitina na função plaquetária. Aqui está demonstrado que a melitina possui uma conformação randômica em condições fisiológicas e que sua estrutura tri-dimensional sofre alterações de acordo com as condições ambientais. Ainda, foi demonstrada uma nova atividade biológica do peptídeo melitina. O peptídeo é capaz de induzir a 8 agregação plaquetária de forma dose-dependente e de interagir diretamente com a superfície de plaquetas. A correlação entre a conformação da melitina e suas atividades biológicas é discutida. Os resultados aqui apresentados podem ser valiosos no entendimento do papel da melitina nas coagulopatias induzidas por veneno de abelha. O estudo estrutural mostrado aqui pode ser aplicado para explicar as diferentes atividades do peptídeo. / Accidents involving africanized bees are frequently reported, particularly in South America. Bee stings cause localized and systemic reactions and the symptoms of envenomation include nausea, vomiting, hemolysis, kidney failure and disseminated intravascular coagulation. For a long time, all toxic reactions were ascribe to the presence of a phospholipase A2, despite of being the venom a complex mixture of substances. Melittin, the most abundant and the major toxic component of bee venom, is a 26 amino acid peptide with the ability to interact and disrupt cell membranes. Melittin is also able to modulate many proteins, enhancing the wide range of the peptide biological activities. The biologically active tridimensional structure of the peptide, which has a helical conformation, has been described until now as a tetramer. In this work we evaluated the conformation of melittin and its oligomeric states in solution by molecular dynamics simulations and performed studies of melittin effect on platelet function. Here we demonstrate that melittin has a random conformation under physiological conditions and its tridimensional structure changes under different environmental conditions. Moreover, here we describe a new biological activity of melittin. The peptide is able to induce platelet aggregation in a dose10 dependent manner and can interact directly with the platelet surface. The correlation between melittin conformation and biological activity is discussed. Our results might contribute to elucidate the role of melittin in bee venom induced coagulopathies. The structural data gathered in this work may explain the different activities of the peptide.

The European honey bee, Apis mellifera, is a primarily beneficial insect for mankind. It has been utilized by humans for thousands of years for the products and services it provides. Crop pollination and honey production are two of the most economically beneficial activities of the honey bee. Though they have been important for many centuries and immeasurable amounts of effort have been expended investigating the methods and means to harness their natural abilities, a far lesser amount of attention has been directed towards exploring their molecular makeup. These experiments involve identifying modification of gene transcription as a result of exposure to a pheromone or a parasite. This data will provide information on the general types of transcripts involved in the biochemical response of the honey bee to the two stimuli and will also provide specific candidates for further investigation of their potential role in downstream behavioral events.

Microsporidia are a group of obligate intracellular fungal parasites that infect a wide range of vertebrates and invertebrates, and are of economic and academic interest. Some areas of their economic impact are in aquaculture where they can infect salmon and other fish species. In agriculture they have been considered as control agents for insect pests, but more importantly as likely contributing to colony collapse disorder of bees. As an academic topic, microsporidia are fascinating because they are the smallest and simplest eukaryotic cells and require eukaryotic host cells in order to complete their life cycle. Therefore one research avenue that moves forward both economic and academic interests is to use cultures of animal cells to support the growth and development of the microsporidia life cycle, including the production of spores. Although the use of animal cell cultures for studying the microsporidia of insect and mammals has a fairly large literature, fish cell cultures have been employed less often but have had some successes as reviewed in this thesis. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable for describing the growth and development of the microsporidia and for documenting the responses of fish cells to infection. In this thesis, cell lines from warm water fish, goldfish, fathead minnow and zebrafish, and a coldwater species, rainbow trout, were explored as potential cellular hosts of two microsporidia species that have never been grown or associated with fish before. One is Anncaliia algerae, which is an aquatic microsporidium that most commonly infects mosquitoes. This microsporidia is one of the easiest species to grow in mammalian cells, with the rabbit kidney cell line, RK 13, being the most documented culture system. The other is Nosema apis, which is a pathogen of bees and for which few cell culture systems exist. The ability of warm water fish cell lines to support the life cycle of A. algerae was investigated first. Spores were purified from RK-13 cultures and added to cell lines from three warm water species as well as to an insect cell line. The cell lines were GFSK-S1 and GFB3C- W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27 °C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish and insects. As ZEB2J was the most characterized of the fish cell lines and supported good A. algerae growth, this cell line was used in further studies described below to compare the efficacy of antimicrosporidial drugs and to test whether fish cells could support N. apis growth, but first A. algerae growth at lower temperatures was explored with cell lines from a coldwater fish. Cultures of cell lines from rainbow trout gill, RTgill-W1, and brain, RTbrain-W1, at 9, 18 and 21°C were evaluated for their ability to support the development of A. algerae. For up to 8 days after the addition of spores, living and DAPI stained cultures were examined by phase-contrast microscopy, allowing the identification of the meront, sporont, and spore stages in cultures at 18 and 21 °C. Meronts and sporonts were both spindle-shaped, but relative to meronts, sporonts were darker under phase contrast and brighter after DAPI staining. Spores were egg-shaped, phase- bright and intensely DAPI stained. These stages could not be identified conclusively in cultures at 9 °C, but their appearance at 18 °C sets a new low temperature for the growth of this species. The growth of A. algerae at room temperature allowed living cultures to be observed conveniently and videoed with a proprietary instrument, the Riveal microscope (www.quorumtechnologies.com). With this microscope, the development of A. algerae life cycle stages at room temperature was confirmed plus for the first time meront division and intracellular germination were captured on video. Spore germination in the absence of host cells and in response to 3 percent hydrogen peroxide was also observed by Riveal microscopy and for first time an abnormal germination phenomenon was clearly documented: polar tubes were extruded but the spore bodies retained the nuclei. ZEB2J cultures that had been infected with Anncaliia algerae spores were used as an in vitro test system to evaluate the curative actions of albendazole, fumagillin, and three fluoroquinolones; ciprofloxacin, norfloxacin, and ofloxacin. For each drug at concentrations above 50 µg/ml, the viability of ZEB2J cell declined sharply so concentrations of 10 and 20 µg/ml were studied. At these concentrations the drugs had little effect on the morphology and germination A. algerae spores. Each of the fluoroquinolones failed to prevent A. algerae from infecting ZEB2J cells and from growing to the same extent as in untreated ZEB2J cultures. Adding albendazole or fumagillin to cultures did not prevent A. algerae from infecting ZEB2J cells but impeded the growth and accumulation of A. algerae life-cycle stages. However, albendazole treatments caused a significant fraction of the ZEB2J cells to have nuclear abnormalities. Fumagillin reduced the intensity of infections within a ZEB2J cell, although the number of infected cells in a culture was not reduced. Over 5 days of infection with A. algerae the accumulation of ZEB2J cells in cultures was reduced but fumagillin treatment restored the accumulation to control levels. These results suggest that fumagillin has some potential as a treatment for A. algerae infections. ZEB2J was exposed to Nosema apis spores from the western honey bee (Apis mellifera). Bees were collected from hives that had been naturally infected and confirmed polymerase chain reaction (PCR) to have N. apis. Frozen bees were crushed in water to yield a mixture of bee parts, pollen grains, yeast, and microsporidial spores. The mixture was filtered and then centrifuged through Percoll to produce a pellet of spores that was resuspended in L-15 with 10 percent fetal bovine serum (FBS). Aliquots of this were added to ZEB2J cultures. Cultures were observed periodically for up to 24 days with a combination of phase contrast microscopy and of fluorescence microscopy, usually after staining with 4’,6-diamidino-2-phenylindole (DAPI). Although earlier life cycle stages were not observed, structures that were concluded to be either sporonts, sporoblasts and/or spores were seen, but these were in less than 5 percent of the fish cells. These N. apis life cycle stages had grown in ZEB2J because some appeared to be inside the cells and often they were arranged around the nucleus of the host cell rather than being randomly distributed in cultures. Despite repeated rinsing over a three week period, all cultures were ultimately lost due to yeast from the original spore preparations over growing the fish cell cultures. The overarching observation of this thesis is that fish cells in culture have been shown for the first time to support the growth A. algerae, and possibly N. apis. This suggests that the cells of vertebrates might support the growth of a wide range of microsporidia species that normally are associated with insects. In turn this suggests restriction of a microsporidial species to a particular animal group is unlikely accomplished at the cellular level but through physiological systems expressed at the organismal level and disturbances in these systems might lead to infections in new groups of animal hosts. The overarching observation of this thesis has two general implications for future studies. Firstly, for studying the expression of antimicrosporidia mechanisms in fish cells, the ZEB2J/A. algerae co-culture system promises to be useful. Secondly, for microsporidia species that are difficult to grow in culture, cell lines from a wide range of vertebrate and invertebrate species should be explored and one possibility for N. apis is fish cells.

The European honey bee, Apis mellifera, is a primarily beneficial insect for mankind. It has been utilized by humans for thousands of years for the products and services it provides. Crop pollination and honey production are two of the most economically beneficial activities of the honey bee. Though they have been important for many centuries and immeasurable amounts of effort have been expended investigating the methods and means to harness their natural abilities, a far lesser amount of attention has been directed towards exploring their molecular makeup. These experiments involve identifying modification of gene transcription as a result of exposure to a pheromone or a parasite. This data will provide information on the general types of transcripts involved in the biochemical response of the honey bee to the two stimuli and will also provide specific candidates for further investigation of their potential role in downstream behavioral events.

Originally described from the Asian honey bee, Apis cerana, the microsporidian Nosema ceranae is an obligate, intracellular parasite that has recently been discovered infecting the western honey bee, Apis mellifera. More research on the biology of N. ceranae as well as on the impact it may have on A. mellifera is greatly needed. However, conducting studies on N. ceranae is not only dependent on seasonal availability of Nosema spores, but also on reliable methods for determining spore viability. This study presents the results of using cryogenics to provide long term storage of viable N. ceranae spores and a differential staining procedure that details how to use bright field microscopy with the fluorescent viability dye, propidium iodide (PI), and the fluorescent stain, 4', 6-diamidino-2-phenylindole (DAPI) to differentiate viable and nonviable spores. Using these methods, it was found that freezing N. ceranae at -70 °C in 10% glycerol yielded the lowest mean rate of spore mortality after thawing (24.2% ± 2.2).

In this project I studied the importance of pollinators in the reproduction of Gastrodia ‘long column’ and the preferences of New Zealand’s native and exotic insect pollinators. This was done in order to determine the specialisation of insect pollination in urban Christchurch. This knowledge can contribute to crop pollination, conservation efforts, and an understanding of the main drivers of the common floral traits (small, white flowers) in New Zealand. The strength of the relationship between the common traits of the New Zealand flora and the preferences of the native invertebrate fauna is not known. Traditionally it had been thought that New Zealand’s insects lack strong preferences, however recent work has not supported this. Changing landscapes worldwide have led to declines in pollinator numbers. Additionally, in New Zealand Apis mellifera numbers have declined as a result of the arrival of the varroa mite and it is important that alternative pollinating species for wild and agricultural pollination are identified. To address these needs I examined the abundance and preferences of insect pollinators in modified landscapes in and around Christchurch testing: the reliance upon pollinators by the undescribed native orchid Gastrodia ‘long column’, the preferences of New Zealand’s native and introduced insect pollinators in an extensive observational study, the results of which I further tested using a subsequent manipulative experiment of petal colour (according to human vision) at the Christchurch Botanical Gardens Sampling at the Christchurch Botanical Gardens, University of Canterbury, and Port Hills across a four month observation period (January – April 2012), revealed that native pollinators preferred white native flowers and exotic pollinators preferred not-white exotic flowers when data were grouped according to insect provenance. A more detailed visit-level analysis found that two native bees, Hylaeus and Leioproctus, showed a significant preference for native flowers; the exotic bees, Apis mellifera and Bombus species, preferred purple over white flowers; and Melangyna novaezelandiae (a native hoverfly) preferred white over yellow. However, a series of experimental arrays to present controlled choices between pairs of flower types at the Christchurch Botanical Gardens (14 December 2012 – 22 January 2013) did not find significant preferences by native or exotic insect pollinators between white and yellow flowers. This may have been a result of the plant species chosen, as a correlation between pollinator preference and plant species has been shown elsewhere. Visitation to experimental arrays was both low and highly skewed, with over half of all visits made by Lasioglossum bees and 615 of the 669 visits made by native species. This may have meant that lack of significant results were representing the choices of native insects generally and Lasioglossum bees specifically. A bagging experiment from 30 January 2012 – 16 February 2012 demonstrated the dependence of the undescribed native orchid species, Gastrodia ‘long column’, upon pollinating animals. Fruit set of most plants worldwide depends upon pollination (by wind or animal-transfer of pollen). In this case study final fruit sets were significantly reduced on bagged inflorescences, while open flowers had surprisingly high natural fruit sets (>75%). A week of observations (29 January 2012 – 6 February 2012) revealed that Gastrodia ‘long column’ was predominantly visited by Lasioglossum bees, and remarkably bee numbers were high enough in a residential property in the middle of Christchurch city for high fruit set on unmanipulated plants. The results of my thesis indicate that pollinators are important in the reproductive system of Gastrodia ‘long column’, suggesting that other previously overlooked plant species may also rely upon insect pollinators. Furthermore, the relative importance of native pollinating insects is high for native plants even when examined in an urban setting. New Zealand pollinators have preferences for certain floral traits which show trends when grouped broadly, but vary when considered at the insect species level. This contrasts with traditional views of unspecialised insect pollinators which lack preferences in regards to the plants they visit. Further work which serves to increase current understanding of the underlying mechanisms of pollination specialisation in New Zealand may wish to focus on single insect species. By identifying particular preferences of pollinators and the underlying ‘native’ traits selected for, alternative options to crop pollination may be found, targeted management strategies implemented, and the strength of the relationship between pollinator preferences and the traits of the plants they visit determined.

Under vintrarna 2006/2007 och 2007/2008 vittnade man i USA om storskaliga förluster avhonungsbin (Apis mellifera). Gemensamma symptom för dessa förluster var att arbetarbinförsvann från kolonierna fastän det fanns rikligt med mat och inga tecken på infektion av någonparasit. Fenomenet fick namnet Colony Collapse Disorder (CCD) eftersom det slutgiltigastadiet var att kolonierna kollapsade. Denna översiktstudie ämnar till att reda ut begreppetCCD och se över vilka potentiella orsaker den rådande forskningen föreslår och vad man harkommit fram till sedan utbrottet 2006. Resultaten indikerar att det antagligen inte är en ensamfaktor bakom CCD utan att flera faktorer samverkar. Det finns ett antal hot mot bina medsymptom som liknar CCD och som antas bidra till försvinnandet. Ett hot är det parasitiskakvalstret Varroa destructor som förutom att det suger binas hemolymfa även agerar vektor åtflera CCD-förknippade patogener som Nosema ceranae och Israeli acute paralysis virus(IAPV). Dessutom sänker V. destructor binas immunförsvar vilket gör att virus som överförsvia kvalstret replikeras snabbare och blir dödligare. Man har även visat att vanligt förekommandebekämpningsmedel kan göra bina mer mottagliga för sjukdomar. Där hittade man ettsignifikant samband med just N. ceranae vilket är i enlighet med teorin om att flera faktorersamverkar. I nuläget bidrar CCD fortfarande till bidöd men är inte lika akut som 2006/2007.Istället är det andra hot såsom biodlares svårighet att behandla invasion av varroakvalster,kalla vintrar och att bisamhällen svälter ihjäl för att de invintras i för små populationer, som ärett större hot än CCD.

Microsporidia are a group of obligate intracellular fungal parasites that infect a wide range of vertebrates and invertebrates, and are of economic and academic interest. Some areas of their economic impact are in aquaculture where they can infect salmon and other fish species. In agriculture they have been considered as control agents for insect pests, but more importantly as likely contributing to colony collapse disorder of bees. As an academic topic, microsporidia are fascinating because they are the smallest and simplest eukaryotic cells and require eukaryotic host cells in order to complete their life cycle. Therefore one research avenue that moves forward both economic and academic interests is to use cultures of animal cells to support the growth and development of the microsporidia life cycle, including the production of spores. Although the use of animal cell cultures for studying the microsporidia of insect and mammals has a fairly large literature, fish cell cultures have been employed less often but have had some successes as reviewed in this thesis. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable for describing the growth and development of the microsporidia and for documenting the responses of fish cells to infection. In this thesis, cell lines from warm water fish, goldfish, fathead minnow and zebrafish, and a coldwater species, rainbow trout, were explored as potential cellular hosts of two microsporidia species that have never been grown or associated with fish before. One is Anncaliia algerae, which is an aquatic microsporidium that most commonly infects mosquitoes. This microsporidia is one of the easiest species to grow in mammalian cells, with the rabbit kidney cell line, RK 13, being the most documented culture system. The other is Nosema apis, which is a pathogen of bees and for which few cell culture systems exist. The ability of warm water fish cell lines to support the life cycle of A. algerae was investigated first. Spores were purified from RK-13 cultures and added to cell lines from three warm water species as well as to an insect cell line. The cell lines were GFSK-S1 and GFB3C- W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27 °C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish and insects. As ZEB2J was the most characterized of the fish cell lines and supported good A. algerae growth, this cell line was used in further studies described below to compare the efficacy of antimicrosporidial drugs and to test whether fish cells could support N. apis growth, but first A. algerae growth at lower temperatures was explored with cell lines from a coldwater fish. Cultures of cell lines from rainbow trout gill, RTgill-W1, and brain, RTbrain-W1, at 9, 18 and 21°C were evaluated for their ability to support the development of A. algerae. For up to 8 days after the addition of spores, living and DAPI stained cultures were examined by phase-contrast microscopy, allowing the identification of the meront, sporont, and spore stages in cultures at 18 and 21 °C. Meronts and sporonts were both spindle-shaped, but relative to meronts, sporonts were darker under phase contrast and brighter after DAPI staining. Spores were egg-shaped, phase- bright and intensely DAPI stained. These stages could not be identified conclusively in cultures at 9 °C, but their appearance at 18 °C sets a new low temperature for the growth of this species. The growth of A. algerae at room temperature allowed living cultures to be observed conveniently and videoed with a proprietary instrument, the Riveal microscope (www.quorumtechnologies.com). With this microscope, the development of A. algerae life cycle stages at room temperature was confirmed plus for the first time meront division and intracellular germination were captured on video. Spore germination in the absence of host cells and in response to 3 percent hydrogen peroxide was also observed by Riveal microscopy and for first time an abnormal germination phenomenon was clearly documented: polar tubes were extruded but the spore bodies retained the nuclei. ZEB2J cultures that had been infected with Anncaliia algerae spores were used as an in vitro test system to evaluate the curative actions of albendazole, fumagillin, and three fluoroquinolones; ciprofloxacin, norfloxacin, and ofloxacin. For each drug at concentrations above 50 µg/ml, the viability of ZEB2J cell declined sharply so concentrations of 10 and 20 µg/ml were studied. At these concentrations the drugs had little effect on the morphology and germination A. algerae spores. Each of the fluoroquinolones failed to prevent A. algerae from infecting ZEB2J cells and from growing to the same extent as in untreated ZEB2J cultures. Adding albendazole or fumagillin to cultures did not prevent A. algerae from infecting ZEB2J cells but impeded the growth and accumulation of A. algerae life-cycle stages. However, albendazole treatments caused a significant fraction of the ZEB2J cells to have nuclear abnormalities. Fumagillin reduced the intensity of infections within a ZEB2J cell, although the number of infected cells in a culture was not reduced. Over 5 days of infection with A. algerae the accumulation of ZEB2J cells in cultures was reduced but fumagillin treatment restored the accumulation to control levels. These results suggest that fumagillin has some potential as a treatment for A. algerae infections. ZEB2J was exposed to Nosema apis spores from the western honey bee (Apis mellifera). Bees were collected from hives that had been naturally infected and confirmed polymerase chain reaction (PCR) to have N. apis. Frozen bees were crushed in water to yield a mixture of bee parts, pollen grains, yeast, and microsporidial spores. The mixture was filtered and then centrifuged through Percoll to produce a pellet of spores that was resuspended in L-15 with 10 percent fetal bovine serum (FBS). Aliquots of this were added to ZEB2J cultures. Cultures were observed periodically for up to 24 days with a combination of phase contrast microscopy and of fluorescence microscopy, usually after staining with 4’,6-diamidino-2-phenylindole (DAPI). Although earlier life cycle stages were not observed, structures that were concluded to be either sporonts, sporoblasts and/or spores were seen, but these were in less than 5 percent of the fish cells. These N. apis life cycle stages had grown in ZEB2J because some appeared to be inside the cells and often they were arranged around the nucleus of the host cell rather than being randomly distributed in cultures. Despite repeated rinsing over a three week period, all cultures were ultimately lost due to yeast from the original spore preparations over growing the fish cell cultures. The overarching observation of this thesis is that fish cells in culture have been shown for the first time to support the growth A. algerae, and possibly N. apis. This suggests that the cells of vertebrates might support the growth of a wide range of microsporidia species that normally are associated with insects. In turn this suggests restriction of a microsporidial species to a particular animal group is unlikely accomplished at the cellular level but through physiological systems expressed at the organismal level and disturbances in these systems might lead to infections in new groups of animal hosts. The overarching observation of this thesis has two general implications for future studies. Firstly, for studying the expression of antimicrosporidia mechanisms in fish cells, the ZEB2J/A. algerae co-culture system promises to be useful. Secondly, for microsporidia species that are difficult to grow in culture, cell lines from a wide range of vertebrate and invertebrate species should be explored and one possibility for N. apis is fish cells.

Texte remanié de: Thesis--University of Amsterdam, 1984. / Contient le texte égyptien du papyrus Vindobonensis 3873 avec sa trad. anglaise. Bibliogr. p. [XI]-XVIII. Index.

Acidentes envolvendo abelhas africanizadas são freqüentemente relatados, particularmente na América do Sul. As picadas de abelhas causam reações localizadas e sistêmicas e os sintomas do envenenamento incluem náuseas, vômitos, hemólise, falência renal e coagulação intravascular disseminada. Durante muito tempo todas as reações tóxicas eram atribuídas à presença de uma fosfolipase A2, mesmo sendo o veneno uma mistura complexa de substâncias. A melitina, o componente mais abundante e tóxico do veneno de abelha, é um peptídeo de 26 aminoácidos com a habilidade de interagir e danificar membranas celulares. A melitina é também capaz de modular muitas proteínas, aumentando a diversidade de atividades biológicas do peptídeo. Até recentemente, acreditava-se que a estrutura tri-dimensional biologicamente ativa do peptídeo, que possui conformação em hélice, era um tetrâmero. Neste trabalho avaliamos a conformação da melitina e seus estados oligoméricos em solução por dinâmica molecular e a interferência da melitina na função plaquetária. Aqui está demonstrado que a melitina possui uma conformação randômica em condições fisiológicas e que sua estrutura tri-dimensional sofre alterações de acordo com as condições ambientais. Ainda, foi demonstrada uma nova atividade biológica do peptídeo melitina. O peptídeo é capaz de induzir a 8 agregação plaquetária de forma dose-dependente e de interagir diretamente com a superfície de plaquetas. A correlação entre a conformação da melitina e suas atividades biológicas é discutida. Os resultados aqui apresentados podem ser valiosos no entendimento do papel da melitina nas coagulopatias induzidas por veneno de abelha. O estudo estrutural mostrado aqui pode ser aplicado para explicar as diferentes atividades do peptídeo. / Accidents involving africanized bees are frequently reported, particularly in South America. Bee stings cause localized and systemic reactions and the symptoms of envenomation include nausea, vomiting, hemolysis, kidney failure and disseminated intravascular coagulation. For a long time, all toxic reactions were ascribe to the presence of a phospholipase A2, despite of being the venom a complex mixture of substances. Melittin, the most abundant and the major toxic component of bee venom, is a 26 amino acid peptide with the ability to interact and disrupt cell membranes. Melittin is also able to modulate many proteins, enhancing the wide range of the peptide biological activities. The biologically active tridimensional structure of the peptide, which has a helical conformation, has been described until now as a tetramer. In this work we evaluated the conformation of melittin and its oligomeric states in solution by molecular dynamics simulations and performed studies of melittin effect on platelet function. Here we demonstrate that melittin has a random conformation under physiological conditions and its tridimensional structure changes under different environmental conditions. Moreover, here we describe a new biological activity of melittin. The peptide is able to induce platelet aggregation in a dose10 dependent manner and can interact directly with the platelet surface. The correlation between melittin conformation and biological activity is discussed. Our results might contribute to elucidate the role of melittin in bee venom induced coagulopathies. The structural data gathered in this work may explain the different activities of the peptide.