Thesis (Ph.D.)--McMaster University, 2004. / Supervisor: Michael J. O'Donnell. Includes bibliographical references (leaves 193-208).

Malpighian tubules of A. dorsalis mosquito larvae, studied in vitro, actively transported magnesium at high rates against concentration gradients as large as 16-fold and transepithelial potential gradients of approximately -l5mV. Fluid secretion rates, determined over 90 minute periods, in the presence and absence of cAMP, indicated that A. dorsalis tubules were viable and had secretion rates of the same magnitude as those reported for A. taeniorhynchus tubules. Having characterized the in vitro preparation of Malpighian tubules, the main hypothesis that Mg²⁺ transport is driven predominately by counter transport with Na⁺ was tested. This hypothesis was not supported by kinetic, Na-substitution, or inhibitor studies. Kinetic and Bumetanide studies suggest backflux of K drives J mg; however, this was not consistently found in other studies. / Science, Faculty of / Zoology, Department of / Graduate

No description available.

Orientador: Maria Luiza Silveira Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T20:32:58Z (GMT). No. of bitstreams: 1 Mampumbu_AndreRoberto_D.pdf: 659647 bytes, checksum: 40d0a48fa1d02750dffb30ae80b25445 (MD5) Previous issue date: 2006 / Resumo: O gênero Melipona (abelhas sem ferrão) tem sido dividido em dois grupos, com base no seu conteúdo em heterocromatina revelada com a técnica de banda-C em cromossomos mitóticos. Melipona quadrifasciata e Melipona rufiventris apresentam, respectivamente, níveis baixos e altos de heterocromatina. Na suposição de que cromatina condensada possa ser rica em seqüências de DNA metiladas, M. quadrifasciata e M. rufiventris poderiam então apresentar diferenças em conteúdo de seqüências CpG metiladas. Se isso acontecesse, as diferenças poderiam ser reveladas pela comparação de valores Feulgen-DNA obtidos por análise de imagem de células tratadas com as enzimas de restrição Msp I e Hpa II, que distinguem entre seqüências metiladas e não metiladas. Msp I e Hpa II clivam as seqüências ¿CCGG-, porém não há clivagem pela Hpa II se a citosina do dinucleotídeo central CG for metilada. Neste trabalho, túbulos de Malpighi de larva de último estádio de M. quadrifasciata e M. rufiventris submetidos à reação de Feulgen precedida pelo tratamento com Msp I e Hpa II tiveram suas células analisadas por microespectrofotometria de varredura automática. Para esse material houve necessidade do desenvolvimento prévio de um ajuste metodológico que tornasse a reação de Feulgen reveladora apenas de DNA, visto que ocorria reação plasmal; isto foi conseguido com um tratamento por boridreto de sódio a 5% e acetona/clorofórmio (1:1, v/v) antecedendo a reação de Feulgen. Também, embora a definição de altos e baixos conteúdos de heterocromatina em Melipona pela técnica de banda-C não fosse extensível à cromatina de núcleos interfásicos dos túbulos de Malpighi dessas abelhas, demonstrou-se que a depurinação do DNA em M. quadrifasciata era mais rápida do que a de M. rufiventris, confirmando, maiores teores de cromatina condensada em M. rufiventris. Os valores Feulgen-DNA para a heterocromatina de Melipona rufiventris e para a pouca heterocromatina somada a alguns domínios de eucromatina de Melipona quadrifasciata diminuíram após tratamento com Msp I, porém ficaram inalterados após tratamento com Hpa II. Conclui-se que seqüências CpG metiladas podem estar contidas em diferentes compartimentos cromatínicos, conforme a espécie do gênero Melipona considerada, e que os seus efeitos silenciadores possam atuar induzindo uma mesma fisiologia celular / Abstract: The genus Mellipona has been divided into two groups based on its heterochromatin content revealed by C-banding pattern in mitotic chromosomes. Melipona quadrifasciata and Melipona rufiventris show low and high heterochromatin content, respectively. Supposing that condensed chromatin may be rich in DNA methylated sequences, M quadrifasciata and M. rufiventris could, thus, show differences regarding their content of CpG methylated sequences. In this situation, such differences could be revealed by comparing the Feulgen-DNA values acquired after image analysis of cells treated with restriction enzymes Msp I and Hpa II, which distinguish between methylated and nonmethylated sequences. Msp I and Hpa II break the CCGG sequences. Nevertheless, Hpa II is ubable to break the DNA strand if the cytosine from the central nucleotide pair CG is methylated. In this work, Malpighian tubules from larvae from the last stage of M. quadrifasciata and M. rufiventris, subjected to the Feulgen reaction after by treatment with Msp I and Hpa II, were analysed in automatic scanning microspectrophotometry. Since a plasmal reaction was observed in this material, it was previously necessary the development of a methodological adjustement to make the Feulgen reaction specific to DNA. This was achieved by treatment of material with 5% sodium borohydrade followed by acetone-chloroform (1:1, v/v) before the Feulgen reaction. Also, although the definition of high and low heterochromatin content in Melipona after C-banding technique is not applicable to the chromatin of interphasic nuclei in Malpighian tubules of bees, it was demonstrated that DNA depurination in M. quadrifasciata was faster than that of M. rufiventris, thus confirming that this species has a higher condensed chromatin content. The Feulgen-DNA values for the heterochromatin of Melipona rufiventris, and for the heterochromatin besides some euchromatic domains of Melipona quadrifasciata, decreased after treatment with Msp I, remaining, however, unaltered after treatment with Hpa II. In conclusion, methylated CpG sequences may be part of different chromatin compartments, according to the considered species of the genus Melipona, and that their silencing effects may act by inducing the same cell physiology / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

<p>Insects are continually exposed to potentially toxic endogenous compounds and xenobiotics that require rapid elimination from the body. Xenobiotic resistance in insects has evolved predominantly by increasing the activity of detoxification enzymes and/or by increasing toxin excretion via the Malpighian (renal) tubules. The tubules have long been known to transport organic anions at high rates. This thesis examines the mechanisms of excretion and detoxification of the organic anion methotrexate (MTX) by isolated tissues of the fruit fly <em>Drosophila melanogaster</em>. A radioisotope tracer technique and the Ramsay assay were used to measure MTX secretion. Quantitative PCR (qPCR) was used to evaluate the expression of the genes for putative organic anion transporters. My results show that MTX transport across the Malpighian tubule epithelium is active, saturable, Na⁺-independent and inhibited by a wide range of organic anions including MK-571, probenecid and Texas Red. Pharmacological studies and qPCR analyses suggest multiple transporters are involved in the movement of MTX across the Malpighian tubules. Moreover, chronic exposure of larvae to dietary MTX or salicylate dramatically increases the transepithelial transport of MTX by isolated Malpighian tubules, suggesting that excretion of MTX is upregulated by exposure to these organic anions in the diet. In addition, treatments known to increase expression of specific detoxification enzymes, such as the P450 monoxygenases (P450s) and the glutathione-S-transferases (GSTs), also led to an increase in expression levels of multidrug efflux transporter (MET), multidrug resistance like protein 1 (dMRP) as well as to increased secretion of MTX by the tubules. This latter finding suggests a coordinated response to toxin exposure, so that when detoxification pathways are increased, there is a corresponding increase in the capacity for elimination of the products of P450 and GST enzymes. Finally, the last section of this thesis has shown that RNAi knockdown of a single organic anion transporter gene in the principal cells of <em>D. melanogaster</em> Malpighian tubules is associated with reductions in the expression of multiple, functionally-related genes. Importantly, these results indicate that dMRP andMET are not the dominantMTX transporters in the tubules when flies are reared onMTX-enriched diets. However, reductions in the expression of organic anion transporting polypeptide (OATP) are associated with reduced secretion of the organic anionsMTX, fluorescein and Texas Red. Taken together, these results suggest that OATP and at least one additional transporter, as yet unidentified, are required forMTX secretion. In conclusion, the results of my research contribute to our understanding of the mechanisms of organic anion detoxification and excretion in flies exposed to dietary toxins.</p> / Doctor of Science (PhD)

Insect haemolynph typically contains very high levels of free amino acids 50 1 00 times that which is normal for mammalian plasma. This study examines the modulatory effects of amino acids on fluid secretion and ion transport by isolated MTs of Rhodnius prolixus and Drosophila melanogaster. The results show that the secretion rates of isolated Malpighian tubules of both Rhodnius and Drosophila are modulated by the presence of specific amino acids in the bathing saline. Some amino acids are stimulatory, some are inhibitory and others have little or no effect. Glutamine appears to be particularly important as a stimulant of fluid secretion. As well, secreted fluid pH and Na +concentration increase and K+ concentration decreases in response to glutamine. Amino acids do not appear to be important as metabolite. in Rhodnius tubules, nor do they act to draw significant amounts of water into the lumen by osmosis. Significant stimulation of fluid secretion can be achieved by physiological levels of particular amino acids, whereas those amino acids that inhibit fluid secretion only do so at concentrations much above those at which they occur naturally in the haemolymph. Amino acids are known to be compatible osmolytes and may be acting to maintain cell homeostasis and thus to sustain fluid secretion. The passive movement of amino acids may result in cell volume changes, and some form of osmosensor is may be coupled to activation of specific kinases to produce the observed increases in fluid secretion. The effects of several kinase inhibitors were therefore examined. The glutamine dependent increase in MT fluid secretion is blocked by two inhibitors of the stress activated protein kinase (SAPK) pathway, SP600125 and dicumoral. Inhibitors of other kinases (PKA, PKC, PKG, PI-3, p38, ERK and MEK), did not block glutamine's effects on fluid secretion rate. Alterations in cytoskeletal structure appear not to be required because cytoskeletal disrupting agents did not block the glutamine dependent inc~ease in fluid secretion, nor was the increase dependent upon protein synthesis. Results of this study are the first to suggest a role for the SAPK pathway in the control of fluid secretion rates by insect Malpighian tubules. / Thesis / Master of Science (MS)

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.

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.

Les cellules souches adultes assurent le renouvellement des cellules différenciées, un mécanisme indispensable à la régénération des organes soumis au vieillissement et aux agressions extérieures. Des cellules souches ont été identifiées dans les tubules de Malpighi (TM) qui assurent les fonctions rénales chez la Drosophile. Ces cellules souches rénales (CSR) proviennent à l'origine de précurseurs des cellules souches intestinales (CSI) qui migrent et colonisent les TM pendant la métamorphose. Nous montrons que le facteur de transcription shavenbaby (svb) est exprimé au sein des CSR. Svb est connu pour contrôler la morphogenèse épidermique durant le développement de la Drosophile tandis que son homologue chez l'Homme, OvoL, est impliqué dans la transition épithélio-mésenchymateuse et est dérégulé dans certains cancers. Nous avons découvert que la principale fonction de svb au sein des CSR est de prévenir l'apoptose. En effet, la perte de fonction de svb, spécifiquement au sein des CSR adultes, induit leur disparition progressive et cet effet est abolit par l'expression d'inhibiteurs d'apoptose. Tout comme dans l'épiderme, nous avons pu observer que la fonction de svb est supportée par sa maturation protéolytique induite par les gènes polished-rice et ubr3. De plus, nous démontrons que Svb interagit avec Yorkie (Yki) un membre de la voie de signalisation Hippo, connue pour contrôler le nombre de CSI. Ce complexe Svb/Yki régule l'expression de l'inhibiteur apoptotique DIAP1 afin de maintenir un nombre normal de CSR. Ces travaux ont donc permis d'identifier un nouveau membre de la voie de signalisation Hippo et de découvrir un mécanisme inattendu de protection des cellules souches contre la mort cellulaire qui pourrait expliquer leur capacité de résistance à l'apoptose. / Throughout adult life, homeostasis of fundamental functions requires cell renewal to compensate for tissue damage and cell death. The renal (Malpighian) tubules in Drosophila are responsible for excretion of metabolic waste like kidney in vertebrates. Although Malpighian tubules are thought to be very stable during development, evidence of adult cell renewal have been showed and stem cells identified. Renal and nephric stem cells (RNSC) derived from intestinal stem cell precursors that colonize Malpighian tubules during metamorphosis. We showed that the gene shavenbaby (svb) which encodes a transcription factor belonging to the OVO-Like family (OVOL) is expressed in RNSCs. In vertebrates, OvoL factors act as guardian of epithelial integrity, while in Drosophila, Svb is well identified for its role in epidermal cell shape remodeling. The transcriptional activity of Svb requires its proteolytic processing mediated by the SmORF peptides Pri encoded by the polished-rice gene. Here, we show that this processing occurs also in RNSC and that the main function of svb in these cells is to protect them from apoptosis. Svb loss of function induces a progressive disappearance of RNSC that can be rescued by blocking programmed cell death. At the molecular level, we found that Svb physically interacts with Yorkie, the downstream effector of the hippo pathway to favor the expression of the inhibitor of apoptosis DIAP1. To conclude, our work identified a new member of the hippo pathway and uncovered an additional way to protect stem cell from apoptosis that may explain their capacity to resist apoptosis.

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.