Η ΒΥΠ διαθέτει αντίτυπο της διατριβής σε έντυπη μορφή στο βιβλιοστάσιο διδακτορικών διατριβών που βρίσκεται στο ισόγειο του κτιρίου της. / Η δεσμίνη, το μυοειδικό μέλος της οικογένειας των ενδιάμεσων ινιδίων, εκφράζεται σε καρδιακό, γραμμωτό και λείο μυ. Στις ώριμες γραμμωτές μυϊκές ίνες, το δίκτυο των ενδιάμεσων ινιδίων δεσμίνης περιβάλλει τους Ζ-δίσκους, ενώνοντας τον ένα με τον άλλο και φαίνεται να συνδέει ολόκληρη τη συσταλτή συσκευή με το σαρκείλημμα, διάφορα μεμβρανώδη οργανίδια και τον πυρήνα. Σε μια προσπάθεια να κατανοηθεί η μοριακή φύση αυτών των αλληλεπιδράσεων, το εργαστήριο ξεκίνησε έναν έλεγχο χρησιμοποιώντας το σύστημα διπλού υβριδίου ζύμης, χρησιμοποιώντας διαφορετικά τμήματα της δεσμίνης, συμπεριλαμβανόμενων των περιοχών της κεφαλής και της ουράς του μορίου. Χρησιμοποιώντας την περιοχή του Ν-τελικού άκρου της δεσμίνης, βρέθηκαν αρκετές πιθανές πρωτεΐνικές αλληλεπιδράσεις. Αυτές περιλάμβαναν τη Smp3 mannosyltransferase, μια πρωτεΐνη με PDZ περιοχές και την prosaposin. Η Smp3 mannosyltransferase προσθέτει την 4η μαννόζη στη φωσφατιδυλοϊνοσιτόλη κατά τη μετα-μεταφραστική τροποποίηση των γλυκοζυλοφωσφτιδυλοϊνοσιτολίων (GPIs). Η πρωτεΐνη με PDZ περιοχές (η οποία φαίνεται να είναι η MUPP1) περιέχει 13 PDZ περιοχές και είναι γνωστό ότι συνδέει τις διαμεμβρανικές περιοχές των στενοσυνδέσμων με ενδοκυττάρια μηνυματοφόρα μόρια. Η prosaposin είναι μια γλυκοπρωτεΐνη 511 αμινοξέων, η οποία αποτελεί πρόδρομο μόριο για τις τέσσερις saposins (sphingolipid activator proteins A, B, C, D). Ο σκοπός της παρούσας διπλωματικής εργασίας είναι η επιβεβαίωση των αλληλεπιδράσεων αυτών και η περαιτέρω μελέτη της πιο σημαντικής. Χρησιμοποιώντας τη μέθοδο GST pull down βρέθηκε ότι το αμινοτελικό άκρο της δεσμίνης όντως αλληλεπιδρά με την Smp3 mannosyltransferase, την πρωτεΐνη PDZ και την prosaposin. Περαιτέρω ανάλυση με την prosaposin έδειξε ότι το αμινοτελικό άκρο της δεσμίνης αλληλεπιδρά με το τμήμα της prosaposin που αντιστοιχεί στη saposin D και όχι με το καρβοξυτελικό άκρο της prosaposin, το οποίο περιέχει μια saposin-like περιοχή. Τα παραπάνω αποτελέσματα αν επιβεβαιωθούν in vivo, προτείνουν έναν πιθανό ρόλο της δεσμίνης στη συγκρότηση πρωτεϊνικών συμπλόκων, τον έλεγχο της αναγέννησης και ανακύκλωσης μεμβρανών, τη μεταγωγή σήματος, το μεταβολισμό των λιπιδίων και τη μεταφορά στα υποκυτταρικά οργανίδια. / Desmin, the muscle-specific member of the intermediate filaments family, is expressed in cardiac, skeletal and smooth muscle. In the mature striated myofibrils, the network of desmin intermediate filaments surrounds Z-discs, extends from one Z-disc to the other and seems to associate with the nucleus, the plasma membrane and other membranous organelles, including mitochondria and sarcoplasmic reticulum. In an effort to understand the molecular nature of these interactions, the laboratory has started a global yeast two-hybrid screen using different desmin fragments, including the head and tail domains of the molecule. Using the desmin N-terminal domain, several potential interacting proteins were found. These include prosaposin, a protein with PDZ domains and Smp3 mannosyltransferase. Smp3 mannosyltransferase adds the 4th mannose to phosphatidyloinositol during the post-translation modification of GPIs. The protein with PDZ domains, which seems to be MUPP1, has 13 PDZ domains and is known to connect the transmembrane domains of the tight junctions with messenger molecules. Prosaposin is a glycoprotein with 511 amino acids, which is cleaved in four mature saposins (sphingolipid activator proteins), when it reaches the lysosomes. The purpose of this work is to verify these interactions and further study the most important one. Using the GST pull down assays, it was verified that the amino terminus of desmin interacts indeed with Smp3 mannosyltransferase, protein PDZ and prosaposin. Further analysis with prosaposin demonstrated that the amino terminus of desmin interacts with the part of prosaposin that corresponds to saposin D and not with the downstream carboxy terminus of prosaposin that contains a saposin-like domain. All the above data, if verified in vivo could suggest a possible role of desmin in the assembly and stabilization of protein-complexes, in the control of biogenesis and regeneration of membranes, in signal transduction, in lipid metabolism and trafficking towards subcellular organelles.

Bovine lactoferrin (BLF) and bovine transferrin (BTF) are major-iron transport and regulation proteins found in bovine whey. BLF and BTF must interact with the eukaryotic cell surface to mediate their biological function of iron delivery and cellular functions of inflammatory and immunological modulation. As common components of the eukaryotic cell surface, gangliosides were used for affinity purification of BLF and BTF. Bovine gangliosides were isolated from fresh buttermilk and covalently immobilized onto controlled-pore glass beads (66 μg/g beads). After the matrix was loaded with whey protein (WPI or WPC), lactoferrin was eluted with 1 M NaCl and lll identified by N-terminal protein sequencing. Pretreated whey isolate (1 % wt/vol) showed the highest lactoferrin purity with 40% among protein sources, and whey protein isolate (10% wt/vol) showed the highest recovery with 105%. Bovine transferrin was eluted with sodium phosphate buffers at pH 7 after the immobilized matrix was loaded with a 2% (wt/vol) whey solution. The ganglioside column resulted in a 74.2% recovery of BTF from whey, and the BTF was enriched to 61% purity after Mono-Q chromatography. Bovine transferrin was identified by SDS-PAGE analysis, Western analysis, and isoelectrofocusing. In conclusion, immobilized gangliosides can be used to purify BLF and BTF from bovine whey.

The aim of the work described in this thesis is to investigate the nature and mechanisms of action of repellent cues for growing axons. In particular I try to resolve the controversy in the literature regarding the need for protein synthesis in the growth cone in response to external guidance cues. My results resolve the conflicting data in the literature on Semaphorin-3A signalling, where differing labs had shown that inhibiting protein synthesis either blocks or has no effect upon repulsion. They demonstrate the presence of at least two independent pathways, protein synthesis-dependent mTOR activation and -independent GSK3? activation. The higher sensitivity of the synthesis-dependent pathway, and its redundancy at higher concentrations where synthesis-independent mechanisms can evoke a full collapse response alone, resolve the apparent conflict. My experiments also demonstrated that Nogo-?20, a domain of Nogo-A, requires local protein synthesis to cause collapse. Unlike Semaphorin-3A, the dependence of collapse upon protein synthesis is concentration-independent and does not involve guanylyl cyclase, but it does share a dependence upon mTOR activity and the synthesis of RhoA, sufficient to cause collapse downstream of Semaphorin-3A. The other axon-repelling domain of Nogo-A, Nogo-66, is partially dependent upon the proteasome instead. It does not share a common pathway with Nogo-?20, except that both are RhoA-dependent. I further attempted to identify the nature of a repulsive activity found in grey matter, ruling out a previously suggested candidate identity. Finally, I examined the phenomenon of nitric oxide-induced growth cone collapse. My experiments revealed that S-nitrosylated glutathione causes growth cone collapse through the activity of protein disulphide isomerase. This mechanism shows only a partial dependence upon soluble guanylyl cyclase, but I argue that it has total dependence upon an S-nitrosylated donor. Coupled with its apparent relation to S-palmitoylation, the reciprocal of S-nitrosylation, I propose that nitric oxide causes collapse by crossing the cell membrane to inhibit S-palmitoylation-determined localisation of proteins. These results reveal some of the many pathways involved in growth cone collapse, whose further characterisation may provide new targets for the treatment of injuries of the central nervous system.