<p> This research was designed to gain a better understanding of the relationships between nutrition, aging and oogenesis. Cohorts of 1800 inbrod, adult house flies were maintained on various undefined and chemically defined diets. From these flies samples for histology were removed periodically, and the rest were analyzed for their survival and their ability to develop and lay viable eggs. The survival of the females was more affected by nutrition and other factors than that of the males. In females fed sugar water, oogenesis was arrested at an early stage, but survival was lower than on a milk diet. From adult emergence until the completion of one or more ovarian cycles, the larval and adult fatbodies and the ovary were compared as to their histology, histochemistry and cytology. / Thesis / Master of Science (MSc)

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.

Les associations entre organismes eucaryotes et micro-organismes sont fréquemment observées dans la nature et s’étendent le long du continuum parasitisme-mutualisme. Si de nombreuses associations symbiotiques ont été bien caractérisées, leur mise en place et leur évolution ont été rarement étudiées. Les bactéries intracellulaires Wolbachia induisent des effets très différents, variant du parasitisme de la reproduction chez les Arthropodes au mutualisme chez les Nématodes. Nous nous sommes ici intéressés à l’hyménoptère Asobara tabida, rare espèce où Wolbachia est obligatoire pour l’ovogenèse de son hôte Arthropode, de façon à étudier les mécanismes à l’origine d’une transition évolutivement récente. Nous avons tout d’abord étudié la variabilité de la dépendance vis-à-vis de Wolbachia au sein du genre Asobara. Par diverses approches de transcriptomique, nous avons ensuite caractérisé les mécanismes moléculaires impliqués dans la dépendance chez A. tabida, et mis en évidence des processus impliqués dans la mort cellulaire programmée, l’immunité (sens large) et le développement. Enfin, nous avons étudié l’impact de Wolbachia sur la physiologie de son hôte, en étudiant le métabolisme du fer et plus généralement l’immunité dans diverses associations symbiotiques. Ces études montrent que la dépendance n’est pas forcément associée à l’apport de nouvelles fonctions et pourrait au contraire être le reflet de processus compensatoires mis en place par l’hôte, en réponse aux perturbations physiologiques induites par le symbiote. Ces résultats appellent à considérer les effets et les conséquences de ces symbiotes au delà des mécanismes qui permettent leur maintien dans les populations / Associations between eukaryotes and micro-organisms are frequently observed in nature and range along the continuum between parasitism and mutualism. Numerous associations have already been well described; however the origin and the evolution of these associations are rarely studied. We focused on the intracellular bacterium Wolbachia, which induces very different phenotypic effects, ranging from facultative reproductive parasitism in Arthropods to obligatory mutualism in Nematodes. Here we studied the hymenopteran Asobara tabida, a rare species in which Wolbachia is necessary for oogenesis completion of its Arthropod host, in order to investigate the mechanisms underlying an evolutionarily recent transition. We first studied the variability of dependence to Wolbachia within the Asobara genus. Using various transcriptomic approaches, we next characterized molecular echanisms involved in dependence between A. tabida and Wolbachia, and highlighted processes implicated in programmed cell death, immunity (broad sense) and development. Finally, we examined to what extent Wolbachia impacts host physiology, by studying iron metabolism and global immunity in various symbiotic associations. These studies highlight that dependence is not always linked with the provision of a new function. It could rather reflect host compensatory mechanisms in response to physiological perturbations induced by the presence of symbiont. More generally, these results invite to consider the effects and the consequences of symbionts over the mechanisms allowing their persistence within populations

Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae) tornou-se um psilídeo de grande importância para a citricultura paulista após a constatação da bactéria Candidatus Liberibacter sp., causadora do Huanglongbing (greening). Sabe-se que esse inseto abriga microrganismos endossimbiontes, os quais desempenham papel fundamental em sua ecologia nutricional, sendo transmitidos verticalmente à progênie. Dessa forma, propomos caracterizar a morfologia do aparelho reprodutor feminino durante o seu desenvolvimento para embasar a identificação do processo de migração dos simbiontes do bacterioma aos tecidos reprodutivos. D. citri possui ovário do tipo telotrófico, com ovaríolos organizados em bouquet e características gerais semelhantes às observadas para outros Sternorrhyncha. Os trofócitos parecem ser desprovidos de delimitação por membrana no ovaríolo desenvolvido. Um único oócito se desenvolve por ciclo no vitelário, o qual mantém-se em contato com a câmara trófica por um prolongamento citoplasmático, denominado cordão trófico. As informações morfo-estruturais do aparelho reprodutor de D. citri obtidas indicam similaridades importantes a de outros membros de Sternorryncha. Nesse contexto, a migração de simbiontes do bacterioma para os oócitos em maturação de D. citri, ocorre de modo semelhante ao descrito para aleirodídeos, caracterizandose pela migração de bacteriócito intacto. Este último, atravessa o epitélio de revestimento do oócito, formado por células foliculares, e invade o oócito, liberando as bactérias nele contidas. Entretanto, os simbiontes associados ao sincício do bacterioma, são liberados na hemocele através de uma pequena abertura formada no epitélio de revestimento dessa estrutura, invadindo o oócito por um mecanismo distinto. Os simbiontes contidos no oócito, formam um agrupamento de aspecto arredondado (= symbiont ball) na região posterior do oócito, próximo ao pedicelo. / Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae) became a serious problem to the citrus industry in São Paulo State once the Huanglongbing disease (greening), which is caused by the bacteria Candidatus Liberibacter sp., was detected. Psyllids are known to harbor endosymbiont microorganisms, which are vertically transmitted to the progeny and play a key role in the nutritional ecology of their hosts. Therefore, we aimed to characterize the morphology of the reproductive system during D. citri development as a tool for further investigation on the symbiont migration from the bacteriome to the reproductive tissues. D. citri has telotrophic ovaries with ovarioles organized in a bouquet, sharing all other characteristics with the remaining Sternorrhyncha. In developed ovarioles, trophocytes seems to lack any membrane delimitation. Only one oocyte develops at a time in the vitellarium, remaining in communication with the trophic chamber by a citoplasmatic brigde, named trophic cord. The morphostructural information reported in here on the D. citri reproductive system shows important similarities with other Sternorryncha. Symbionts associated to the bacteriome of D. citrus migrate to the ovaries and invade the oocytes during ovary maturation, as previously reported for aleyrodids. In this case, symbionts will move within the bacteriocyte as it detaches from the bacteriome and moves through the oocyte follicular epithelium, releasing the contained bacteria into the oocyte. However, symbionts associated to the bacteriome syncitium are relased into the hemocoel through small openings on the bacteriome epithelium, invading the oocyte by a different mechanism. All symbionts that invaded or were discharged into the oocyte aggregate into a balllike symbiont structure at the posterior pole close to the egg pedicel.

Au cours de l’ovogenèse, les stades vitellogéniques nécessitent une énergie considérable, et leur formation doit être ajustée en fonction d’autres besoins physiologiques. En utilisant la drosophile comme modèle, j’ai montré que la signalisation Insuline régule une transition du cycle cellulaire, mitose/ endocyle (M/E), une étape critique qui contrôle l’entrée des follicules en vitellogenèse. Mes travaux montrent que la transition M/E porte le rôle d’un point de contrôle nutritionnel. La carence protéique induit un blocage de cette transition au travers d’une interaction entre FoxO, Cut et Notch, empêchant une perte d’énergie. Ce blocage reste réversible, autorisant la reprise de l’ovogenèse sous retour à une alimentation normale. Ce travail montre qu’un point de contrôle nutritionnel au cours de l’ovogenèse permet de coupler des signaux métaboliques et développementaux pour protéger les tissus des dommages liés à la carence. D’autre part, j’ai montré que la signalisation Insuline contrôle la migration d’une cohorte de cellules d’origine épithéliale pour assurer la fertilité de l’ovocyte. L’insuline participe à la formation d’extensions cytoplasmiques riches en actine. Lors de ce processus, la signalisation Insuline contrôle notamment l’expression de chickadee, qui code pour la Profiline, une protéine nécessaire pour la polymérisation de l’actine qui permet la motilité des cellules. L’ensemble de ce travail montre que des tissus somatiques assurent l’homéostasie de l’ovogenèse malgré des conditions de nutritions fluctuantes. Ces travaux posent les bases de l’étude de nouveaux aspects de l’ovogenèse, potentiellement conservés chez les mammifères. / How oogenesis is controlled upon nutrient challenge is a key biological question to understand the balance between reproduction and adult fitness. During Drosophila oogenesis, vitellogenic stages are highly energy consuming so their formation has to be balanced with other physiological needs. We reveal the role of the Insulin pathway and FoxO in regulating the transition from Mitotic-to-Endocycle, a critical step controlling the entry of egg chambers into vitellogenesis. We show that the M/E switch functions as a nutrient checkpoint, blocking the entry into vitellogenesis upon starvation and therefore protecting adults from energy loss. Pausing of the M/E switch involves a previously unknown crosstalk between FoxO, Cut and Notch, a fully reversible process ensuring rapid resuming of oogenesis upon re-feeding. This work reveals a FoxO-dependent nutrient checkpoint integrating metabolic cues with reproduction and protecting tissues from starvation-induced damages. In addition, we show that the Insulin pathway regulates the migration of a subset of epithelial cells to ensure oocyte fertilization. We demonstrate that Insulin signaling regulates the formation of actin-rich cellular extensions in invasive cells. During this process, FoxO represses chickadee expression, which encodes Profilin. Insulin signaling activity leads to the inhibition of FoxO and subsequent Profilin accumulation, which further allows actin polymerization, necessary for cell motility. Altogether, data reveal a crucial role for the conserved Insulin signaling pathway in regulating ovarian follicles through somatic tissues, a process which is likely to share much in common with oogenesis in mammals.

Crumbs is a transmembrane protein and apical determinant in Drosophila epithelial cells. Its cytoplasmic tail contains a PDZ and a FERM domain-binding site through which Crumbs interacts with the FERM proteins Yurt, Moesin and Expanded. Recent evidence suggests that Crumbs can also interact with the uncharacterised FERM proteins CG34347 and Cdep. The main objective of my thesis was to generate mutations in CG34347 and Cdep to facilitate the functional analysis of these genes. I generated a mutation for Cdep that remains to be characterised and two mutant lines for CG34347; one lacking the first exon and one lacking the entire gene, using a FRT-based recombination strategy. Both CG34347 mutants cause severe ovarian defects. The most consistent defect is a multilayering of the interfollicular stalk. These defects are also observed when Notch, Hippo, Wingless and Hedgehog signalling pathways are overactive in ovaries suggesting that CG34347 participates in one of those pathways.

Crumbs is a transmembrane protein and apical determinant in Drosophila epithelial cells. Its cytoplasmic tail contains a PDZ and a FERM domain-binding site through which Crumbs interacts with the FERM proteins Yurt, Moesin and Expanded. Recent evidence suggests that Crumbs can also interact with the uncharacterised FERM proteins CG34347 and Cdep. The main objective of my thesis was to generate mutations in CG34347 and Cdep to facilitate the functional analysis of these genes. I generated a mutation for Cdep that remains to be characterised and two mutant lines for CG34347; one lacking the first exon and one lacking the entire gene, using a FRT-based recombination strategy. Both CG34347 mutants cause severe ovarian defects. The most consistent defect is a multilayering of the interfollicular stalk. These defects are also observed when Notch, Hippo, Wingless and Hedgehog signalling pathways are overactive in ovaries suggesting that CG34347 participates in one of those pathways.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.

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Η διδακτορική μου διατριβή εστιάζει στη λειτουργική ανάλυση του μοριακού μηχανισμού λειτουργίας της ιντεγκρινο-συνδεόμενης κινάσης (ILK) κατά την ανάπτυξη στη Drosophila. Έγινε διερεύνηση: α) της λειτουργικής συντήρησης της ILK κατά την εξέλιξη, β) του πιθανού ρόλου συγκεκριμένων αμινοξικών μοτίβων στον κυτταρικό εντοπισμό και τη λειτουργία της πρωτεΐνης και γ) των λειτουργικών ιδιοτήτων της ILK όταν είναι ομοιοπολικά συζευγμένη στην πλασματική μεμβράνη. Παράλληλα, αναζητήσαμε νέους λειτουργικούς ρόλους της ILK κατά την ανάπτυξη: α) σε άλλους ιστούς εκτός από το μυϊκό σύστημα και β) στην ωογένεση. Η ILK στο επίπεδο της αμινοξικής αλληλουχίας παρουσιάζει 60% ταυτότητα και 75% ομολογία με την αντίστοιχη πρωτεΐνη των θηλαστικών. Με βάση αυτή την ομολογία, ελέγξαμε την πιθανή φυλογενετική συντήρηση της λειτουργίας της ILK. Για το σκοπό αυτό κατασκευάστηκαν διαγονιδιακά στελέχη που περιείχαν την κωδική περιοχή της ILK του ανθρώπου (hILK) και του ποντικού (mILK) αντίστοιχα. Η ILK του ποντικού και του ανθρώπου ακολουθεί παραπλήσιο πρότυπο υποκυτταρικής κατανομής με την ενδογενή πρωτεΐνη στα μυϊκά κύτταρα Drosophila. Οι δυο ετερόλογες πρωτεΐνες υποκαθιστούν τη λειτουργία της ILK στη Drosophila. Ωστόσο, η ILK του ανθρώπου παρουσιάζει μειωμένη δυνατότητα πρόσδεσης με την parvin της Drosophila. Προκειμένου να διερευνήσουμε το μοριακό μηχανισμό ρύθμισης και δράσης της ILK κατά την ανάπτυξη, ελέγξαμε εάν η φωσφορυλίωση των αμινοξέων S176 και T180 υπεισέρχεται στη ρύθμιση της λειτουργίας της ILK. Σε πειράματα κυτταρικών σειρών έχει δειχθεί ότι στις θέσεις αυτές η φωσφορυλίωση ελέγχει την υποκυτταρική κατανομή της πρωτεΐνης στον πυρήνα. Ωστόσο, αποδείξαμε ότι η πιθανή φωσφορυλίωση στις ισχυρά συντηρημένες θέσεις S176 και T180 δεν είναι απαραίτητη για τον εντοπισμό της ILK στις μυοτενοντικές συνδέσεις και τη λειτουργία της ILK. Ένα άλλο αμινοξικό κατάλοιπο που είναι απαραίτητο για τον εντοπισμό της ILK στις εστιακές θέσεις προσκόλλησης είναι το F436, το οποίο εδράζεται στην τελευταία α έλικα του καρβοξυτελικού λοβού της περιοχής κινάσης. Ο υποκυτταρικός εντοπισμός της ILK και η λειτουργία της δεν επηρεάζονται από τη σημειακή μεταλλαγή F436Α σε αντίθεση με πειράματα σε κυτταρικά μοντέλα. Η σημειακή μεταλλαγή F436A αποδυναμώνει την ικανότητα αλληλεπίδρασης της ILK με την parvin. Εξετάσαμε αν η μεμβρανοδεσμευόμενη ILK μέσω παλμυτυλίωσης ή φαρνεσυλίωσης μπορεί να υποκαταστήσει την απουσία της ενδογενούς, καθώς και εάν μπορεί να προσελκύσει πρωτεΐνες του συνδεοσώματος ανεξάρτητα των ιντεγκρινών. Κατασκευάστηκαν δυο εναλλακτικές μορφές μεμβρανοδεσμευμένης ILK, οι GAP-ILK-GFP και ILK-GFP-HRAS εντοπίζονται με επιτυχία σταθερά στην πλασματική μεμβράνη των εμβρυϊκών μυϊκών κυττάρων. Επίσης, οι GAP-ILK-GFP και ILK-GFP-HRAS υποκαθιστούν πλήρως την ενδογενή ILK σε όλα τα αναπτυξιακά στάδια της ζωής της μύγας. Τέλος, η GAP-ILK-GFP συγκεντρώνει στις ΜΤΣ τα άλλα δυο μέλη του IPP συμπλόκου αλλά και την talin στις ΜΤΣ εμβρύων τελικού σταδίου τόσο αγρίου τύπου όσο και ομοζυγώτων για aPS2. Παράλληλα, μελετήσαμε , τόσο σε γενετικό όσο και σε μοριακό επίπεδο, το ρόλο της ILK στη μορφογένεση του ωοθυλακίου, την οργάνωση και ομοιόσταση του θυλακώδους επιθηλίου κατά τη διάρκεια της ωογένεσης στη Drosophila. Αποσιωπήσαμε την ilk κατασκευάζοντας γενετικά μωσαϊκά αλλά και ιστοειδικά διασωσμένα άτομα. Παρατηρήσαμε ότι η ILK είναι απαραίτητη για τη διαδικασία της ωογένεσης στη μύγα. Η απουσία της ILK προκαλεί διαταραχές στο σχηματισμό των διαθυλιακών μίσχων και ανωμαλία στο διαχωρισμό των νεοσχηματιζόμενων διαδοχικών ωοθυλακίων (σιαμαία ωοθυλάκια). Επίσης, τα πειράματά μας αποκάλυψαν ότι η ILK είναι απαραίτητη για την οργάνωση των ινιδίων ακτίνης κατά τα τελευταία στάδια της ωογένεσης και για την ομοιόσταση του κυτταροσκελετού ακτίνης κατά τον ακραιο-βασικό άξονα του κυττάρου. Ακόμη, η ILK είναι απαραίτητη για την οργάνωση και διατήρηση των βασο-πλευρικών κυτταρικών συνδέσεων στα θυλακιοκύτταρα, αλλά όχι των συνδέσεων ζώνης. Η απουσία της ILK διαταράσσει τον εντοπισμό των ιντεγκρινών στα άκρα των ινιδίων τάσης της ακτίνης στα θυλακιοκύτταρα των τελευταίων σταδίων. Επιπλέον, η ILK συμμετέχει στη ρύθμιση της δυναμικής της F-ακτίνης μειορρυθμίζοντας το Dia και αυξορρυθμίζοντας την profilin. H ILK εμπλέκεται στον έλεγχο της συσταλτότητας των ινιδίων ακτο-μυοσίνης στα θυλακιοκύτταρα των τελευταίων σταδίων, πιθανότατα μέσω της διαταραχής στον υποκυτταρικό εντοπισμό του RhoI, καθώς και μέσω της εκτοπικής συσσώρευσης της μυοσίνης (zipper). Τέλος, η ilk αλληλεπιδρά γενετικά με τη dpak στο επιθήλιο του ωοθυλακίου. Η ΙLK επηρεάζει τον εντοπισμό της dPAK στα θυλακιοκύτταρα τελευταίων σταδίων. Επίσης, η dPAK είναι απαραίτητη για τον εντοπισμό των ιντεγκρινών και της ILK στα άκρα των ινιδίων τάσης της ακτίνης. Ενώ, η απουσία της dpak, όπως και της ilk, διαταράσσoυν την οργάνωση και των ινιδίων τάσης της ακτίνης σε θυλακιοκύτταρα τελευταίων σταδίων. / My thesis is focused on the functional analysis of the molecular mechanism of the integrin-linked kinase (ILK) during development in Drosophila. We studied: a) the functional conservation of ILK in evolution, b) the possible role of specific amino acid motifs in the subcellular localization and function of ILK and c) the functional properties of ILK, when covalently bound to the plasma membrane. Furthermore, we sought new functional roles for ILK during development: a) in other tissues besides muscle system and b) in oogenesis. ILK protein sequence shares 60% identity and 75% similarity with the mammalian ILK. Based on these data, we tested the possible phylogenetic conservation of ILK function. For this purpose, we generated transgenic lines carrying the coding sequence of either human ILK (hILK) or mouse ILK (mILK). The mammalian ILK has localizes similarly to the endogenous protein, in the muscle cells of Drosophila. Both mammalian proteins can substitute for the ILK function in Drosophila. However, human ILK binds to Dparvin with reduced affinity compared to the fly ILK. In order to investigate the molecular mechanism through which ILK regulates and acts during development, we tested whether the phosphorylation on the amino acids S176 and T180 contributes to the regulation of ILK function. It has been shown, in cell culture models, that the phosphorylation on these sites controls the subcellular localization of the protein in the nucleus. However, we proved that the possible pgoshorylation of these highly conserved residues is dispensable for the ILK localization at the muscle attachment sites (MAS) as well as for the function of ILK. Another residue which is necessary to localise ILK at the focal adhesion sites is F436. It is located on the last a helix of the carboxyl-terminal lobe of the kinase-like domain. The subcellular localization and the ILK function are unaffected by the point mutation F436A, in contrast to the experimental data on cell culture models. The point mutation F436A affects the ability of ILK to bind to parvin. We examined, whether membrane-bound ILK, through palmytoylation or farnesylation, is able to substitute the absence of the endogenous ILK, if ii can recruit proteins of the adhesome, independently of integrins. We generated two alternative forms of membrane-bound ILK, GAP-ILK-GFP and ILK-GFP-HRAS, which both localize successfully at the plasma membrane of the embryonic muscle cells. Also, GAP-ILK-GFP and ILK-GFP-HRAS can substitute for the endogenous ILK throughout development. Moreover, GAP-ILK-GFP is able to recruit both PINCH and Parvin, as well as talin at the MAS, in both wild type and aPS2 mutant embryonic muscle cells. Furthermore, we studied, in genetic molecular level, the role of ILK in the morphogenesis of the egg chambers, the organization and the homeostasis during oogenesis in Drosophila. We used two experimental approaches in order to silence ilk: a) we generated genetic mosaics for ilk and b) we used conditionally rescued ilk-/- flies. We observed that ILK is indispensable for the process of oogenesis in the fly. Loss of ILK disrupts the stalk cell formation and the separation of the successive newly formed egg chambers (twin egg chambers). Also, our experiments revealed that ILK is essential for the organization of the actin stress fibers at the late developmental stages of oogenesis and for the homeostasis of the actin cytoskeleton along apico-basal axis of the cell. ILK is indispensable for the organization and the maintenance of the baso-lateral cell junctions in the follicle cells, but not for the adherens junctions. Loss of ILK disrupts the localization of integrins at the tips of the actin stress fibers of the follicle cells at late developmental stages. Moreover, ILK participates in the regulation of the F-actin dynamics by down-regulating Dia and up-regulating profilin. ILK is involved in the control of the contractility of the acto-myosin fibers in the follicle cells at late developmental stages, probably by affecting the subcellular localization of Rho1, and causing ectopic accumulation of myosin (zipper). Finally, ilk interacts genetically with dpak in the follicular epithelium. ILK affects dPAK localization in the follicle cells at late developmental stages. Furthermore, dPAK is essential for the localization of both integrins and ILK at the tips of actin stress fibers. Loss of dpak, similarly to ilk, disrupts the organization of actin stress fibers in follicle cells at late developmental stages.