The Janus kinase (JAK) pathway is an integral part of signaling through a variety of ligands and receptors in mammals. The extensive reutilization and pleiotropy of this pathway in vertebrate development is conserved in other animals as well. In Drosophila melanogaster, JAK signaling is involved in embryonic pattern formation, sex determination, larval blood cell development, wing venation, planar polarity in the eye, and formation of other adult structures. Here we describe several roles for JAK signaling in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired, is expressed specifically in the polar follicle cells, two pairs of somatic cells at the anterior and posterior poles of the developing egg chamber. A primary defect of chambers with reduced JAK activity is fusion of successive chambers. These chambers exhibit an expansion of the polar cell population and concomitant loss of interfollicular stalk cells. Mosaic analysis of both JAK pathway transducers, hopscotch and stat92E, reveals that JAK signaling is specifically required in the somatic follicle cells. Another role of JAK signaling is in oocyte localization. In chambers mosaic for loss of hop activity, oocyte mislocalization results. Proper localization occurs only when the posterior follicle cells are wild type for hop.

The JAK-STAT pathway is an intracellular signaling pathway that is found to have crucial roles in hematopoiesis, immune response and the development of many other tissues in mammals. The pathway is conserved in Drosophila melanogaster, and is much simpler: there is only one Drosophila JAK (Hopscotch, Hop) and STAT (STAT92E) respectively, while there are at least 4 JAKs and 7 STATs in mammals. The pathway has been intensively studied in Drosophila, and has been implicated in many tissue development and cellular processes. In this work, I present several roles of JAK signaling in oogenesis.First, JAK signaling is required for cell differentiation within a specific lineage of follicle cells – stalk cells and polar cells. Unpaired (upd), which encodes the known ligand for the pathway, is expressed specifically in the polar cells in the developing egg. Reduced function of Upd or Hop results in fusions of egg chambers, which is primarily caused by improper formation of stalk cells, while general activation of the pathway in the egg chamber produces an extra number of stalk cells and sometimes eliminates polarfollicle cells. Based on the known function of the Notch pathway in oogenesis, we propose a model that Notch signaling determines a pool of precursors for the polar and stalk cells while JAK activity determines their specific fates within that pool.Second, JAK signaling is also involved in epithelial follicle cell differentiation. Consistent with the expression pattern of upd in the ovary, there is a gradient of JAK activity expanding from the poles, and this JAK activation gradient is both required and sufficient to suppress the main body follicle cell fate. Also, different levels of JAK activity are required and sufficient to determine both anterior and posterior terminal follicle cell fates. Consistent with these data is a model that a gradient of JAK activity triggered by Upd from the poles pre-patterns the epithelium into three domains and pre-determines sub-populations of terminal follicle cell fates prior to the EGFR activation, and cooperates with EGFR activity later to define posterior terminal follicle cell fates. This provides the first evidence for a morphogenic function of the JAK-STAT pathway in any organism.

A produção in vitro de embriões, no Brasil, ocupa lugar de destaque dentre as biotecnologias da reprodução animal e o atual desafio é torná-la cada vez mais acessível, para tanto, é fundamental aproximar os resultados de produção e sobrevivência dos embriões produzidos in vitro daqueles desenvolvidos naturalmente. Boa parte desta variação do potencial de desenvolvimento do embrião está relacionada à características do oócito. Há a possibilidade de essa capacidade estar relacionada ao perfil lipídico do líquido folicular e das células do folículo, neste sentido, este projeto teve como objetivo principal avaliar a correlação do potencial de desenvolvimento de oócitos com o conteúdo do perfil lipídico no líquido folicular e das células foliculares, baseado no sistema de ativação partenogenética, cultivo individual e analise retrospectiva. Não existe, até o momento, uma caracterização detalhada do microambiente folicular, um componente importante dos fluídos são os lipídios e recentemente tem sido demonstrado que eles estão envolvidos em vários aspectos do metabolismo além de também funcionarem como moléculas sinalizadoras ou até mesmo serem capazes de induzir modificações na cromatina. Os íons oriundos do estudo do perfil lipídico foram avaliados em comparação ao potencial de desenvolvimento do oócito. No conjunto de dados do MALDI observou-se maior concentração de triacilglicerídeos, principalmente contendo ácidos graxos como o oleico, linoleico e esteárico nos líquidos foliculares e células do folículo provenientes do ambiente folicular de oócitos que clivaram. No perfil lipídico do líquido folicular feito por LC-MS/MS os lipídios identificados foram de classes diversas, com participação expressiva no modo positivo de triacilgliceróis, fosfatidilcolinas, esfingomielinas, monoacildiacilglicerídeos entre outros e no modo negativo a classe lipídica que prevaleceu foi dos lipídios mitocondriais, as cardiolipinas. Também foram detectados lipídios citosólicos como o CE e alguns pouco descritos no líquido folicular como o PIP2 e PIP3, além disso, alguns lipídios foram característicos dos grupos clivados e blastocistos. O perfil lipídico do líquido folicular e das células do folículo é complexo e alguns lipídios deste ambiente tem potencial para serem biomarcadores de qualidade oocitária. / In vitro embryo production in Brazil occupies a prominent place among the animal reproduction biotechnology, the current challenge is to make it increasingly accessible so it is essential to approach the results of production and survival of in vitro produced embryos of those developed naturally. Much of this potential change of embryo development is related to the oocyte characteristics. There is the possibility of this ability is related to the lipid profile of follicular fluid and follicle cells , in this sense, this project aimed to evaluate the correlation of the oocytes development potential with the content of the lipid profile in follicular fluid and cells follicular, based on parthenogenetic activation , individual cultivation and retrospective analysis system. There isn\'t, to date, a detailed characterization of the follicular microenvironment, an important component of the follicular fluid and cels are the lipids and recently has been shown that they are involved in various aspects of metabolism in addition also function as signaling molecules or even be capable of inducing changes in chromatin. Derived ions from the study of lipid profile were evaluated compered with oocyte developmental potential. In MALDI dataset observed triacylglycerides higher concentration, mainly containing fatty acids such as oleic, linoleic and stearic acids in follicular fluid and follicle cells from follicular environment of cleaved oocytes. In follicular fluid lipid profile done by LC-MS/MS lipids were identified in diferent classes, with significant participation in positive mode of triacylglycerols, phosphatidylcholines, sphingomyelins, monoacildiacilglicerídeos among others and in negative mode prevailed lipid class was of mitochondrial lipids, the cardiolipin. Cytosolic lipids were also detected as CE and some little follicular fluid as described in the PIP2 and PIP3, and others lipids were characteristic of cleaved and blastocysts groups. The lipid profile of follicular fluid and follicle cells is complex and some of this lipid environment has the potential to be biomarkers of oocyte quality.

A produção in vitro de embriões, no Brasil, ocupa lugar de destaque dentre as biotecnologias da reprodução animal e o atual desafio é torná-la cada vez mais acessível, para tanto, é fundamental aproximar os resultados de produção e sobrevivência dos embriões produzidos in vitro daqueles desenvolvidos naturalmente. Boa parte desta variação do potencial de desenvolvimento do embrião está relacionada à características do oócito. Há a possibilidade de essa capacidade estar relacionada ao perfil lipídico do líquido folicular e das células do folículo, neste sentido, este projeto teve como objetivo principal avaliar a correlação do potencial de desenvolvimento de oócitos com o conteúdo do perfil lipídico no líquido folicular e das células foliculares, baseado no sistema de ativação partenogenética, cultivo individual e analise retrospectiva. Não existe, até o momento, uma caracterização detalhada do microambiente folicular, um componente importante dos fluídos são os lipídios e recentemente tem sido demonstrado que eles estão envolvidos em vários aspectos do metabolismo além de também funcionarem como moléculas sinalizadoras ou até mesmo serem capazes de induzir modificações na cromatina. Os íons oriundos do estudo do perfil lipídico foram avaliados em comparação ao potencial de desenvolvimento do oócito. No conjunto de dados do MALDI observou-se maior concentração de triacilglicerídeos, principalmente contendo ácidos graxos como o oleico, linoleico e esteárico nos líquidos foliculares e células do folículo provenientes do ambiente folicular de oócitos que clivaram. No perfil lipídico do líquido folicular feito por LC-MS/MS os lipídios identificados foram de classes diversas, com participação expressiva no modo positivo de triacilgliceróis, fosfatidilcolinas, esfingomielinas, monoacildiacilglicerídeos entre outros e no modo negativo a classe lipídica que prevaleceu foi dos lipídios mitocondriais, as cardiolipinas. Também foram detectados lipídios citosólicos como o CE e alguns pouco descritos no líquido folicular como o PIP2 e PIP3, além disso, alguns lipídios foram característicos dos grupos clivados e blastocistos. O perfil lipídico do líquido folicular e das células do folículo é complexo e alguns lipídios deste ambiente tem potencial para serem biomarcadores de qualidade oocitária. / In vitro embryo production in Brazil occupies a prominent place among the animal reproduction biotechnology, the current challenge is to make it increasingly accessible so it is essential to approach the results of production and survival of in vitro produced embryos of those developed naturally. Much of this potential change of embryo development is related to the oocyte characteristics. There is the possibility of this ability is related to the lipid profile of follicular fluid and follicle cells , in this sense, this project aimed to evaluate the correlation of the oocytes development potential with the content of the lipid profile in follicular fluid and cells follicular, based on parthenogenetic activation , individual cultivation and retrospective analysis system. There isn\'t, to date, a detailed characterization of the follicular microenvironment, an important component of the follicular fluid and cels are the lipids and recently has been shown that they are involved in various aspects of metabolism in addition also function as signaling molecules or even be capable of inducing changes in chromatin. Derived ions from the study of lipid profile were evaluated compered with oocyte developmental potential. In MALDI dataset observed triacylglycerides higher concentration, mainly containing fatty acids such as oleic, linoleic and stearic acids in follicular fluid and follicle cells from follicular environment of cleaved oocytes. In follicular fluid lipid profile done by LC-MS/MS lipids were identified in diferent classes, with significant participation in positive mode of triacylglycerols, phosphatidylcholines, sphingomyelins, monoacildiacilglicerídeos among others and in negative mode prevailed lipid class was of mitochondrial lipids, the cardiolipin. Cytosolic lipids were also detected as CE and some little follicular fluid as described in the PIP2 and PIP3, and others lipids were characteristic of cleaved and blastocysts groups. The lipid profile of follicular fluid and follicle cells is complex and some of this lipid environment has the potential to be biomarkers of oocyte quality.

The blue crab, Callinectes sapidus Rathbun, 1896, was studied because of its high dollar value to Florida's commercial and recreational fisheries. The purpose of this study was to describe the structure of the ovary and oogenesis in the blue crab and the morphological changes in the female reproductive developmental stages over time. Histological techniques for high-resolution light microscopy were used to determine sexual maturity of female blue crabs. The ovarian morphology, oogenesis, and changes through the annual reproductive cycle of blue crabs in Tampa Bay were investigated for a period of two years, from January 2005 to January 2007. Ovarian structure was assessed by analyzing histological sections embedded in plastic epoxy resin, which provided a higher resolution than any other embedding material previously used in research on blue crab reproduction. Qualitative analyses of female gonads were made by describing the structure of the oocytes and determining the developmental stage of the oocytes from oogonia to full-grown oocytes. This study developed and introduced a new reproductive staging criteria for the species. Morphological characteristics of ovarian tissues and oocytes were determined to develop a classification for oocyte maturation stages. Morphological changes in the oocytes are well defined, and these were used to develop the staging schema. In this study, it was found that carapace width is not a good indicator of maturity or developmental stage. Examination of the annual reproductive cycle indicates that late secondary growth occurs from July to March, and gravid crabs were found during November and December. Histological examination of ovarian tissue is essential for determining maturity in female blue crabs. By observing ovarian characteristics and by establishing the length of secondary growth during oogenesis in blue crabs of Tampa Bay, a more thorough understanding of the cyclic reproductive aspects of this species was obtained and specifically that animals at a carapace width between 100 mm and 125 mm may have mature oocytes, yet external features may not indicate that they are mature.

More than 300 billion cells die in the human body every day. Although there are over a dozen different death paradigms, they all produce the same result - dead and dying cells. As they are no longer actively maintained, persistent corpses can proceed to a secondary necrotic state in which its cell membrane ruptures thus releasing its contents to the extracellular milieu. As many of the intracellular contents act as damage associated molecular patterns (DAMPs), they pose a potential danger to the rest of the surrounding tissue and organism. Excessive cell death has been correlated with diseases such as atherosclerosis, Alzheimer’s, and autoimmune disorders. To avoid damage and disease associated with cell corpses, two classes of cells evolved to clear them away – professional and nonprofessional phagocytes. A professional phagocyte's primary function is to clear away dying cells and other debris. Nonprofessional phagocytes, however, have a primary role other than clearance. When nonprofessional phagocytes encounter a cell corpse, their phagocytic machinery is engaged to clear it away. Interestingly, a recent study demonstrated that most, if not all, tissues contain nonprofessional phagocytes. To investigate nonprofessional phagocytes, the model organism Drosophila melanogaster is ideal. Drosophila is a useful model system as they have orthologs for 70% of human disease genes, a simplified immune system, and a host of genetic tools. Their ovaries have three morphologically distinct cell types – 15 nurse cells and an oocyte all surrounded by an epithelial follicle cell layer. As the ovaries are immunoprivileged, the follicle cell layer acts as the ovaries’ sole phagocytes. During late stage oogenesis, a small subsection of the follicle cell layer – the stretch follicle cells – murder the nurse cells in order to produce a fully developed oocyte. As past studies of cell corpse clearance have predominantly concentrated on the professional phagocytosis in the context of apoptotic cell corpses, there are still many gaps in our knowledge of nonprofessional phagocytosis and non-apoptotic death. This dissertation focuses on the molecular mechanisms that regulate the transition of nonprofessional phagocytes from their primary role as epithelial cells to their phagocytic role in the context of a newly characterized form of non-autonomous cell death known as phagoptosis. To gain a global view of these changes, two large scale experiments were performed – a classic genetic screen of kinases using RNAi and a high-throughput translatome study. The kinase screen identified dozens of kinase genes required for proper clearance. Of the 27 kinase genes that demonstrated a severe phenotype when knocked down, two were previously uncharacterized and six produced an “undead” phenotype, a phenotype that had only been previously witnessed when genes were perturbed in the germline. A follow up study was performed on Gprk2, one of the genes that induced a severe phenotype. By comparing the phenotypes of Gprk2 knockdowns and those of the two canonical clearance pathways, a third clearance pathway was discovered. The translatome study identified over 400 genes that were statistically significantly differentially expressed between primary state and phagocytic state follicle cells, including groups affecting calcium signaling and muscle contraction. This dissertation further describes the expansion of the molecular network of nonprofessional phagocytes driven by these large-scale experiments and their follow up studies.

The development of multi-cellular organisms requires extensive cell-cell communication to coordinate cell functions. However, only a handful of signaling pathways have emerged to mediate all the intercellular communications; therefore, each of them is under an array of regulations to achieve signaling specificity and diversity. One such signaling pathway is the Janus Kinase/ Signal Transducer and Activator of Transcription (JAK/STAT) pathway, which is the primary signaling cascade responding to a variety of cytokines and growth factors in mammals and involved in many developmental processes. This signaling pathway is highly conserved between mammals and Drosophila, but the Drosophila JAK/STAT pathway possesses only three ligands: Unpaired (Upd), Upd2 and Upd3. Co-localized expression patterns of the ligands at several developmental stages raise the possibility that they physically interact. This work was aimed at testing the protein-protein interactions between Upd-family ligands and exploring possible outcomes of ligand oligomerization. Physical interactions between Upd-family ligands were tested using a Bimolecular Fluorescence Complementation (BiFC) assay. The data suggested that homotypic interactions of Upd2 and Upd3 were stronger than their respective heterotypic interactions with Upd, and the homotypic interaction between Upd molecules was the weakest. In addition, the homotypic interaction of Upd3 was confirmed using yeast two-hybrid interaction assays. To identify protein domains critical for Upd3/Upd3 interaction, a series of poly-alanine substitutions were made to target the 6 conserved domains of Upd3. All 6 substitutions altered the strength of Upd3/Upd3 interaction and drastically reduced Upd3-induced JAK signaling activity. In addition, poly-alanine substitutions of some domains also affected Upd3 extracellular localization or protein accumulation. Potential outcomes of interactions between Upd-family ligands were tested both in vitro and in vivo. The interaction between Upd and Upd3 did not significantly change the level of JAK signaling activity. However, loss of Upd3 restricted the distribution of Upd in egg chambers and consequently altered the follicle cell composition. Therefore, Upd/Upd3 interaction is likely to affect the range rather than the intensity of JAK signaling in egg chambers. In summary, this study suggested the possibility of ligand oligomerization as a mechanism for regulating signaling pathways in order to achieve signaling specificity and diversity during development.

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.