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This study evaluated a recombinant aquaporin 1 protein of Rhipicephalus (Boophilus) microplus (RmAQP1) as antigen in a vaccine against R. sanguineus. Five dogs were immunized with RmAQP1 (10 µg) + adjuvant (Montanide) (G1), and five were inoculated with adjuvant only (G2), three times. Twenty-one days after the last immunization, animals of both groups were challenged with R. sanguineus larvae, nymphs and adults, and their biotic potential was compared. Blood samples were collected before each immunization and every 28 days after the last immunization for 10 weeks. Serum antibody titers (IgG) were assessed by ELISA. We observed that: engorgement period of adult females from G1 was 12% shorter than G2; larvae from G1 had 8.7% longer engorgement period than G2 and weighed 7.2% less; nymphs from G1 had 4.5% shorter engorgement period than G2 and weighed 3.6% less; although the antibody titers increased following the second immunization, they rapidly decreased after the third immunization. Results indicated low immunoprotection of RmAQP1 against adult R. sanguineus ticks, and possible efficacy on larvae and nymphs fed on immunized dogs. Further studies should be performed for a full evaluation of the immunoprotection of RmAQP1 against R. sanguineus infestations in dogs. Resumo Este estudo avaliou a proteína recombinante (aquaporina) do carrapato Rhipicephalus (Boophilus) microplus como antígeno em vacina contra Rhipicephalus sanguineus. Cinco cães foram imunizados com RmAQP1 (10 µg) + adjuvante (G1) e cinco foram inoculados apenas com adjuvante (G2), três vezes. 21 dias após a última imunização todos os animais foram desafiados com larvas, ninfas e adultos de R. sanguineus, e potencial biótico dos carrapatos foi comparado. Amostras de sangue foram coletadas antes de cada imunização e a cada 28 dias após a última imunização, durante 10 semanas. Títulos de anticorpos dos soros dos cães foram avaliados por ELISA. Resultados: o período de ingurgitamento das fêmeas do G1 foi 12% mais curto que o período de ingurgitamento de G2; o período de ingurgitamento das larvas do G1 8,7% foi mais longo e o peso 7,2% menor que no caso de G2; o período de ingurgitamento das ninfas do G1 4,5% foi mais curto e peso 3,6% menor que no caso do G2; aumento dos títulos de anticorpos do G1 após a segunda imunização e declínio após a terceira imunização. Os resultados indicaram baixo potencial de imunoproteção de RmAQP1 contra R. sanguineus adultos, e possível eficácia contra larvas e ninfas, na dose testada. Sugere-se desenvolver novos estudos para melhor avaliação da eficácia de RmAQP1 contra R. sanguineus em cães.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The age polyethism of Apis mellifera workers is related to morphological and physiological changes in many systems, including the digestive system, and to diet change of the bees, younger workers consuming mainly pollen and older bees consuming honey. The aquaporins are membrane proteins that have been identified in different organs of the digestive tract of insects and are responsible for transmembrane transport of water. This study evaluated whether there are differences in the expression of aqp-4-like, a gene for aquaporin in A. mellifera bees between nurses and foragers workers. The Malpighian tubules, crop, midgut, ileum and rectum of workers with different tasks were used to perform qPCR. The results showed that in nurse bees aqp-4-like mRNA was greater expressed in Malpighian tubules and ileum, while in the forager the highest expression occurred in the crop and rectum. The midgut was the only organ that showed no difference in expression between nurses and foragers, but it was the organ with the highest expression of aqp-4-like compared to the Malpighian tubules. These data show that AQP-4-like is differentially expressed in various organs of the digestive tract of nurses and foragers workers due to its different diets and physiology. The differential expression of the gene is related to osmoregulation in the Malpighian tubules, ileum and rectum. Also, the dehydration of the food content in the crop and midgut by this aquaporin avoids the digestive enzymes dilution, ensuring the completion of digestion. O polietismo etário das operárias de Apis mellifera está relacionado a alterações morfofisiológicas de diversos sistemas, incluindo o sistema digestório, e à mudança da dieta das abelhas, sendo que operárias mais jovens consomem principalmente pólen e as mais velhas mel. As aquaporinas são proteínas de membrana identificadas em diferentes órgãos do trato digestório de insetos e são responsáveis pelo transporte transmembrana de água. Este trabalho avaliou se há diferença na expressão de aqp-4-like, um gene para aquaporina de A. mellifera, entre abelhas nutridoras e campeiras. Os túbulos de Malpighi, papo, intestino médio, intestino fino e reto de operárias exercendo diferentes funções foram utilizados na realização de qPCR. Os resultados demonstraram que em abelhas nutridoras houve maior expressão de mRNA para aqp-4-like nos túbulos de Malpighi e no intestino fino, enquanto que nas campeiras ocorreu maior expressão no papo e reto. O intestino médio foi o único órgão que não apresentou diferença de expressão entre nutridoras e campeiras, mas foi o órgão com maior expressão de aqp-4-like quando comparado aos túbulos de Malpighi. Estes dados mostram que a AQP-4-like é diferencialmente expressa em vários órgãos do trato digestório de operárias nutridoras e campeiras devido às diferentes dietas e fisiologia. A expressão diferencial do gene está relacionada à osmorregulação nos túbulos de Malpighi, intestino fino e reto. A desidratação do alimento no papo e intestino médio por esta aquaporina evita a diluição de enzimas digestivas, garantindo a realização da digestão.

[spa] La hidratación de los oocitos de teleósteos marinos que producen huevos pelágicos es clave para la supervivencia de los embriones en el océano. Estudios previos han descu-bierto el papel de una acuaporina específica de teleósteos (Aqp1b) durante este proceso, pero se carece todavía de evidencias experimentales directas, así como información sobre la regulación molecular de la Aqp1ab. En la presente tesis, estudios iniciales con el fletan Atlántico (Hippoglossus hippoglossus) confirman que los parálogos aqp1aa y -1ab de teleósteos han surgido probablemente por duplicación génica en tándem, y han demostrado el papel esencial de la Aqp1ab durante la hidratación del oocito. Para investigar el control transcripcional del gen aqp1ab en los oocitos de la dorada (Sparus aurata) se ha aislado su región promotora, la cual contiene elementos cis-reguladores de unión al receptor nuclear de progestinas (Pgr), como la 17α,20β-dihidroxi-4-pregnen-3-ona (17,20β-P), y factores de transcripción Sox. Estudios de localización subcelular indican que el Pgr aparece en el citoplasma de las oogonias, así como en el núcleo de oocitos en crecimiento primario (pre-vitelogénicos) coincidiendo con la activación de la expresión de aqp1ab. En este estadio también se expresan cuatro isoformas diferentes del Pgr, dos de las cuales pueden inhibir la transcripción mediada por el Pgr de forma dominante negativa. Las oogonias también expresan sox3 y -8b, mientras que el sox9b aparece en el estadio de alveolo cortical, cuando se reduce la expresión de aqp1ab. Ex-perimentos de transactivación indican que el Pgr activa la transcripción de aqp1ab, con Sox3 y -8b actuando de forma sinérgica, mientras que el Sox9b reprime este mecanismo. El papel del Pgr se ha investigado sobre explantes ováricos pre-vitelogénicos incubados in vitro, lo cual ha demostrado que la 17,20β-P, producida en las células de la granulosa en respuesta a la hormona folículo estimulante, activa el promotor de aqp1ab en el oocito induciendo la síntesis de Aqp1ab. Los resultados de esta tesis revelan por primera vez una estricta regulación transcripcional del gen aqp1ab durante la oogénesis de teleósteos marinos, lo cual remarca la función fisiológica esencial de este canal de agua durante la formación de los huevos pelágicos, [eng] In marine teleosts that spawn pelagic eggs (pelagophils), the process of oocyte hydration that occurs during meiosis resumption is a key physiological process for the survival of the eggs in the ocean. Previous studies have discovered the role of a teleost-specific aquaporin water channel (Aqp1b) during fish oocyte hydration, but direct experimental evidence for the function of Aqp1b in oocytes is still lacking. In addition, the molecular regulation of the Aqp1b-mediated mechanism remains poorly understood. In this context, the main objectives of the present thesis were to investigate the evolutionary origin of aqp1ab in teleosts, to provide functional evidence of the role of Aqp1b during oocyte hydration, and to begin to dissect the molecular mechanisms involved in the transcriptional regulation of aqp1b in the oocyte of marine teleosts. By integrating the molecular phylogeny with synteny and structural analyses we show that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication, and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut (Hippoglossus hippoglossus), a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. Using immunological inhibition of Aqp1ab in halibut oocytes and artificial expression of the halibut paralog Aqp1aa, we demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. To investigate the aqp1ab transcriptional regulation in oocytes, we isolated the 5’-flanking region of the gilthead seabream (Sparus aurata) aqp1ab gene which contains regulatory cis-elements for the nuclear progestin receptor (Pgr) and SOX transcription factors. The Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oo-gonia, whereas in primary growth oocytes, when aqp1ab mRNA and protein are synthesized, the Pgr is translocated into the nucleus. In contrast, sox9b is highly expressed in more advanced oocytes showing the depletion of aqp1ab transcripts. In the seabream, four different pgr transcript variants are expressed in primary growth ovaries which are generated by alternative pre-mRNA splicing. Seabream wild-type Pgr shows the highest transactivation response to progestins such as 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), whereas two of the N-terminally truncated Pgr isoforms regulate novel nuclear and cytosolic mechanisms of dominant-negative repression of Pgr-mediated transcription. Transactivation assays on the aqp1ab promoter demonstrated that aqp1ab transcription is dependent on wild-type Pgr, with Sox3 and -8b acting synergistically, while Sox9b acts as a repressor. Incubation of primary ovarian explants in vitro with 17,20β-P, followed by chromatin immunoprecipitation, confirmed that 17,20β-P-activated Pgr enhanced aqp1ab promoter activation. The production of 17,20β-P in seabream primary growth ovaries in vivo was consistent with the expression of P450c17-II (Cyp17a2) and 20β-hydroxysteroid dehydrogenase (Cbr1), enzymes needed for progestins synthesis, in granulosa cells associated with primary growth oocytes, and with a high concentration of 17,20β-P. Incubation of primary ovarian explants with recombinant piscine follicle-stimulating hormone (rFsh) in vitro stimulated 17,20β-P synthesis, which was reduced in the presence of Cbr1 inhibitors. The rFsh-mediated production of 17,20β-P correlated with the up-regulation of cyp17a2 and cbr1 transcription, as well as of wild-type pgr mRNA and protein levels. Altogether, these data suggest that aqp1ab transcription in seabream primary growth oocytes is under Fsh regulation through the synthesis of progestins. The results of this thesis show that the Aqp1ab mediated mechanism for oocyte hy-dration is likely conserved in marine teleosts. In addition, the tight transcriptional reg-ulation of Aqp1ab during oogenesis highlights the essential physiological role of this water channel and opens new research avenues for understanding the molecular basis of egg formation in marine fish.

O diabetes insipidus nefrogênico (DIN) é uma doença rara caracterizada pela incapacidade do rim de concentrar a urina, a despeito de concentrações normais ou aumentadas do hormônio antidiurético arginina-vasopressina (AVP). Recentes avanços da fisiopatologia renal mostraram que, após a ligação do AVP ao seu receptor AVPR2 (receptor de vasopressina tipo 2), uma cascata de eventos culmina com a reabsorção de água no túbulo coletor, por meio de canais permeáveis exclusivamente à água e localizados nas membranas apicais do túbulo coletor, sendo o mais importante deles a aquaporina-2 (AQP2). A identificação, caracterização e análise mutacional dos genes AVPR2 e AQP2 permitiram estabelecer as bases moleculares de vários tipos hereditários de diabetes insipidus nefrogênico. Aproximadamente 90% desses pacientes apresentam mutações do AVPR2, 8% apresentam mutações no AQP2 e o restante não tem causas identificadas. Nessa revisão apresentamos exemplos de alterações genéticas e sugerimos que o uso de técnicas de biologia molecular pode minimizar as complicações dessa doença heterogênea mas com fenótipo bastante semelhante. Nephrogenic diabetes insipidus (NDI) is a rare disease characterized by the kidney failure to concentrate urine despite normal or increased plasma concentrations of the antidiuretic hormone arginine vasopressin (AVP). Recent advances in kidney physiopathology demonstrate that, after AVP binding to its receptor AVPR2 (vasopressin type 2 receptor), a cascade of events leads to water reabsorption in the collecting duct through channels exclusively permeable to water and localized at the apical membranes of these collecting ducts, the most important being aquaporin-2 (AQP2). The identification, characterization and mutational analysis of genes AVPR2 and AQP2 helped to establish the molecular basis of the various types of hereditary nephrogenic diabetes insipidus. Approximately 90% of patients present with mutations of AVPR2, 8% have mutations of AQP2 and the remaining do not have any identifiable causes. In this review, we present examples of such genetic alterations and suggest that the use of molecular biology techniques can minimize the complications of this heterogeneous disease that presents with similar phenotype.