Efficient removal of apoptotic cells is critical for development, tissue remodelling, maintenance of homeostasis, and response to injury. Phagocytosis of apoptotic cells is mediated by many phagocytic receptors, soluble bridging molecules, and pro-phagocytic ligands on the surface of apoptotic cells. Macrophage phagocytosis in general is controlled by stimulatory and inhibitory mechanisms. An example of the latter mechanism is that mediated by the cell surface glycoprotein CD47, which by binding to the inhibitory receptor Signal Regulatory Protein alpha (SIRPα) on macrophages, is known to inhibit phagocytosis of viable host cells. The studies of the present thesis aimed at investigating possible changes to CD47 on apoptotic cells, which could influence their elimination by macrophages. The endoplasmatic protein calreticulin (CRT), in conjunction with Low density lipoprotein Receptorrelated Protein 1 (LRP1) on the phagocyte, can act as a receptor for collectin family members and mediate uptake of apoptotic cells. However, CRT itself was found to also be expressed on the surface of many viable cell types, and the CRT expression increased on apoptotic cells. By using antibodies to LRP1 or receptor‐associated protein (RAP), an antagonist blocking LRP1 ligand binding, we found that CRT on target cells could interact in trans with LRP1 on a phagocyte and stimulate phagocytosis. CD47 on the target cell inhibited LRP1‐mediated phagocytosis of viable cells (e.g. lymphocytes or erythtocytes), but not that of apoptotic cells. The inability of CD47 on apoptotic cells to inhibit LRP1‐ mediated phagocytosis could be explained in two ways: 1) Some apoptotic cell types (fibroblasts and neutrophils, but not Jurkat T cells) lost CD47 from the cell surface, or 2) CD47 is evenly distributed on the surface of viable cells, while it was redistributed into patches on apoptotic cells, segregated away from areas of the plasma membrane where the pro‐phagocytic ligands CRT and phoaphatidylserine (PS) were concentrated. Apoptotic murine thymocytes also showed a patched distribution of CD47, but no significant loss of the receptor. However, both PS‐independent and PS‐dependent macrophage phagocytosis of apoptotic CD47‐/‐ thymocytes was less efficient than uptake of apoptotic wild‐type (wt) thymocytes. This contradictory finding was explained by the fact that CD47 on apoptotic thymocytes did no longer inhibit phagocytosis, but rather mediated binding of the apoptotic cell to the macrophage. These effects could in part be dependent on the apoptotic cell type, since uptake of experimentally senescent PS+ wt or CD47‐/‐ erythrocytes by macrophage in vitro, or by dendritic cells (DC) in vivo, were the same. In vivo, PS+ erythrocytes were predominantly trapped by marginal zone macrophages and by CD8+ CD207+ DCs in the splenic marginal zone. DCs which had taken up PS+ erythrocytes showed a slight increase in expression levels of CD40, CD86 and MHC class II. These findings suggest that PS+ erythrocytes may be recognized by splenic macrophages and DCs in ways similar to that reported for apoptotic T cells. Uptake of senescent erythrocytes by DCs may serve as an important mechanism to maintain self‐tolerance to erythrocyte antigens, and defects in this function may facilitate development of AIHA. Glucocorticoids are used to treat inflammatory conditions and can enhance macrophage uptake of apoptotic cells. We found that the glucocorticoid dexamethasone time‐ and dose‐dependently stimulated macrophage cell surface LRP1 expression. Dexamethasone‐stimulated macrophages also showed enhanced phagocytosis of apoptotic thymocytes and unopsonized viable CD47‐/‐ erythrocytes. In summary, LRP1 can mediate phagocytosis of both viable and apoptotic cells by binding CRT on the target cell. Macrophage expression of LRP1 is increased by glucocorticoids, which could be one explanation for the anti‐inflammatory role of glucocorticoids. While CD47 on viable cells efficiently inhibits phagocytosis in macrophages, CD47 on apoptotic cells does not and can sometimes even promote their removal.

Leishmania donovani is a blood-borne tropicial parasite, which infects humans through bites by Phlebotomus sandflies. The parasite survives and multiplies inside macrophages in inner organs, and causes the deadly disease visceral leishmaniasis (Kala-Azar). Macrophages and dendritic cells (DC) are professional antigen-presenting cells involved in the initiation of immune responses. Immature DC are present in all tissues where they internalise and process antigen, in response to which they migrate from tissue, into draining lymphoid organs, undergo maturation and present antigens to lymphocytes. Control measures for leishmaniasis include testing of new diagnostics and development of affordable and effective vaccines for humans. Lipophosphoglycan (LPG) is the major surface component of Leishmania donovani promastigotes. LPG comprises a membrane-anchoring lysophosphatidylinositol part and an extracellular chain of disaccharide phosphates. These repetitions are crucial for parasite survival inside macrophages following phagocytosis. LPG has several specific effects on the host cell including inhibition of protein kinase C (PKC) activity, and inhibition of phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. Confocal microscopy and image analysis were used to follow F-actin dynamics in single macrophages during phagocytosis of L. donovani promastigotes and LPG-coated particles. F-actin did not depolymerize, but instead progressively polymerized around phagosomes with LPG-containing prey. This correlated with reduced translocation of PKCα to the phagosome and blocked phagosomal maturation. LPG also inhibited cortical actin turnover, which could be the underlying cause of the reduced uptake of LPG-containing prey. Extracellular- and intracellular calcium was necessary for phagocytosis, periphagosomal F-actin breakdown and phagosomal maturation in macrophages interacting with unopsonized prey,and for the action of LPG. We also studied F-actin turnover in macrophages overexpressing dominant-negative (DN) PKCα. DN PKCα macrophages showed increased amounts of cortical F-actin, decreased phagocytic capacity, inhibition of periphagosomal F-actin breakdown and defective phagosomal maturation. When DN PKCα macrophages interacted with LPG-containing prey, phagocytosis was almost completely blocked. Moreover, we found that Leishmania promastigotes and particularly LPG inhibit DC maturation and detachment from distinct surfaces. Thus, LPG from Leishmania donovani could directly inhibit DC migration to lymphoid organs, antigen-presentation and development of immunity.

Neutrophil granulocytes play an important role in the host defence against invading microorganisms and constitute the frontline of defence within the innate immune system and are among the first cells to arrive at the site of inflammation. Effective phagocytosis and killing of invading pathogens by neutrophils is of significant importance for successful resistance to infectious diseases. An important complication in diabetes mellitus is an increased sensitivity to infections and increased tissue damage, leading to many secondary diseases. This may in part be explained by an impaired function of neutrophil granulocytes. Since the exact mechanisms underlying defective neutrophil function in diabetes mellitus are not fully understood, the aim of the present study was to investigate the effects of elevated glucose and insulin concentrations on phagocytosis of opsonized yeast and on production of reactive oxygen metabolites (ROS) in normal human neutrophils. Elevated D-glucose concentrations (15-25 mM) inhibited the phagocytosis of C3bi- or IgG-opsonized yeast particles, which was neither an osmotic effect nor an effect due to reduced binding of opsonized yeast particles to the neutrophils. Inhibition of protein kinase C (PKC) by GF109203X or Go6976 could completely reverse the inhibitory effect of 25 mM D-glucose on phagocytosis. Diacylglycerol (DAG) dose-dependently inhibited phagocytosis and suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect. Elevated concentrations of insulin (80-160 μU/ml) also inhibited neutrophil phagocytosis, an effect shown in part to be due to a delayed phagocytosis process. Insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. The inhibition of phagocytosis by either glucose or insulin resulted in an expected reduction of intracellular respiratory burst. However, the extracellular release of ROS during phagocytosis was increased by insulin, but inhibited by glucose. The ability of insulin to enhance ROS production was found to be F-actin dependent. Data suggests that glucose inhibited intracellular respiratory burst activation by interfering with intracellular signaling downstream of PKC activation, whereas extracellular release of ROS was inhibited by glucose upstream of PKC signaling. Taken together these results suggest that both hyperglycemia and hyperinsulinemia inhibit complement receptor and Fc receptor-mediated phagocytosis in human neutrophils. Insulin, but not glucose, also induced an enhanced extracellular release of ROS during phagocytosis. The combination of reduced phagocytosis and alterations in ROS production may possibly explain both the increased sensitivity to infections and tissue damage seen in type 2 diabetes.

<p><I> Trypanosoma congolense </i> is a single-cell blood parasite and an important pathogen causing African trypanosomiasis, also called ngana, in livestock. Ngana in cattle is a chronic disease associated with anemia, cachexia and increased susceptibility to secondary infections. Infection of mice can be used as an experimental model to study the host-parasite relationship. As determined by their survival time, BALB/c mice are highly susceptible to <i> T. congolense </i> infection, whereas C57BL/6 mice are relatively resistant. The surfaces of African trypanosomes are covered with a layer of a single species of glycoprotein, called variant surface glycoprotein (VSG). Production of antibodies to the VSG of African trypanosomes is one of the major immune responses leading to control of parasitemia. The reaction of antibodies with VSG of trypanosomes, for presently unknown reasons, predominantly activates the alternative complement pathway rather than the classical pathway of complement. IgM antibodies are the first and predominant class of anti-trypanosomal antibodies in infected animals. Antibody-mediated phagocytosis of <i> T. congolense </i> by macrophages is considered a major mechanism of control of parasitemia, besides antibody/complement-mediated lysis and cytotoxic effect by macrophage-derived nitric oxide (NO). The receptor(s) on macrophages that recognizes IgM antibody-coated trypanosomes and enables their phagocytosis is unknown. Interaction of antibodies with the VSG of trypanosomes not only causes phagocytosis of trypanosomes by macrophages, but also leads to the release of sVSG from the trypanosomes. sVSG has been found to modulate various functions of the host: induction of polyclonal B cell activation and modulation of macrophage functions, such as the induction of TNF-á synthesis and the inhibition of IFN-ã-induced nitric oxide production. The objectives of this thesis are:</p> <p> 1) to test whether CR3 (Mac-1; CD11b/18) is involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages </p> <p> 2) to test the effects of anti-VSG antibody and complement on the release of soluble VSG from <i> T. congolense </i> </p> <p>1) When the trypanosomes were incubated with IgM anti-VSG antibody and fresh mouse serum, fragments of complement component C3 were found to be deposited onto <i> Trypanosoma congolense </i>. Thus, it was assessed whether complement receptor CR3 (CD11b/CD18; receptor for iC3b) might be involved in IgM anti-VSG mediated phagocytosis of <i> T. congolense </i>. In the presence of fresh mouse serum, there was significantly and markedly less phagocytosis of IgM-opsonized <i> T. congolense </i> by CD11b-deficient macrophages compared to phagocytosis by normal macrophages (78% fewer <i> T. congolense </i> were ingested per macrophage). There also was significantly less TNF-á (38% less), but significantly more NO (63% more) secreted by CD11b-deficient macrophages that had engulfed trypanosomes than by equally treated normal macrophages. It was concluded that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages. It was further concluded that signaling via CR3, associated with IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i>, either directly or indirectly, enhances synthesis of disease-producing TNF-á and inhibits the synthesis of parasite-controlling NO.</p> <p> 2) This investigation revealed that there was more sVSG released from <i> T. congolense </i> by interaction with IgM anti-VSG than by interaction with equal amounts of IgG2a anti-VSG. The release of sVSG occurred in an antibody dose-dependent pattern. It was also found that IgM anti-VSG, after interacting with the surface of <i> T. congolense </i>, formed soluble immune complexes with released sVSG. The results also showed that antibody-induced release of sVSG can occur without complement, but is enhanced by complement. It was further tested whether fresh sera from either relatively resistant C57BL/6 mice or highly susceptible BALB/c mice, which differ in their complement cascade, had different effects on the release of sVSG from <i> T. congolense </i>. The results showed that antibody-induced shedding of sVSG was higher in the presence of fresh C57BL/6 serum than in the presence of fresh BALB/c serum. All these data suggest that the concentration of anti-VSG antibody, antibody class and source of complement can affect the release of sVSG from <i> T. congolense </i></p>.

<p><I> Trypanosoma congolense </i> is a single-cell blood parasite and an important pathogen causing African trypanosomiasis, also called ngana, in livestock. Ngana in cattle is a chronic disease associated with anemia, cachexia and increased susceptibility to secondary infections. Infection of mice can be used as an experimental model to study the host-parasite relationship. As determined by their survival time, BALB/c mice are highly susceptible to <i> T. congolense </i> infection, whereas C57BL/6 mice are relatively resistant. The surfaces of African trypanosomes are covered with a layer of a single species of glycoprotein, called variant surface glycoprotein (VSG). Production of antibodies to the VSG of African trypanosomes is one of the major immune responses leading to control of parasitemia. The reaction of antibodies with VSG of trypanosomes, for presently unknown reasons, predominantly activates the alternative complement pathway rather than the classical pathway of complement. IgM antibodies are the first and predominant class of anti-trypanosomal antibodies in infected animals. Antibody-mediated phagocytosis of <i> T. congolense </i> by macrophages is considered a major mechanism of control of parasitemia, besides antibody/complement-mediated lysis and cytotoxic effect by macrophage-derived nitric oxide (NO). The receptor(s) on macrophages that recognizes IgM antibody-coated trypanosomes and enables their phagocytosis is unknown. Interaction of antibodies with the VSG of trypanosomes not only causes phagocytosis of trypanosomes by macrophages, but also leads to the release of sVSG from the trypanosomes. sVSG has been found to modulate various functions of the host: induction of polyclonal B cell activation and modulation of macrophage functions, such as the induction of TNF-á synthesis and the inhibition of IFN-ã-induced nitric oxide production. The objectives of this thesis are:</p> <p> 1) to test whether CR3 (Mac-1; CD11b/18) is involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages </p> <p> 2) to test the effects of anti-VSG antibody and complement on the release of soluble VSG from <i> T. congolense </i> </p> <p>1) When the trypanosomes were incubated with IgM anti-VSG antibody and fresh mouse serum, fragments of complement component C3 were found to be deposited onto <i> Trypanosoma congolense </i>. Thus, it was assessed whether complement receptor CR3 (CD11b/CD18; receptor for iC3b) might be involved in IgM anti-VSG mediated phagocytosis of <i> T. congolense </i>. In the presence of fresh mouse serum, there was significantly and markedly less phagocytosis of IgM-opsonized <i> T. congolense </i> by CD11b-deficient macrophages compared to phagocytosis by normal macrophages (78% fewer <i> T. congolense </i> were ingested per macrophage). There also was significantly less TNF-á (38% less), but significantly more NO (63% more) secreted by CD11b-deficient macrophages that had engulfed trypanosomes than by equally treated normal macrophages. It was concluded that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages. It was further concluded that signaling via CR3, associated with IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i>, either directly or indirectly, enhances synthesis of disease-producing TNF-á and inhibits the synthesis of parasite-controlling NO.</p> <p> 2) This investigation revealed that there was more sVSG released from <i> T. congolense </i> by interaction with IgM anti-VSG than by interaction with equal amounts of IgG2a anti-VSG. The release of sVSG occurred in an antibody dose-dependent pattern. It was also found that IgM anti-VSG, after interacting with the surface of <i> T. congolense </i>, formed soluble immune complexes with released sVSG. The results also showed that antibody-induced release of sVSG can occur without complement, but is enhanced by complement. It was further tested whether fresh sera from either relatively resistant C57BL/6 mice or highly susceptible BALB/c mice, which differ in their complement cascade, had different effects on the release of sVSG from <i> T. congolense </i>. The results showed that antibody-induced shedding of sVSG was higher in the presence of fresh C57BL/6 serum than in the presence of fresh BALB/c serum. All these data suggest that the concentration of anti-VSG antibody, antibody class and source of complement can affect the release of sVSG from <i> T. congolense </i></p>.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 164-204.

Τα ουδετερόφιλα κύτταρα του ανθρώπινου περιφερικού αίματος, ανήκουν σε μία κατηγορία κυττάρων, τα οποία ονομάζονται επαγγελματίες φαγοκύτταρα και τα οποία είναι υπεύθυνα για την πρόσληψη και θανάτωση των παθογόνων μικροοργανισμών (Bessler et al.2002). Από παλαιότερες έρευνες της ερευνητικής μας ομάδας, έχει δειχθεί η συμμετοχή των σηματοδοτικών μορίων FAK, Elk-1 και MAP κινασών στη ρύθμιση της κυτταροφαγίας από τα εξειδικευμένα φαγοκύτταρα (πλασματοκύτταρα), που βρίσκονται στην αιμολέμφο της μύγας της Μεσογείου, Ceratitis capitata. (Lamprou et al. 2007, Mamali et al. 2008). Είναι επίσης γνωστό ότι κατά την κυτταροφαγία παράγονται διάφορες μορφές δραστικού οξυγόνου (ROS) και αζώτου (RNS), με κύριες το Η2Ο2 και ΝΟ αντίστοιχα. Οι παραγόμενες ROS και RNS συμμετέχουν ως μόρια μεταγωγής σήματος σε διάφορα ενδοκυτταρικά σηματοδοτικά μονοπάτια που ρυθμίζουν την κυτταροφαγία από τα λευκοκύτταρα. Όμως τα βιβλιογραφικά δεδομένα στο πεδίο αυτό είναι λιγοστά. Στην παρούσα εργασία, μελετήθηκε η συμμετοχή των FAK, Elk-1, MAP κινασών και Η2Ο2 στην κυτταροφαγία των βακτηρίων E.coli και S.aureus από τα ουδετερόφιλα κύτταρα του ανθρώπου καθώς και η αλληλεπίδραση μεταξύ των FAK, Elk-1, MAP κινασών και του Η2Ο2 που παράγεται από τα κύτταρα λόγω της παρουσίας των βακτηρίων E.coli και S.aureus. Με κυτταρομετρία ροής και τη χρήση του εξειδικευμένου φθοροχρώματος διυδροροδαμίνη (DHR), πιστοποιήθηκε η παραγωγή Η2Ο2 από τα ουδετερόφιλα κύτταρα του αίματος, παρουσία E.coli και S.aureus. Η ενεργός συμμετοχή του παραγόμενου Η2Ο2 αποδείχτηκε όταν η μείωση της παραγωγής υπεροξεικών ανιόντων με τη χρήση του εξειδικευμένου αναστολέα Ν-εθυλεν-μαλεϊμίδιο (NEM), μείωσε την πρόσληψη βακτηρίων από τα κύτταρα. Η συμμετοχή των FAK και Elk-1 στην κυτταροφαγία ελέγχθηκε με καταστολή της γονιδιακής τους έκφρασης με τη χρήση μορίων siRNA, ενώ αντίστοιχα για τις MAP κινάσες χρησιμοποιήθηκαν αναστολείς της φωσφορυλίωσης τους, οι οποίοι καταστέλλουν την ενεργότητα τους. Με τη χρήση των ίδιων μεθόδων αναστολής για τα μόρια FAK, Elk-1 και MAPKs, μελετήθηκε και ο ρόλος τους στην παραγωγή Η2Ο2 κατά την κυτταροφαγία των βακτηρίων E.coli και S.aureus από τα ουδετερόφιλα κύτταρα του ανθρώπου. Τέλος, ερευνήθηκε και ο ρόλος του Η2Ο2, σαν σηματοδοτικό μόριο κατά τη διαδικασία της κυτταροφαγίας, μελετώντας με ανάλυση κατά western, την επίδραση της αναστολής της παραγωγής του Η2Ο2, στην φωσφορυλίωση (ενεργοποίηση) των MAP κινασών. / Neutrophil cells from human peripheral blood belong in a group of cells, that are called professional phagocytes and are responsible for the uptake and killing of pathogen microorganisms (Bessler et al.2002). Research data from our lab, has shown that the signaling molecules FAK, Elk-1 and MAP kinases participate in the regulation of phagocytosis from the phagocytes of the Mediterranean fly, C.capitata (Lamprou et al. 2007, Mamali et al. 2008). It is also known, that during phagocytosis phagocytes produce different forms of reactive oxygen species (ROS) and nitrogen species (RNS), mainly Η2Ο2 and ΝΟ, respectively. Produced ROS and RNS, act as signaling molecules in different intracellular signaling pathways that regulate phagocytosis in leykocytes. . In the present work, we examined the participation of FAK, Elk-1, MAPKs and Η2Ο2 in the phagocytosis of bacteria E.coli and S.aureus and also the interaction between FAK, Elk- 1, MAPKs and Η2Ο2 that is produced from the cells, due to the presence of the bacteria E.coli and S.aureus. Using flow cytometry and the specialized fluorescent dye Dihydrorodamine-123 (DHR), we certified the production of Η2Ο2 from the human neutrophil cells, in the presence of E.coli and S.aureus. The participation of the produced Η2Ο2 was shown, when the reduction of the produced Η2Ο2 with the use of the inhibitor Ν-etheylen-maleimide (NEM), reduced the uptake of bacteria from the cells. The participation of FAK and Elk-1 in phagocytosis was examined by suppressing their expression with the use of siRNA molecules, while for the MAP kinases we used specific activation inhibitors. By using the same inhibitng methods for the molecules FAK, Elk-1 and MAPKs we studied their role in the production of Η2Ο2 during the phagocytosis of bacteria E.coli and S.aureus from human neutrophil cells. Finally, by reducing the production of superoxide anions, thereby reducing the production of H2O2, by using the specific inhibitor Ν-etheylen-maleimide (NEM), we examined the expression of the phosphorylated forms of MAPKs, by western analysis, in human neutrophils in the presence of the bacteria E.coli and S.aureus.

Ο προγραμματισμένος κυτταρικός θάνατος είναι μία θεμελιώδης διαδικασία τόσο για την ανάπτυξη όσο και για την άμυνα και διατήρηση της δομής και της οργάνωσης όλων των πολυκύτταρων οργανισμών. Τα αιμοκύτταρα της μύγας της Μεσογείου υφίστανται απόπτωση με φυσιολογικούς μηχανισμούς κατά την μεταμόρφωση του εντόμου από το 3ο προνυμφικό στάδιο μέχρι και την νυμφοποίηση. Η απόπτωση των αιμοκυττάρων είναι δυνατό επίσης να επάγεται από εξωγενή χημικά ή φυσικά ερεθίσματα (κυκλοεξαμίδιο, ανισομυκίνη, λιποπολυσακχαρίτη, σταυροσπορίνη, θερμοκρασιακό σοκ). Η έκφραση και φωσφορυλίωση των κινασών FAK, Src, ERK, PI-3K και Akt εξαρτάται από τo στάδιο ανάπτυξης, ενώ το μέγιστο της έκφρασης και φωσφορυλίωσής τους παρατηρείται στο στάδιο της λευκής νύμφης, όπου σημειώνεται και το μέγιστο της απόπτωσης. Η απόπτωση φαίνεται να επηρεάζεται από τα σηματοδοτικά μονοπάτια των Ras/ERK, FAK/Src και PI-3K/Akt. Ενώ ακόμα, πειράματα αποσιώπησης του RNA της κινάσης των εστιών προσκόλλησης (FAK) έδειξαν ότι στη διαδικασία της αναπτυξιακά ρυθμιζόμενης απόπτωσης σημαντική είναι η συμμετοχή της FAK καθώς και των στόχων που έπονται αυτής (downstream), τα σηματοδοτικά μόρια Src, ERK, PI-3K p85a, και Akt. Η κυτταροφαγία είναι μία βασική ενδογενής ανοσολογική απόκριση των οργανισμών έναντι των παρασίτων και άλλων παθογόνων. Τα αιμοκύτταρα των εντόμων αναγνωρίζουν μία πληθώρα «ξένων» για τον οργανισμό βιοτικών και αβιοτικών παραγόντων και αποκρίνονται ενεργοποιώντας διάφορες σηματοδοτικές οδούς μέσω των επιφανειακών υποδοχέων τους με τελικό αποτέλεσμα την κυτταροφαγία. Πειράματα αποσιώπησης της έκφρασης της β υπομονάδας των ιντεγκρινών έδειξαν ότι στα αιμοκύτταρα της μύγας της Μεσογείου η κυτταροφαγία των βακτηρίων E. coli πραγματοποιείται μέσω των ιντεγκρινών, ενώ στη συνέχεια ενεργοποιείται το σηματοδοτικό μονοπάτι FAK/MAP. Επίσης προσδιορίστηκε ότι η FAK ρυθμίζει την κυτταροφαγία των βακτηρίων E. coli μέσω των MAP κινασών και της Src. Τα σηματοδοτικά μόρια των FAK, Src και ERK βρέθηκαν να συμμετέχουν σε κοινό πρωτεϊνικό σύμπλοκο. Τελικός αποδέκτης των παραπάνω μηχανισμών σηματοδότησης που διέπουν τις ενδογενείς ανοσοαποκρίσεις των αιμοκυττάρων φαίνεται να είναι ο μεταγραφικός παράγοντας Elk-1-like, ο οποίος φωσφορυλιώνεται κατά την κυτταροφαγία των βακτηρίων E. coli καθώς και των σφαιριδίων λάτεξ. Η φωσφορυλίωση του Elk-1-like πραγματοποιείται μέσω των FAK, Src και ΜΑΡ κινασών. Πειράματα οσμωτικής ένθεσης αντισωμάτων έναντι του Elk-1 έδειξαν ότι η έκφραση της Elk-1-like πρωτεΐνης στα αιμοκύτταρα ρυθμίζει την κυτταροφαγία των βακτηρίων της E. coli. Μετά την πιστοποίηση της λειτουργικής σχέσης μεταξύ της FAK και της Elk-1-like πρωτεΐνης κατά την κυτταροφαγία, βρέθηκε ότι ο Elk-1- like μεταγραφικός παράγοντας εντοπίζεται στους ίδιους υπo-κυτταρικούς χώρους με την FAK και ότι τα δύο παραπάνω μόρια συμμετέχουν σε κοινό πρωτεϊνικό σύμπλοκο. Ακολούθησε η διερεύνηση της σχέσης των δύο παραπάνω μηνυματοφόρων μορίων, FAK και Elk-1 στα HK-2 κύτταρα του ανθρώπου. Διαπιστώθηκε ότι τα δύο μηνυματοφόρα μόρια FAK και Elk-1 συνεντοπίζονται, σχηματίζοντας πρωτεϊνικό σύμπλοκο κοντά στην περιφέρεια του πυρήνα των HK-2 κυττάρων, παρουσία υψηλής συγκέντρωσης γλυκόζης (25mM). Αναστέλλοντας την έκφραση του Elk-1 στα HK-2 κύτταρα με την τεχνική αποσιώπησης γονιδίων σημειώθηκε αύξηση της απόπτωσης καθώς επίσης και σημαντική μείωση της έκφρασης των FAK και MAP κινασών. Τα παραπάνω ευρήματα συνηγορούν στο συμπέρασμα ότι ο Elk-1 ρυθμίζει την απόπτωση όπως επίσης φαίνεται να ρυθμίζει και την έκφραση των σηματοδοτικών μορίων των FAK/Src και ΜΑΡΚ μονοπατιών στα HK-2 κύτταρα. / Programmed cell death (apoptosis) is an evolutionary conserved process, crucial for the development and maintenance of multicellular organisms. Medfly hemocytes undergo apoptosis during larval-pupal transformation. Hemocytes also undergo apoptosis in response to external chemical or physical stimuli (cycloheximide, anisomycin, lipopolysaccharide, staurosporine and heat shock). Expression and phosphorylation of FAK, Src, ERK, PI-3K and Akt appear to be dependent upon developmental stages. The maximum expression and phosphorylation of the above signalling molecules appeared in hemocytes of the white pupal stage, which is also the peak of apoptosis levels. FAK/Src, Ras/ERK and PI-3K/Akt signalling pathways are involved in the regulation of apoptosis. Furthermore, FAK silencing experiments show that FAK, as well as, its downstream targets (Src, ERK, PI-3K, Akt) are involved in the developmentally regulated apoptosis. Phagocytosis is an important innate immune response against pathogens and parasites. Insect hemocytes, responsible for cellular defense responses, recognize a variety of foreign biotic and abiotic targets and respond by activating several intracellular signalling pathways via hemocyte surface receptors, leading to phagocytosis. Integrin beta-subunit silencing experiments demonstrated that the uptake of bacteria by insect hemocytes is regulated by integrins and results to the activation of FAK/MAPKs signalling pathways. Focal adhesion kinase and its downstream targets (MAPKs and Src) are also implicated in the process of phagocytosis. In addition, experimental results strongly support a physical association between FAK, Src and ERK. The above signalling cellular innate immune responses result to the phosphorylation of Elk-1-like transcription factor in E. coli/latex beads-challenged hemocytes. Elk-1-like protein is a downstream target for FAK/Src and MAPKs pathways, which is phosphorylated via activation of MAPKs. Osmotic loading experiments using Elk-1 antibodies demonstrated the dependence of phagocytosis from Elk-1-like protein. In light of the functional association of FAK with Elk-1-like protein, confocal microscopy analysis shows that these two signalling molecules are localized in the same subcellular compartments, where co-localization also occurs. In light of the functional and physical association of FAK with Elk-1-like protein in medfly hemocytes, we explored whether an analogous functional and physical association between these two essential molecules also exists in mammalian cell systems, in particular, in HK-2 human cells. Indeed, FAK and Elk-1 are co-localized in the periphery of the nuclear membrane in glucose activated cells. Elk-1 silencing in HK-2 cells increased apoptosis, whereas the levels of FAK and MAPKs were significantly decreased. The above results suggest that Elk-1 appears to affect apoptosis via regulating the expression of FAK and MAPKs.

Η FAK, οι MAPKs και ο μεταγραφικός παράγοντας Elk-1 έχει αναφερθεί ότι εμπλέκονται στις ίδιες κυτταρικές διαδικασίες, παρ’ όλα αυτά, η άμεση ή έμμεση αλληλεπίδρασή τους και οι ενδεχόμενες λειτουργίες τους δεν έχουν ακόμα τεκμηριωθεί. Σκοπός αυτής της διατριβής ήταν η περαιτέρω διερεύνηση των σηματοδοτικών μονοπατιών που ρυθμίζουν τη διαδικασία της κυτταροφαγίας και ειδικότερα την αποκάλυψη της φυσικής διασύνδεσης των παραπάνω σηματοδοτικών μορίων. Αρχικά, μελετήσαμε την υποκυτταρική κατανομή των FAK, ERK και Elk-1 στα αιμοκύτταρα της μύγας της Μεσογείου, παρουσία ή απουσία E. coli. Πειράματα ανοσοφθορισμού έδειξαν ότι οι FAK, MAPΚs και Elk-1 κατανέμονται ομοιόμορφα σε όλο το κύτταρο, όμως παρουσία E. coli, φωσφορυλιώνονται και σταδιακά εισέρχονται στον πυρήνα. Στη συνέχεια, με συνεστιακή μικροσκοπία δείχθηκε ότι παρουσία E. coli, οι FAK και ERK φωσφορυλιώνονται και συνεντοπίζονται στην περιφέρεια του κυττάρου. Βιοχημική προσέγγιση επιβεβαίωσε ότι μόνο οι φωσφορυλιωμένες FAK και ERK συμπλοκοποιούνται. Ακολούθως, η φυσική διασύνδεση της FAK και του Elk-1 διερευνήθηκε με ανοσοσυγκατακρήμνιση, αντίστροφη ανοσοσυγκατακρήμνιση, ανάλυση κατά Western και ανοσοφθορισμό. Από τα πειράματα αυτά φάνηκε συμπλοκοποίηση μεταξύ του Elk-1 ή του pSer383Elk-1 και της FAK όχι όμως και της pTyr397FAK. Το σύμπλοκο FAK/Elk-1 εντοπίζεται, κυρίως, στον πυρήνα. Τέλος, πειράματα συνεστιακής μικροσκοπίας έδειξαν ότι η ERK συν-εντοπίζεται με τον Elk-1 σε μικρό βαθμό, όμως ανοσοσυγκατακρήμνιση, αντίστροφη ανοσοσυγκατακρήμνιση και ανάλυση κατά Western δεν αποκάλυψαν κάποια φυσική διασύνδεση μεταξύ της ERK και του Elk-1. / Focal adhesion kinase (FAK), mitogen-activated protein kinases (MAPKs) and the nuclear transcription factor Elk-1 have been reported to be implicated in the same cellular processes, however, their direct or indirect interaction and potential function(s) has not been documented. The goal of this study was to explore further the signaling pathways that regulate the process of phagocytosis and specifically to show the physical association of the above signaling molecules. Initially, we explored the subcellular distribution of FAK, ERK and Elk-1 in medfly haemocytes, treated in the presence or absence of E. coli. Immunofluorescence analysis demonstrated that FAK, MAP kinases and Elk-1 appear to localize throughout the cytoplasm but in the presence of E. coli they are phosphorylated and gradually imported in the nucleus. Furthermore, confocal analysis showed that in the presence of E. coli, FAK and ERK are phosphorylated and co-localized in the periphery of the cell. Biochemical approaches confirmed that only phosphorylated FAK and ERK are physical associated. Moreover, the physical association of FAK and Elk-1 was explored, using co-immunoprecipitation, reciprocal co-immunoprecipitation, Western blot and immunofluorescence analysis. These experiments revealed an association between Elk-1 or pSer383Elk-1 and FAK but not with pTyr397FAK. The FAK/Elk-1 complex is found, mainly, in the nucleus. Finally, confocal analysis demonstrated that ERK co-localizes with Elk-1 at a low level but co-immunoprecipitation, reciprocal co-immuno-precipitation and Western blot did not reveal a physical association between ERK and Elk-1.

This thesis is an investigation of the interactions of insulin resistance (IR), metabolic markers, neutrophil function, and reproductive health in peripartum dairy cows, including the evaluation of a hand-held glucometer for diagnosis of IR. The neutrophil functions of interest were oxidative burst and phagocytosis capacity, and reproductive diseases were endometritis and cervicitis. A total of 81 Holstein cows were enrolled 3 wk prior to expected calving date from November 2010 until October 2011, and were followed until 5 wk postpartum. Known markers of IR, neutrophil function, and disease were monitored through this period. The hand-held glucometer was identified as a useful alternative to laboratory measurements of glucose. Markers of IR influenced phagocytosis capacity and reproductive disease. High haptoglobin concentrations were associated with increased risk of reproductive disease and diminished oxidative burst function. Metabolically related inhibition of neutrophil function may be important in development of reproductive disease. / National Sciences and Engineering Research Council of Canada, Ontario Graduate Scholarship Program