Made available in DSpace on 2016-06-02T20:20:33Z (GMT). No. of bitstreams: 1 4406.pdf: 2884968 bytes, checksum: a40d69433ee7cef0093f66b8c32c1bb8 (MD5) Previous issue date: 2012-03-06 / Universidade Federal de Minas Gerais / Bioluminescence in beetles is dependent on luciferase which evolved from AMP/CoA ligases. The cDNA of a luciferase-like enzime was cloned from the Malpighian tubules of Zophobas morio mealworm (Coleoptera: Tenebrionidae). The gene product of this cDNA displays weak luminescence and it is composed of 528 aminoacids residues with N-terminal and C-terminal sequences signal addressed to smooth endoplasmic reticulum membrane. Although having a low identity (26-32%) with beetle luciferases, this enzyme is a reasonable protoluciferase model to investigate the origin and evolution of beetle luciferases. The luciferin binding site is higly conserved among the beetle luciferases. However, in this protoluciferase of Z. morio, most of these residues of this motif are substituted by others. Using a site-directed mutagenesis survey some of aminoacids residues of this protoluciferase, which are located at correspondent luciferin binding site of luciferases, were replaced by the conserved residues of beetle luciferases. Most of the substitutions had negative effect on the luminescent activity, however, the substitution I327T, which is located in a β-hairpin motif close to the luciferin binding site, improved the luminescence activity. Such substitution indicates the importance of this motif for luciferase activity and indicates a possible route for the evolution of bioluminescence function of beetle luciferase. Since this enzyme is located in the Malpighian tubules, which are involved in excretion and metabolization of carboxylic substrates, this enzyme could be involved to excretion the some type of chemical compound. Regardless of the function the results show that the potential for bioluminescent activity is older and probably arose before the divergences of the Coleoptera bioluminescent families. / A bioluminescência em coleópteros é dependente das luciferases, enzimas que evoluíram das AMP-CoA ligases. O cDNA de uma enzima tipo-luciferase foi clonado dos túbulos de Malphighi de larvas de Zophobas morio (Coleoptera: Tenebrionidae). O produto gênico deste cDNA mostra naturalmente uma fraca luminescência na presença de MgATP e luciferina e possui 528 aminoácidos com sequências sinal na região N-terminal e C-terminal endereçadas a membrana do retículo endoplasmático liso. Apesar de ter uma baixa identidade (26-32%) com as luciferases de vaga-lumes, esta enzima é um modelo apropriado de protoluciferase para investigar a origem e evolução das luciferases de besouros. O sítio de ligação da luciferina é altamente conservado entre todas as luciferases de besouros; na protoluciferase de Z. morio porém, a maioria dos resíduos desta região é substituído por outros. Utilizando-se a técnica de mutagênese sitio-dirigida, alguns resíduos de aminoácidos desta protoluciferase, que são localizados na correspondente região do sítio ativo das luciferases, foram substituídos pelos resíduos conservados das luciferases. A maioria das substituições teve um efeito negativo sobre a atividade luminescente. Porém, a substituição I327T, cujo resíduo é localizado em um motivo grampo β, perto do sítio de ligação da luciferina, aumentou sua atividade luminescente. Tal substituição mostra a importância deste motivo para a atividade luciferásica e indica uma possível rota de evolução das luciferases de coleópteros. Uma vez que esta enzima foi extraída dos túbulos de Malpighi, é possível que esteja envolvida com a excreção de algum composto químico. Independente de sua função, os resultados do presente trabalho sugerem que o potencial para atividade bioluminescente é bem antigo nas ligases e provavelmente evoluíram antes da divergência das famílias de coleópteros bioluminescentes.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho utilizou-se a técnica fluorescência de raios X usando radiação síncrotron (SR-TXRF) para estudar, quantitativamente, o transporte de cloro, potássio e cálcio na hemolinfa, urina e túbulos de Malpighi (TM) em ninfas de quinto estágio do Rhodnius prolixus (R. prolixus), considerando a excreção destes elementos em diferentes dias após o repasto sanguíneo. R. prolixus é um dos principais vetores do Trypanosoma cruzi, agente causador da doença de Chagas. R. prolixus fornece um sistema modelo particularmente útil porque seus TMs tanto secretam quanto reabsorvem íons a taxas elevadas. Os TMs filtram a hemolinfa e secretam um líquido que é muitas vezes comparado com a urina primária em vertebrados. Os resultados obtidos mostram que a concentração de potássio na urina é substancialmente maior do que na hemolinfa. A concentração de cloro na hemolinfa é menor do que na urina, mas a diferença não é tão marcada como no caso do potássio. No caso do Rhodnius é razoável interpretar a elevada concentração de potássio na urina como adaptativo para o problema de excreção imediato do inseto. A concentração de cálcio nos TMs é substancialmente maior em comparação com os valores encontrados na hemolinfa e urina. Este resultado mostra que o cálcio é retido no corpo do R. prolixus e pouco eliminado. Os resultados obtidos estão coerentes com a literatura. Avaliou-se também o efeito no transporte de Cl, K e Ca após um repasto de sangue de coelho contaminado com HgCl2 de modo a avaliar o efeito da presença deste metal tóxico no balanço iônico nos fluidos de excreção urina e hemolinfa e também pelo principal órgão de transporte, os túbulos de Malpighi. As excreções de Cl e K pela urina são afetadas pela ingestão. Este resultado é esperado levando-se em consideração a ingestão de excesso de Cl através do HgCl2. O transporte de Cl, K e Ca na hemolinfa do Rhodnius prolixus não é afetada pela ingestão de HgCl2. Nos túbulos de Malpighi, as altas concentrações de Ca obtidas foram comparáveis àquelas encontradas nos insetos controle. Pode-se concluir que SR-TXRF é um método muito promissor para análises diretas, rápidas e confiáveis para a quantificação simultânea de elementos envolvidos na regulação do transporte e em todo o sistema excretor de insetos. Além disso, o estudo do transporte e a excreção de elementos no inseto Rhodnius prolixus abrem oportunidade para a maior compreensão de efeitos da poluição em espécies de invertebrados. / In this work, we investigated changes in the concentrations of Cl, K and Ca, in 5th instar using total reflection X-ray fluorescence Rhodnius prolixus with synchrotron radiation (SR-TXRF). The elements were quantified using urine, hemolymph and Malpighian tubules samples collected on different days after a blood meal. Rhodnius prolixus is one of the most important vectors of the Trypanosoma cruzi, causative agent of Chagas? disease. R. prolixus provides a particularly useful model system because its MTs both secrete and reabsorb ions at high rates. The TMs filter hemolymph and secrete a liquid that is often compared with the primary urine in vertebrates. The experimental results showed that the concentration of potassium in the urine is substantially greater than in the hemolymph. The concentration of chlorine in the hemolymph is generally less than in the urine, but the difference is not so marked as in the case of potassium. In the case of Rhodnius, it is reasonable to interpret the high concentration of potassium in the urine as adaptive to the animals? immediate excretory problem. The concentration of calcium in the TMs is substantially greater than in both the hemolymph and the urine. This result shows that that calcium is retained in the body and not eliminated. These results are in accordance with the literature. We also investigated whether dietary mercury contamination may influence the transport of Cl, K and Ca by the hemolymph, urine and Malpighian tubules of R. prolixus fed on blood containing HgCl2. The results suggested that dietary Hg contamination may influence the Cl and K contents during excretion of the urine. It was expected considering the large amounts of chlorine ingested by Rhodnius prolixus in its meals of blood containing HgCl2. Statistical analysis showed no significant variation in all elements contents for hemolymph samples. The main conclusion which can be drawn from the results is that in all the insects studied calcium is deposited in Malpighian tubules. These observations point out that the analytical approach of the SR-TXRF method can be efficiently used to measure elements involved in the transport regulation into insect Malpighian tubules and also provides useful data concerning the biological effects of pollution on invertebrate species.

Insects maintain blood (haemolymph) Ca2+ concentrations within a narrow range in order to support the health of internal tissues and organs. The Malpighian (renal) tubules play a primary role in haemolymph Ca2+ homeostasis by sequestering excess Ca2+ within calcified biomineral deposits (Ca-rich granules) often located within type I (principal) tubule cells. Using the classic Ramsay assay, the scanning ion-selective microelectrode technique (SIET), and modifications of these two electrophysiological techniques, this thesis begins to unravel the sites and mechanisms of Ca2+ transport by the Malpighian tubules isolated from eight insects, representing seven orders. A segment-specific pattern of Ca2+ flux was observed along the length of the Malpighian tubules isolated from D. melanogaster, A. aegypti and A. domesticus and was uniform along the length in the remaining species. The majority (≥ 90%) of Ca2+ entering the tubule cells is sequestered within intracellular calcium stores in Ca2+-transporting segments of D. melanogaster and A. domesticus tubules, consistent with the presence of Ca-rich storage granules in these tubule segments. In addition, this thesis provides the first measurements of basolateral Ca2+ flux across single principal and secondary tubule cells of T. ni, where Ca2+ uptake occurs only across principal cells. Perhaps the most important finding of this thesis is that increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca2+ in isolated tubules of A. domesticus had opposite effects on tubule Ca2+ transport. The adenylyl cyclase-cAMP-PKA pathway promotes Ca2+ sequestration whereas both 5-hydroxytryptamine and thapsigargin inhibited sequestration. In contrast, tubules of the remaining species were generally insensitive to cAMP or thapsigargin and v rates of tubule Ca2+ transport were often very low. The presence of Ca-rich granules in the cells of the midgut in several of the species with low rates of tubule Ca2+ transport provide evidence for a putative role of the midgut in haemolymph Ca2+ homeostasis. Taken together, these results suggest that the principal cells of the Malpighian tubules contribute to haemolymph calcium homeostasis through neuroendocrine regulated sequestration of excess Ca2+ during periods of high dietary calcium intake. Sequestration of dietary Ca2+ by the midgut may reduce Ca2+ entry into the haemolymph and therefore Ca2+ sequestration by the Malpighian tubules need not be so rapid. Finally, reversible tubule Ca2+ transport may allow internal reserves of Ca2+ (Ca-rich granules) to be returned to the haemolymph allowing insects to survive prolong periods of Ca2+ deficiency (i.e. overwintering). / Thesis / Doctor of Philosophy (PhD) / This thesis contributes to our understanding of how insects regulate the calcium content of their blood (haemolymph). Using electrophysiological techniques with improved spatial resolution (from millimeters to micrometers) this thesis sought to determine the sites, mechanisms and regulation of Ca2+ transport by insect Malpighian (renal) tubules in order to gain insights into the role of Ca-rich granules (similar to those identified in early stages of human kidney stone formation i.e. nephrolithiasis) within these tissues. Using eight insect species this thesis demonstrates that the Malpighian tubules act as dynamic Ca2+ stores that appear to be under neuroendocrine control: actively taking up Ca2+ through calcium entry channels, where the majority (≥ 90%) of excess haemolymph Ca2+ is sequestered within intracellular stores (Ca-rich granules) during period of excess dietary calcium and passively releasing Ca2+ back to the haemolymph during periods of metamorphosis or calcium deficiency (i.e. overwintering).

Présentement, la désorption ionisation laser assistée par la matrice (MALDI) est la méthode d’ionisation préférentielle pour étudier les lipides par l’imagerie par spectrométrie de masse (IMS). Bien qu’il existe les matrices spécifiques aux lipides, tel que la 1,5-DAN pour les phospholipides et la 2,5-DHB pour les triacylglycérols, il est toujours nécessaire d’augmenter la sensibilité de cette technique pour les échantillons atypiques ou certaines classes de lipides. Dans la première étude, nous avons amélioré la sensitivité pour les phospholipides sur les tubes de Malpighi de mouches prélevés par microdissection dans un tampon physiologique à base de sodium et potassium. Un protocole de lavage à deux étapes a était trouvé favorable : un premier rinçage dans le glycérol suivi d’un second rinçage dans l’acétate d’ammonium. Ce protocole permet de réduire au maximum la présence de sels sans délocalisation notoire des phospholipides. La détection et l’imagerie des phospholipides en ionisation négative et positive ont suggéré une distribution uniforme sur toute la longueur des tubes. Ces résultats ont été comparés à ceux obtenus sur des sections tissulaires minces de mouche entière acquis avec les deux polarités. Néanmoins, la structure tridimensionnelle complexe des tubes rénaux suggère que la microdissection est l’approche la plus favorable pour en étudier leur lipidome. Dans la deuxième étude, nous avons déterminé que l’addition de formate d’ammonium (AF) peut améliorer la détection des gangliosides par IMS dans le cerveau. Curieusement, il est nécessaire de rincer l’échantillon dans une solution d’AF avant l’addition de ce même sel suivit d’une conservation de l’échantillon dans un congélateur pendant 24 heures après la déposition de la matrice afin d’obtenir la meilleure augmentation de sensibilité. En moyenne, cette approche a permis d’augmenter l’intensité d’un facteur dix avec trois fois plus d’espèces de gangliosides détectées. De plus, malgré l’étape de lavage, nous n’avons pas observé la délocalisation des gangliosides puisqu’il est toujours possible d’obtenir les résultats d’IMS de qualité avec une résolution spatiale de 20 µm. Finalement, nous avons établi que le nitrate d’argent permet l’analyse des oléfines par IMS, en particulier du cholestérol. En optimisant le protocole de déposition par nébulisation, il est possible de générer une couche mince et homogène de nitrate d’argent ce qui rend la possibilité d’effectuer l’IMS à haute résolution spatiale, jusqu’à 10 µm, sans perte de qualité comparativement aux autres approches publiées. L’ensemble de ce travail démontre l’effet du sel sur la sélectivité et la sensibilité pour cibler les familles de lipides désirées, ce qui nécessite les études ultérieures sur le rôle de ces sels lors du processus de la désorption-ionisation. / Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is currently the ionization method of choice for elucidating the spatial distribution of lipids on thin tissue sections. Despite the discovery of lipid friendly matrices such as 1,5-DAN for phospholipids and 2,5-DHB for triacylglycerols, there is a continued need to improve sensitivity. In the first study, we improved the overall sensitivity for phospholipids of entire fly Malpighian tubules microdissected in PBS with a two-step wash in glycerol followed by ammonium acetate that removed the bulk of the salt with minimal species delocalization and tubule displacement. We were able to detect phospholipids in both positive and negative ion modes and revealed an even distribution of most phospholipids along the length of this organ. We compared the method to the results from whole body fly sections acquired in dual-polarity mode at the same spatial resolution and found it to be more suitable for studying the tubules because of the complex three-dimensional structure of this organ within the fly. In the second study, we observed a marked improvement in ganglioside signals on mouse brain tissue sections with ammonium salt addition. Specifically, when the sample was first desalted in a low concentration ammonium formate solution, spray-coated with the same salt, coated with matrix and finally left in the freezer overnight before data acquisition, we observed an average overall improvement in ganglioside signal intensity by ten-fold and the number of species detected by three-fold. This method also did not affect the spatial distribution of the gangliosides, as high spatial resolution IMS results acquired at 20 µm showed no species delocalization. Finally, we sought to determine if salts could be employed directly as matrices. In this work, we tested silver-based metal salts and discovered that spray depositing silver nitrate alone is a viable method for the IMS detection of olefins, particularly cholesterol. With the optimized dry spray parameter, the overall deposition is homogeneous and composed of microscopic salt crystals that allow for high spatial resolution IMS down to 10 µm while maintaining acceptable overall signal quality comparable to that of previously published protocols. Overall, this thesis demonstrates we can manipulate the local salt distribution to influence the sensitivity and selectivity to target specific lipid subfamilies, opening the door for future research to understanding the role salts play during the laser desorption/ionization process.