Dorsal-ventral (DV) polarity in the Drosophila embryo is defined by spatially regulated activation of the transmembrane receptor Toll, which is uniformly distributed throughout the early embryo's plasma membrane. Ventral activation of Toll is accomplished through the local production of its activating ligand, a processed C-terminal fragment of the Spätzle protein, which is generated in the last step of a proteolytic cascade involving the sequentially-acting proteases Gastrulation Defective (GD), Snake and Easter. Pipe protein, a homologue of vertebrate glycosaminoglycan modifying enzymes, which is expressed during oogenesis in ventral follicle cells adjacent to the developing oocyte, is believed to control the ventrally restricted processing of Spätzle. pipe expression and the sulfation of its enzymatic target in the ventral follicle cells leads to the formation of a stable ventral cue, embedded in the eggshell. Recently the Pipe enzymatic target has been identified as several protein components of the vitelline membrane, the inner layer of the eggshell. Prior to this work, an important piece of information missing from our understanding of Drosophila DV patterning was the identity of the initial step in the protease cascade that requires Pipe activity. Here, I show that the processing of Snake is independent of Pipe activity, while the processing of Easter requires Pipe function, indicating that Easter processing by Snake is the key proteolytic step that is controlled by Pipe activity and presumably the first cleavage event that is spatially regulated. A second key gap in our understanding of Drosophila embryonic DV patterning concerned the role of GD in the protease cascade. While GD is the protease that cleaves and activates Snake, the existence of two distinct classes of complementing gd alleles has suggested that GD provides another, distinct function. Investigations described here indicate that the second function of GD is to promote the ability of activated Snake to process Easter, independent of its Snake-processing function. Finally, I provide evidence for the formation of protein complexes containing various components of the serine protease cascade, which suggest that conformational changes in the complexes, which act to promote productive interactions between the proteins, are an important aspect of their activation. / text

Universidade Federal de Uberlândia / CHAPTER II: Infection in insects stimulates a complex defensive response. Recognition of pathogens may be accomplished by plasma or hemocyte proteins that bind specifically to bacterial or fungal polysaccharides. Several morphologically distinct hemocyte cell types cooperate in the immune response. Hemocytes attach to invading organisms and then isolate them by phagocytosis, by trapping them in hemocyte aggregates called nodules, or by forming an organized multicellular capsule around large parasites. In the current investigation the cellular population in the hemolymph third instar larvae of M. scutellaris has been characterized by means of light microscopy analysis and phagocytosis assays were performed in vivo by injection of 0,5μm fluorescence beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found in 3rd instar larvae: prohemocytes, plasmatocytes, granulocytes and oenocytoids. The results presented plasmatocytes and granulocytes involved in phagocytic response of foreign particles in 3rd instar larvae of M. scutellaris. CHAPTER III: Insects are continuously exposed to potentially pathogenic microorganisms and eukaryotic parasites, but only a few encounters result in infection. Insects possess a complex and efficient system of biological defense against pathogens and parasites. This system involves the following: the integument and gut as physical barriers to infection, coordinated responses of several subpopulations of hemocytes when these barriers are breached, and the induced synthesis of antimicrobial peptides and proteins, primarily by the fat body. The purpose in the present study was to verify a Toll receptor (MsToll) expression in Melipona scutellaris. By semiquantitative RT-PCR we evaluate the MsToll levels at different development stages and in different M. scutellaris workers tissues. The MsToll expression in the immune response was evaluated by real time RT-PCR in workers infected with Escherichia coli (gram-negative). Our data showed lower MsToll expression in the larval stage compared with other development stages. The specific tissue analysis showed that its expression in intestine was significantly higher compared with other tissues analyzed. Furthermore, the MsToll levels in innate immune response of M. scutellaris showed four folds enhanced in bees infected with E. coli compared with control. / CAPITULO II: Infecção em insetos estimula uma resposta defensiva complexa. O reconhecimento de patógenos pode ser realizado pelos hemócitos ou proteínas que se ligam especificamente em microorganismos com padrões moleculares específicos, os chamados (PAMPs). Diferentes células da hemolinfa cooperam na resposta imune. Os hemócitos reconhecem os patógenos e os isolam por fagocitose, formando nódulos ou, cápsula multicelular em torno do parasita. Nesse trabalho foram identificadas as células da hemolinfa da abelha sem ferrão Melipona scutellaris e caracterizados os hemócitos envolvidos no processo de fagocitose utilizando beads de 0,5μm de diâmetro, em média, com fluorescência vermelha. Na hemolinfa do 3° instar larval de M. scutellaris foram distinguidos quatro tipos de hemócitos: prohemócitos, plasmatócitos, granulócitos e oenocitóides. No ensaio de fagocitose foram identificados plasmatócitos e granulócitos, com beads fluorescentes fagocitados no citoplasma. CAPITULO III: Insetos são continuamente expostos a microrganismos potencialmente patogênicos, mas apenas alguns contatos resultam em infecção. Insetos possuem um complexo e eficiente sistema de defesa contra patógenos e parasitas, que envolve o tegumento e intestino como barreiras físicas para infecção; respostas coordenadas de vários tipos de hemócitos quando estas barreiras são violadas e a síntese de peptídeos antimicrobianos e proteínas, principalmente pelo corpo gorduroso. Nosso objetivo foi clonar e sequenciar parcialmente um gene do sistema imune inato MsToll da abelha Melipona scutellaris. Por análises de RT-PCR semiquantitativo avaliou-se os níveis de expressão de MsToll em diferentes estágios do desenvolvimento e em diferentes tecidos de operárias de M. scutellaris. A expressão de MsToll na resposta imune foi avaliada por RT-PCR tempo real em operarias infectadas com Escherichia coli (gram-negativa). Os resultados mostraram menor expressão do gene MsToll nos estágios larvais quando comparados com os demais estágios do desenvolvimento. A análise tecido específico de MsToll mostrou que em intestino sua expressão foi significativamente maior quando relacionado com os demais tecidos analisados. Com relação aos níveis de MsToll na resposta imune observou-se o aumentou de quatro vezes dos níveis desse transcrito em abelhas infectadas com E. coli comparadas com o controle. / Mestre em Genética e Bioquímica