O peptídeo-1 semelhante ao glucagon (GLP-1) é um hormônio incretina secretado pelas células L do trato gastrointestinal e liberado imediatamente após a ingestão de alimento. O GLP-1 estimula a secreção de insulina pós-prandial moderando a elevação precoce da glicose no sangue. Embora primariamente envolvido na homeostase da glicose, o GLP-1 é capaz de induzir a diurese e natriurese, quando administrado em doses farmacológicas em humanos e em roedores. Estudos prévios do nosso laboratório demonstraram que o mecanismo de ação renal do GLP-1, bem como de agonistas sintéticos do receptor GLP-1R, envolve o aumento do fluxo plasmático renal (FPR) e do ritmo de filtração glomerular (RFG) bem como a diminuição da reabsorção de sódio dependente da isoforma 3 do trocador Na?/H? (NHE3) em túbulo proximal renal. Entretanto, até o momento, nenhum estudo investigou se o GLP-1 endógeno exerce efeitos sobre o manuseio renal de sal e água, nem o seu papel fisiológico sobre a regulação da atividade do NHE3. Portanto, o objetivo deste estudo foi testar a hipótese que o GLP-1 endógeno modula a função renal de ratos, ao menos em parte, via inibição da atividade do NHE3 em túbulo renal. Para este fim, ratos Wistar (2-3 meses de idade) foram devidamente anestesiados, submetidos à traqueostomia e tiveram a veia jugular e a bexiga canuladas para infusão de uma solução contendo 100 ug/kg/min do antagonista do receptor GLP-1R exendin-9 (Ex-9, 40 uL/min) por um período de 30 minutos e para a coleta de urina, respectivamente. A infusão sistêmica de Ex-9 diminuiu a concentração de AMPc urinário e atividade da PKA cortical renal consistente com o bloqueio da sinalização deflagrada pela interação GLP-1/GLP-1R no rim. Além disso, a administração sistêmica de Ex-9 reduziu a diurese, natriurese, RFG, FPR, clearance de lítio e pH urinário. Em experimentos de microperfusão estacionária in vivo, não foram observadas diferenças no fluxo de bicarbonato dependente de NHE3 entre os túbulos proximais perfundidos com exendin-9 (2 uM) e os túbulos perfundidos com solução controle. No entanto, a perfusão tubular proximal com Ex-9 foi capaz de bloquear completamente as ações inibitórias do GLP-1 (20 nM) sobre a atividade do NHE3. Por outro lado, a infusão sistêmica do Ex-9 reduziu os níveis de fosforilação da serina 552, sítio consenso para a fosforilação por PKA localizado na região C-terminal do NHE3, e que está associado à inibição da atividade de troca Na+/H+ mediada por este transportador. Baseando-se nos achados que a infusão sistêmica do Ex-9 aumenta a reabsorção de sódio e secreção de H?, reduz o clearance do lítio e os diminui os níveis de fosforilação do NHE3 na serina 552 são consistentes com um aumento na atividade deste transportador na ausência/redução da sinalização mediada pela interação do GLP-1 endógeno com seu receptor no rim. Por sua vez, o fato do Ex-9 não afetar a atividade do NHE3 sob as condições experimentais da microperfusão estacionária in vivo é condizente com o fato do GLP-1 não ser sintetizado no néfron e sugere fortemente que é o GLP-1 filtrado que se liga ao seu receptor no túbulo proximal renal resultando na diminuição da reabsorção de bicarbonato de sódio mediada pelo NHE3. Em conjunto, estes resultados sugerem que o GLP-1 endógeno exerce efeito tônico sobre o manuseio renal de sódio e água, mediando portanto, uma relação funcional entre a homeostase glicêmica e volêmica / The glucagon like peptide-1 (GLP-1) is an incretin hormone secreted by the L-cells of the gastrointestinal tract and released immediately after ingestion of food. GLP-1 stimulates postprandial insulin secretion moderating early increase in blood glucose. Although primarily involved in glucose homeostasis, GLP-1 is capable of inducing diuresis and natriuresis when administered in pharmacologic doses in humans and rodents. Previous studies from our laboratory have shown that the renal mechanism of action of GLP-1 and synthetic agonists of GLP-1R receptor, involves an increase of renal plasma flow (RPF) and glomerular filtration rate (GFR) as well a decrease in reabsorption of sodium mediated by the Na? / H? exchanger (NHE3) isoform 3 in the renal proximal tubule. However, to date, no study has investigated whether endogenous GLP-1 exerts effects on the renal handling of salt and water, or its physiological role in the regulation of the activity of NHE3. Therefore, the aim of this study was to test the hypothesis that endogenous GLP-1 modulates renal function in rats, at least in part, via inhibition of the NHE3 in renal tubule. To this end, male Wistar rats (2-3 months old) were properly anesthetized, tracheostomized and the jugular vein and the bladder were cannulated to the infusion of a solution containing 100 ug / kg / min GLP-1R antagonist receiver exendin-9 (Ex-9, 40 uL/min) for a period of 30 minutes and to collect urine, respectively. Systemic infusion of Ex-9 reduced the urinary concentration of cAMP and the renal cortical PKA activity, consistent with the blockage of the signal triggered by the interaction of GLP-1 / GLP-1R in the kidney. Furthermore, systemic administration of ex-9 reduced diuresis, natriuresis, GFR, RPF, lithium clearance and urinary pH. In experiments of in vivo stationary microperfusion, no differences were observed in the NHE3-mediated net bicarbonate flow between proximal tubules perfused with exendin-9 (2 mM) and perfused tubules with control solution. However, the tubular proximal perfusion with Ex-9 was able to completely block the inhibitory actions of GLP-1 (20 nM) on the activity of NHE3. On the other hand, systemic infusion of Ex-9 reduced phosphorylation levels of serine 552, a consensus site for phosphorylation by PKA located in the C-terminal region of NHE3, which is associated with inhibition of exchange activity of Na+/H+ mediated by this transporter. Collectively, the findings that systemic infusion of Ex-9 increases sodium reabsorption and secretion of H+, reduces the lithium clearance and decreases the NHE3 phosphorylation at serine 552 levels are consistent with the idea that NHE3 activity is upregulated in the absence/reduction of the signaling cascade mediated by the interaction of the endogenous GLP-1 with its receptor in the kidney. In turn, the fact Ex-9 does not affect the activity of NHE3 under the experimental conditions of stationary microperfusion in vivo is consistent with the fact that GLP-1 is not synthesized in the nephron. Besides, it strongly suggests that is the filtrated GLP-1 that binds to its receptor in renal proximal tubule, resulting in a decrease in NHE3-mediated sodium bicarbonate reabsorption. Taken together, these results suggest that endogenous GLP-1 exerts a tonic effect on renal sodium and water handling, mediating therefore a functional relationship between volume and glucose homeostasis

O presente estudo objetivou testar experimentalmente a existência da possível correlação entre a penetração de cimento nos túbulos dentinários e a qualidade do selamento. Foram utilizados 60 incisivos centrais superiores humanos que formaram um único grupo experimental. Após a eliminação das porções coronárias, as raízes foram padronizadas em 13 mm de comprimento. A instrumentação dos canais foi realizada no sentido coroa-ápice, e o comprimento de trabalho estabelecido a 1 mm aquém do forame apical. Como solução irrigadora foi empregado o NaOCl a 5,25% e ao final, EDTA a 17%. Em seguida, todos os canais foram obturados com guta-percha e cimento AH Plus marcado com um corante fluorescente. Para determinar a qualidade do selamento das obturações endodônticas, as amostras foram submetidas ao modelo de infiltração de glicose sob pressão. As raízes foram montadas em um dispositivo de dupla-câmara selada para permitir a infiltração da glicose. Como controle negativo foram utilizados 4 dentes hígidos, e como controle positivo, 2 dentes instrumentados porém, não obturados. Foram utilizados 0,75 mL de solução de glicose a 1 mol/L na câmara superior e 0,75 mL de água deionizada na câmara inferior. Os dispositivos foram conectados a um sistema de distribuição de pressão desenvolvido com o objetivo de permitir a infiltração de 32 amostras em uma mesma etapa. A solução de glicose foi forçada apicalmente sob uma pressão de 15 psi durante 1 hora. Uma alíquota de 50 L foi coletada da câmara inferior para quantificar a glicose infiltrada. A concentração de glicose foi determinada através de um método enzimático com o auxílio do Kit Glucose HK e de um espectrofotômetro em um comprimento de onda de 340 nm. Na sequência, as amostras foram desacopladas dos corpos de prova, embutidas em resina epóxi e cortadas em 3 secções transversais. Uma sequência de preparação metalográfica padrão foi realizada para permitir a observação da penetração de cimento nos túbulos dentinários por meio de microscopia confocal e óptica. Os dados obtidos nos 2 experimentos foram cruzados pelo teste de correlação de Spearman, o qual revelou a inexistência de qualquer possibilidade de correlação (r = 0,12). Com base nesses resultados, o presente trabalho concluiu que, dentro das condições experimentais usadas, a quantidade de cimento presente dentro dos túbulos dentinários não teve relação com a qualidade do selamento produzido. / The purpose of the present study was to experimentally test the potential correlation between tubular dentin sealer penetration and sealability. Sixty human maxillary central incisors were selected in order to compose a single experimental group. After removing of crowns, the roots were standardized at 13 mm in length. A crown-down root canal instrumentation was performed 1mm short of the apical foramen (working length). How irrigating solution was used 5.25% NaOCl and final, 17% EDTA. Then, all root canals were filled with gutta percha and AH Plus sealer labeled with a fluorescent dye. To determine the sealability pattern, samples were submitted to glucose leakage model under pressure. Four intact teeth were used as negative control while further 2 instrumented but nonfilled teeth was used as positive control. The roots were mounted in a double-chamber apparatus where 0,75 mL of 1 mol/L glucose solution was placed into the superior chamber and 0,75 mL of deionized water was placed into the inferior chamber. The devices were connected to a pressure distribution system that was developed in order to allow testing 32 specimens at the same experimental stage. The glucose solution was forced apically under a pressure of 15 psi during 1 hour. A sample of 50 L was taken from the inferior chamber and the glucose concentration was measured following an enzymatic reaction using a Glucose HK Kit readed under spectrophotometry (wave-length of 340 nm). After that, the samples were removed from the apparatus, embedded in epoxy resin and cut into 3 sections. Standard metallographic preparation was performed prior the observation of the sealer penetration into dentinal tubules by confocal and optical microscopy. Data provided by the evaluations were submitted to Spearman correlation test which revealed a lack of correlation between the two variables (r = 0.12). Therefore, the present study concluded that there was no correlation between tubular sealer penetration and sealability in non-bonding conventional root-fillings.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Apesar de muito se discutir sobre os efeitos citotÃxicos dos venenos ofÃdicos, pouco ainda à conhecido sobre os mecanismos de aÃÃo sobre as diversas cÃlulas, e em especial, sobre as cÃlulas renais. No caso particular da citotoxicidade dos venenos crotÃlicos, tem-se postulado a participaÃÃo de diversos metabÃlitos da hidrÃlise de lipÃdios de membrana, e, mais recentemente, da disfunÃÃo mitocondrial. O presente trabalho teve como objetivo estudar o efeito da fosfolipase A2 (FLA2) isolada do veneno da Crotalus durissus terrificus sobre rim isolado de rato assim como estudar a toxicidade e as alteraÃÃes da funÃÃo mitocondrial induzidas pelas FLA2s de pÃncreas de porco (PFLA2) e de veneno da C. d. terrificus (VSFLA2) em suspensÃes de tÃbulos proximais (TP). No rim isolado foi observado aumento no fluxo urinÃrio, no ritmo de filtraÃÃo glomerular (RFG) e na pressÃo de perfusÃo (PP) enquanto ocorreram decrÃscimos nos percentuais de transporte total de sÃdio (%TNa+), de potÃssio (%TK+) e de cloreto (%TCl-). Na anÃlise histopatolÃgica foi observada a deposiÃÃo de material proteinÃceo nos tÃbulos de rins perfundidos com FLA2. No estudo de suspensÃes de TP tratadas com PFLA2 e com VSFLA2 foi observado que estas induziram injÃria celular, sugerida pelo aumento na liberaÃÃo de lactato desidrogenase (LDH), promoveram aumentos nos nÃveis de Ãcidos graxos nÃo esterificados (AGNE) e diminuÃram o potencial de membrana mitocondrial (ΔΨm), sem, no entanto, alterar os nÃveis de ATP. Em relaÃÃo ao ΔΨm, a PPFLA2 nÃo produziu efeitos nas concentraÃÃes mais elevadas, apesar de ter aumentado, significativamente, na menor concentraÃÃo. As concentraÃÃes mais elevadas da FLA2 crotÃlica, entretanto, induziram um decrÃscimo significativo no ΔΨm. A adiÃÃo de BSA reverteu completamente os efeitos das FLA2s sobre o ΔΨm. No estudo da permeabilidade mitocondrial de transiÃÃo (PMT) foi observado que a PFLA2 e a VSFLA2 promoveram a liberaÃÃo da safranina O e, por entanto, induziu a formaÃÃo de PMT, apesar da leve edema mitocondrial produzido. Conclui-se que as fosfolipases A2 de pÃncreas de porco e do veneno da C. d. terrificus produziram um efeito citotÃxico em preparaÃÃes de tÃbulos proximais evidenciado pelo aumento na liberaÃÃo de LDH, alÃm de promoverem alteraÃÃes no potencial mitocondrial de membrana, o que sugere alteraÃÃo da funÃÃo mitocondrial por essas enzimas. Em rim isolado, foi observado que a FLA2 crotÃlica promoveu alteraÃÃes da funÃÃo renal / Although the increasing interest on biological effects of snake venoms, their cytotoxic as well as their nephrotoxic mechanisms are still unknown. In the particular case of crotalic venoms, it has been postulated the participation of several metabolites from membrane phospholipids hydrolysis and more recently, mitochondrial dysfunction. The present work investigated the renal effects promoted by the phospholipase A2 (PLA2) isolated from Crotalus durissus terrificus venom in the isolated rat kidney. Addition of PLA2 increased UF, GFR and PP, while reducing %TNa+, %TK+ and %TCl-. The histological analysis showed a mild amount of a proteinaceous substance in the renal tubules of kidneys perfused with PLA2. In the present study also showed that the phospholipase A2 isolated from porcine pancreas (PPLA2) and from C d terrificus snake venom (SVPLA2) produced cellular injury suggested by the increase in LDH release and increased nonesterified fatty acid (NEFA) levels from rabbit proximal tubules in suspension. Furthermore, the SVPLA2 induced a decrease in mitochondrial membrane potential (ΔΨm) assessed by both JC-1 uptake and safranin O uptake. PPLA2 produced no effects on ΔΨm with the highest concentrations, and an unexpected increase in the group treated with the lowest concentration. Addition of BSA completely reversed the effects induced by phospholipases on ΔΨm. It was observed no changes in cell ATP levels. Finally, to determine whether mitochondrial membrane permeability was affected by PPLA2 and SVPLA2, we measured the change safranin O uptake to assess both changes in mitochondrial volume and in ΔΨm. The latter was affected by both PLA2s although small alterations in the mitochondrial volume were observed. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic PLA2s disturbed the membrane integrity as well as the mitochondrial function. Furthermore, crotalic PLA2 altered renal function in the isolated rat kidney preparation.

O Pseudohermafroditismo masculino disgenético (Anomalia da diferenciação sexual 46,XY ADS 46,XY) é definido como ambigüidade genital num paciente com testículos e/ou cariótipo 46,XY com uma das seguintes características: alteração histológica testicular, ausência ou hipoplasia das células de Leydig em tecido previamente estimulado com gonadotrofina coriônica humana(hCG), falta de resposta de testosterona ao estímulo com hCG sem acúmulo de precursores, ausência de células germinativas, presença de derivados müllerianos indicando inadequada produção do hormônio antiMülleriano (HAM) ou resistência de seus receptores. Esse estudo apresenta uma avaliação clínica, laboratorial, anátomopatológica, morfométrica e molecular de 10 pacientes com ADS 46,XY; dois pacientes apresentaram mutação no SF1 (fator esteroidogênico 1), duas mutações no domínio hingee uma terceira produziu um stop códon na posição 404; três pacientes com deleção da cópia do DAZ2. A morfometria testicular mostrou todos os diâmetros tubulares médios (DTM) moderado a gravemente diminuídos e os índices de fertilidade tubular leve a moderadamente diminuídos. Devido à dificuldade do diagnóstico diferencial e etiológico, o estudo morfométrico e molecular deve sempre acompanhar esses casos de ADS 46,XY. / The dysgenetic male pseudohermaphroditism 46,XY ; disorders of sex development (DSD 46,XY) is defined as sexual ambiguity in patients with testis and/or 46,XY karyotype and one of the characteristics: hystologic alteration of the testis; absence or hypoplasia of Leydig cells; a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors; absent germ cells; presence of müllerian duct derivatives showing inappropriate production of antimüllerian hormone (AMH) or resistance to its receptors. This study shows the clinic, laboratory, histologic, morphometric and molecular evaluation of 10 patients with DSD 46,XY; two patients showed mutations in the SF1 gene (steroidogenic factor-1); two in the hinge domain and one stop codon at the position 404 of the protein; three patients exhibited deletion of DAZ2. The testis morphometry showed reduction: marked to severe of all mean tubular diameter (MTD) while the reduction of the tubular fertility index (TFI) were slight to marked. Due to difficulties establishing the differential diagnosis and the etiology, the morphometric and molecular evaluation must be always done in the patients with DSD 46,XY.

O objetivo foi determinar se aldosterona e corticosterona agem sobre a acidificação do túbulo proximal e se esses efeitos são genômicos e/ou não-genômicos. A reabsorção de HCO3- foi avaliada por microperfusão estacionária. Aldosterona e corticosterona perfundidas na luz tubular causaram aumento significante do JHCO3-. Na presença de etanol, actinomicina D, cicloheximida ou espironolactona, o JHCO3- foi estatisticamente igual ao valor controle (2,84 ± 0,079 nmol.cm-2.s-1). RU486 sozinho inibiu o efeito estimulador da aldosterona e corticosterona. Losartan não alterou o JHCO3-. Concanomicina ou S3226 diminuiram o efeito estimulador da corticosterona. A aldosterona perfundida nos capilares peritubulares aumentou o JHCO3-. Assim, a aldosterona e corticosterona tem um efeito rápido, não-genômico, estimulante do JHCO3-, provavelmente com a participação do GR e pela ativação do NH3 e da H+-ATPase luminais. Além disto, a aldosterona e corticosterona endógenas estimulam o JHCO3- no túbulo proximal. / The purpose was to determine if aldosterone and corticosterone act on the acidification of proximal tubule and if these hormonal effects are genomic and/or nongenomic. Bicarbonate reabsorption was evaluated by microperfusion. Aldosterone and corticosterone caused a significant increase in JHCO3-. In the presence of ethanol, actinomycin D, cycloheximide or espironolactone, the JHCO3- was not different from the control value (2.84 ± 0.079 nmol.cm-2.s-1). However, in the presence of RU486 a decrease on JHCO3- was observed. Losartan inhibited the JHCO3-. Concanamicyn or S3226 decreased the stimulatory effect of corticosterone. Aldosterone perfused into peritubular capillaries also increased JHCO3-. Our results indicate that: aldosterone and corticosterone has a rapid, nongenomic, stimulatory effect on JHCO3-; probably, GR participates in this process and; this effect, probably, occurs by activation of luminal NH3 and H+-ATPase. Besides, endogenous aldosterone and corticosterone stimulate JHCO3-.

G protein-coupled receptors (GPCRs) are composed of seven transmembrane domains and play an essential role in regulating physiological functions and mediating responses to environmental stimuli, biogenic amines, neurotransmitters, peptides, lipids, and hormones. The calcitonin-like diuretic hormone 31 (DH31) is known to elicit natriuresis from the Malpighian tubules (MTs) of mosquitoes Anopheles gambiae and Aedes aegypti upon blood feeding. However, the contribution of DH31 cognate receptor, calcitonin receptor 1 (GPRCAL1), has not been evaluated with respect to postprandial fluid regulation or myostimulatory activity in blood feeding insects. Thus, this dissertation has investigated potential roles of AedaeGPRCAL1 in the regulation of fluid homeostasis and hindgut muscle contraction in female A. aegypti mosquito. The full length cDNA encoding AedaeGPRCAL1 was cloned and sequenced. The receptor expression in the MTs and hindgut from female mosquito was analyzed by western blot and immunohistochemistry using anti-AedaeGPRCAL1 affinity purified antibodies, and subsequently its role in fluid transport and hindgut contraction was evaluated by RNA interference (RNAi). The mosquitoes that underwent knock-down of the AedaeGPRcal1 exhibited up to 57% lower rate of MT fluid secretion in presence of Aedae-DH31 in the in vitro assay and a ~30% reduction in the fluid excreted from live females upon blood feeding. The receptor was immunolocalized in principal cells, predominantly towards the distal end of MTs. Analyses of receptor signal probability indicate the receptor is expressed in a gradient-like fashion along the length of the MTs. A striking discovery was the fact that not all principal cells express the receptor, contrary to previous belief. Immunolocalization revealed the AedaeGPRCAL1 is expressed in hindgut circular and longitudinal muscles. The application of DH31 increased the frequency of hindgut contractions in all female mosquitoes, those injected with AedaeGPRcal1 dsRNA and controls, as compared to their basal contraction rate, but the percent change in frequency of hindgut contraction from AedaeGPRcal1 knock-down females was about 2-fold lower than the controls after application of Aedae-DH31. To my knowledge, this is first evidence of RNAi-induced phenotypes in any invertebrate that allowed the quantification of the contribution of single family B GPCR to fluid loss and muscle contractility.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Τα τελευταία χρόνια πολλές ερευνητικές ομάδες εστίασαν στην ταυτοποίηση γονιδίων που ενέχονται σε διάφορες βιολογικές διεργασίες και η Δροσόφιλα αποτέλεσε τον ιδανικό οργανισμό-μοντέλο λόγω της διαθεσιμότητας πολλών γενετικών εργαλείων. Η ανάπτυξη της τεχνολογίας της RNA παρεμβολής ευνόησε ιδιαίτερα την ευρείας κλίμακας γενετική ανάλυση στη Δροσόφιλα. Εκμεταλλευόμενοι τα διαθέσιμα εργαλεία πραγματοποιήσαμε μία μελέτη σάρωσης βασισμένη σε RNAi για γονίδια που εμπλέκονται στη μορφογένεση του συστήματος των σωματικών μυών και του επιθηλίου του φτερού της Drosophila melanogaster. Συνολικά, εξετάσαμε 321 γονίδια με συστημική ή ιστοειδική RNAi σιώπηση στο μεσόδερμα ή με συνδυασμό και των δύο και ταυτοποιήσαμε 58 γονίδια άγνωστης λειτουργίας τα οποία χρειάζονται για την ανάπτυξη και ομοιόσταση του εμβρυϊκού/προνυμφικού μυϊκού συστήματος. Περιέργως, στις μισές σχεδόν περιπτώσεις δεν παρατηρήσαμε φαινότυπο θνησιμότητας πλήρους διεισδυτικότητας, γεγονός που υποδεικνύει ότι ο αριθμός των γονιδίων που συμμετέχουν στη συγκεκριμένη διεργασία είναι μεγαλύτερος από αυτόν που έχει προβλεφθεί με βάσει τα αποτελέσματα μίας ευρείας κλίμακας μελέτης σάρωσης με RNAi στο μυϊκό σύστημα που ολοκληρώθηκε πρόσφατα. Επιπλέον, μελετήσαμε 242 γονίδια με ιστοειδική σιώπηση στα επιθήλια και ταυτοποιήσαμε 32, τα οποία είναι απαραίτητα για τη βιωσιμότητα, και 24, τα οποία είναι αναγκαία για τη μορφοποίηση του ενήλικου φτερού. Από τα γονίδια που έδωσαν θετικό αποτέλεσμα επιλέξαμε ένα το οποίο είναι απαραίτητο και για τις δύο διεργασίες, το chd64. Το chd64 κωδικοποιεί μία πρωτεΐνη που αποτελείται από μία δομική περιοχή με ομολογία στις καλπονίνες (CH) και ένα μοτίβο CLIK23 και παρουσιάζει 42% και 43% ταυτότητα με τις τρανσγελίνες 2 και 3 των θηλαστικών αντιστοίχως. Οι τρανσγελίνες συνιστούν μία οικογένεια πρωτεϊνών που εμφανίζουν υψηλό βαθμό συντήρησης και συμμετέχουν στο σχηματισμό δεσμίδων και στη σταθεροποίηση των ινιδίων ακτίνης. Το γονιδίωμα της Δροσόφιλας εμπεριέχει τρεις διαφορετικούς γενετικούς τόπους: CG4694 ή Mp20, CG14996 ή Chd64 and CG5023, σε πλήρη αναλογία με τα γονιδιώματα των θηλαστικών. Τα προκαταρκτικά αποτελέσματα μας έδειξαν ότι από τα γονίδια που κωδικοποιούν τις τρεις τρανσγελίνες της Δροσόφιλας μόνο το Chd64 είναι απαραίτητο για τη βιωσιμότητα και τη σωστή ανάπτυξη των μυϊκών και επιθηλιακών ιστών. Για να μελετήσουμε τη λειτουργία του γονιδίου ήταναπαραίτητο: α) να διερευνήσουμε το πρότυπο έκφρασης του και β) να αξιολογήσουμε τα αποτελέσματα της απώλειας λειτουργίας του σε διαφορετικούς ιστούς. Έτσι, δημιουργήσαμε διαγονιδιακές μύγες που έφεραν τυχαίες ενθέσεις ενός γενωμικού τμήματος του γονιδίου συζευγμένου με GFP που επέτρεπε την παρατήρηση του ενδογενούς προτύπου έκφρασης του γονιδίου σε ζωντανό οργανισμό κατά τη διάρκεια της ανάπτυξης. Ταυτόχρονα, δημιουργήσαμε ένα αντίσωμα έναντι ολόκληρης της πρωτεΐνης Chd64. Επιπλέον, προμηθευτήκαμε δύο UAS:chd64IR στελέχη και πολλά διαφορετικά GAL4 στελέχη με σκοπό να μελετήσουμε την επίδραση της μειορρύθμισης του chd64 σε διαφορετικούς ιστούς. Τέλος, δημιουργήσαμε δύο ελλείψεις που απομακρύνουν το chd64. Τα δεδομένα που έχουμε ως τώρα δείχνουν ότι το chd64 εκφράζεται έντονα στον εμβρυϊκό/προνυμφικό πεπτικό σωλήνα, στα αιμοκύτταρα, στην τραχεία, στο νευρικό σύστημα, στα περιτραχειακά κύτταρα Inka, στους εμβρυϊκούς δίσκους, στους σιελογόνους αδένες, στην επιδερμίδα και στα τενόντια κύτταρα. Επιπλέον, διαπιστώσαμε έκφραση του γονιδίου στα θυλακοκύτταρα και μισχοειδή κύτταρα των ωοθυλακίων. Σε όλους τους παραπάνω κυτταρικούς τύπους η πρωτεΐνη συσσωρεύεται στο κυτταρόπλασμα και στην περιφέρεια του κυττάρου. Λεπτομερέστερη ανάλυση των μεγάλων κυττάρων των σιελογόνων αδένων έδειξε ότι η πρωτεΐνη εντοπίζεται σε μεβρανικές δομές που αντιστοιχούν στο ενδοπλασματικό δίκτυο και στο φλοιό του κυττάρου, όπου συνεντοπίζεται με την ακτίνη. Οι μελέτες απώλειας λειτουργίας έδειξαν ότι έκφραση του chd64 είναι απαραίτητη για τη βιωσιμότητα και τη μορφολογία ή/και λειτουργικότητα των μαλπιγγιανών σωληναρίων. Συγκεκριμένα, διαπιστώσαμε ότι η αναστολή της ζυγωτικής έκφρασης του γονιδίου οδηγεί σε διόγκωση των μαλπιγγιανών σωληναρίων και σχετίζεται με τη μη φυσιολογική υποκυτταρική κατανομή της DE-καδερίνης. Ωστόσο, ο τρόπος δράσης του γονιδίου στην προαναφερθείσα διαδικασία παραμένει άγνωστος. Η απομάκρυνση της μητρικής προέλευσης Chd64 πρωτεΐνης και η εκτενέστερη εξέταση της διαδικασίας καθορισμού και της πολικότητας των κυττάρων που συγκροτούν τα μαλπιγγιανά σωληνάρια σε αγρίου τύπου και μεταλλαγμένο γενετικό υπόβαθρο με τη χρήση κατάλληλων μαρτύρων θα μας δώσουν νέες πληροφορίες σχετικά με τη λειτουργία του γονίδιου σε ολόκληρο τον οργανισμό. / In the past years many research groups have focused in the identification of genes that are involved in distinct biological processes and Drosophila has provided the ideal model-organism due to the availability of several genetic tools. The introduction of RNAi technology has greatly facilitated the conduction of many large scale genetic analyses in Drosophila. Taking advantage of the available tools we conducted an RNAi-based screen for genes involved in the morphogenesis of the somatic muscle system and wing epithelium of Drosophila melanogaster. Collectively, we tested 321 genes with either systemic or tissue-specific RNAi silencing in the mesoderm or a combination of both and discovered 58 novel genes which are required for proper development and homeostasis of the embryonic/larval muscular system. Surprisingly, in almost half of the cases we did not observe a lethal phenotype of complete penetrance arguing that the number of genes involved in the particular process is greater than the one estimated based on the results of a recently completed genome-wide scale RNAi-based muscle screen. In addition, we tested 242 genes by tissue-specific gene inactivation in the epithelia and identified 32 that are required for adult viability and 24 that are indispensible for proper adult wing morphogenesis. Among our positive hits we selected one that is required for both processes for further examination, namely chd64. Chd64 encodes a protein that consists of a calponin-homology (CH) domain and a CLIK23 motif and exhibits 42% and 43% identity with the mammalian transgelins 2 and 3 respectively. Transgelins comprise a highly conserved family of proteins that have been implicated in the bundling and stabilization of actin filaments. The Drosophila genome bears three distinct loci: CG4694 or Mp20, CG14996 or Chd64 and CG5023 by complete analogy to the mammalian genomes. Our initial results showed that out of the three genes that code for the fly transgelins only chd64 is required for viability and the proper development of muscle and epithelial tissues. In order to gain insight into the function of chd64 it was crucial to: a) explore the expression pattern of the gene and b) evaluate the effects of chd64 loss of function in diverse tissues. Thus, we generated transgenic flies that bear random insertions of a GFP-tagged genomic fragment for the gene that allowed us to visualize the endogenous gene expression pattern in the living organism throughout development. Meanwhile, we developed an antibody against the full-length Chd64 protein. Inaddition, we obtained two different UAS:chd64IR strains and many tissue specific GAL4 strains in order to explore the effects of chd64 knock-down in the specific tissues. Finally we generated two deletions that remove chd64. Our data so far indicate that chd64 is largely expressed in the embryonic/larval gut, hemocytes, trachea, nervous system, peritracheal Inka cells, imaginal discs, salivary glands, epidermis and tendon cells. In addition, we observed chd64 expression in the follicle and stalk cells of egg chambers. In all the above mentioned cell types the protein is accumulated in the cytoplasm and periphery. More detailed analysis using the large salivary gland cells shows that Chd64 is specifically localized in some membranous structures of the cytoplasm corresponding to the ER and in the cell cortex where it colocalises with actin. Our loss of function studies demonstrate that chd64 expression is indispensable for viability and for the normal morphology and/or function of malpighian tubules. We specifically observed that the ablation of chd64 zygotic expression results in the appearance of bloated tubules and is involved in the abnormal subcellular distribution of DE-cadherin. However the mode of the gene’s action in the above mentioned procedure remains unknown. Consequently, we conclude that chd64 exhibits a complicated expression pattern during development and is required for viability. Removal of the maternally derived Chd64 protein and further examination of malpighian tubule cell specification and polarity using specific markers in wild type and mutant backgrounds will provide a novel insight on the gene’s functions in the whole organism.

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.