Η δεσμίνη, η μυοειδική πρωτεΐνη των ενδιαμέσων ινιδίων, δημιουργεί ένα εκτενές δίκτυο που συνδέει τη συσταλτή συσκευή των μυοκυττάρων με την κυτταροπλασματική μεμβράνη -στο επίπεδο των εμβόλιμων δίσκων για τα καρδιομυοκύτταρα- τον πυρήνα και διάφορα μεμβρανώδη κυτταρικά οργανίδια. Σκοπός αυτής της εργασίας ήταν η διερεύνηση του μηχανισμού καρδιοπροστασίας που παρέχει ο κυτταροσκελετός της δεσμίνης in vivo, καθώς και η γενικότερη συμμετοχή της στα μονοπάτια της απόπτωσης. Η αποδιοργάνωση του κυτταροσκελετού είναι μια βασική διαδικασία για την εξέλιξη του μονοπατιού της απόπτωσης. Τόσο τα μικροϊνίδια της ακτίνης, όσο και τα ενδιάμεσα ινίδια -συμπεριλαμβανομένων και των πυρηνικών λαμινών- αποτελούν υπόστρωμα πέψης για διάφορα μέλη της οικογένειας των κασπασών. Η δεσμίνη είναι γνωστό ότι πέπτεται in vitro από ανασυνδυασμένη κασπάση-6, στο 263 ασπαρτικό οξύ. Το αμινοτελικό προϊόν αυτής της πέψης δεν είναι ικανό να συμμετέχει στη συγκρότηση ινιδίων, αλλά αντίθετα παρεμποδίζει αυτή τη διαδικασία σχηματίζοντας συσσωματώματα. Ως in vivo μοντέλο επαγωγής της απόπτωσης χρησιμοποιήθηκαν διαγονιδιακά ποντίκια που υπερεκφράζουν, ειδικά στην καρδιά, τον Παράγοντα Νέκρωσης Όγκων (Tumor Necrosis Factor, TNF-α). O TNF-α έχει βρεθεί ότι επάγει διαφορετικά μονοπάτια κυτταρικού θανάτου ενεργοποιώντας, μεταξύ άλλων, μέλη της οικογένειας των πρωτεολυτικών ενζύμων, των κασπασών. Το μυοκάρδιο των ποντικιών που υπερεκφράζει TNF-α (ΜΗCsTNF) χαρακτηρίζεται αρχικά από ομόκεντρη υπερτροφία των καρδιομυοκυττάρων που ακολουθείται από διάταση τόσο των κόλπων, όσο και των κοιλιών. Τα ποντίκια αυτά πεθαίνουν από καρδιακή ανεπάρκεια σε ποσοστό 25% πριν τη συμπλήρωση του έκτου μήνα ζωής. Στο μυοκάρδιο των ΜΗCsTNF ποντικιών ελέγχθηκε αρχικά ο υποκυτταρικός εντοπισμός της δεσμίνης. Η δεσμίνη στην καρδιά των ΜΗCsTNF ποντικιών έχει χάσει τον εντοπισμό της στους εμβόλιμους δίσκους, αλλά διατηρεί, εν μέρει, την παρουσία της στους Ζ-δίσκους. Προκειμένου να διαπιστωθεί εάν εκτός από τη δεσμίνη, έχει αλλοιωθεί η παρουσία και άλλων πρωτεϊνών των εμβόλιμων δίσκων, μελετήθηκε στο μυοκάρδιο αυτών των ποντικιών, η υποκυτταρική κατανομή της δεσμοπλακίνης, της β-κατενίνης και της κοννεξίνης 43, που είναι χαρακτηριστικές πρωτεΐνες των δεσμοσωμάτων, των συνδέσμων πρόσδεσης και των χασματοσυνδέσμων, αντίστοιχα. Οι πρωτεΐνες αυτές αρχικά φαίνεται να αγκυροβολούνται στους εμβόλιμους δίσκους, παροδικά όμως απομακρύνονται και κατευθύνονται προς την πλευρική επιφάνεια των καρδιομυοκυττάρων. Μελέτη με ηλεκτρονικό μικροσκόπιο αποκάλυψε ότι εκτός από την κατανομή συγκεκριμένων πρωτεϊνών, είναι διαταραγμένη και η γενική αρχιτεκτονική των εμβόλιμων δίσκων. Επιπρόσθετα, πιστοποιήθηκε ότι η δεσμίνη πέπτεται in vivo και συσσωρεύεται σε συσσωματώματα. Η παρουσία συσσωματωμάτων δεσμίνης έχει συσχετιστεί με αρκετές κληρονομικές μυοπάθειες στον άνθρωπο, οι οποίες χαρακτηρίζονται ως Μυοπάθειες Σχετιζόμενες με τη Δεσμίνη (Desmin Related Myopathies, DRM). Μεταλλάξεις, αλλά και τροποποιήσεις στη δεσμίνη ή σε άλλες πρωτεΐνες με τις οποίες αλληλεπιδρά προκειμένου να δημιουργήσει το συνεχόμενο κυτταροσκελετικό δίκτυο, έχουν ως συνέπεια τη συγκέντρωση συσσωματωμάτων στο κυτταρόπλασμα και έχουν ενοχοποιηθεί για τη διατατική μυοκαρδιοπάθεια και την εξέλιξη της καρδιακής ανεπάρκειας. Στο μυοκάρδιο των ΜΗCsTNF ποντικιών πιστοποιήθηκε η παρουσία δύο διαφορετικών ειδών συσσωματωμάτων της δεσμίνης. Τα πρώτα εμφανίζουν διάχυτο κυτταροπλασματικό πρότυπο και η δεσμίνη σε αυτά συνεντοπίζεται με την HSP25, ενώ τα δεύτερα τείνουν να περιοριστούν κοντά στον πυρήνα και εδώ η δεσμίνη συνεντοπίζεται με την ουμπικουϊτίνη. Μελέτη της υπερδομής των συσσωματωμάτων έδειξε ότι ποικίλουν σε μέγεθος και σύσταση, καθώς περιέχουν κυστίδια, μιτοχόνδρια, μεμβρανώδες υλικό και υλικό υψηλής πυκνότητας, που φαίνεται ότι παρεμποδίζουν, εκτός από τη λειτουργία του πρωτεασώματος, την συσταλτική ικανότητα των μυοϊνιδίων και συμβάλλουν στην εξέλιξη της καρδιακής ανεπάρκειας. Προκειμένου να μελετηθεί η σπουδαιότητα της δεσμίνης στην παθολογία του μυοκαρδίου που υπερεκφράζει TNF-α, ΜΗCsTNF ποντίκια διασταυρώθηκαν με ποντίκια που δεν εκφράζουν δεσμίνη (des-/-). Τα des-/- ποντίκια χαρακτηρίζονται από μια παροδική υπερτροφία των καρδιομυοκυττάρων, η οποία ακολουθείται από ανάπτυξη διατατικής μυοκαρδιοπάθειας με εξέχοντα χαρακτηριστικά τις ανωμαλίες στα μιτοχόνδρια, το μαζικό κυτταρικό θάνατο και τις εκτενείς εναποθέσεις ασβεστίου στην εξωτερική επιφάνεια της καρδιάς και κολλαγόνου στο εσωτερικό της. Τα ΜΗCsTNF des-/- ποντίκια παρουσίασαν δραματική μείωση και στις δύο μορφές συσσωματωμάτων, ενώ η δεσμοπλακίνη και η β-κατενίνη -πρωτεΐνες που ανήκουν σε μια ενιαία και διαπλεκόμενη μορφή κυτταρικών συνδέσεων, την “area composita”- διατήρησαν σημαντικά την παρουσία τους στους εμβόλιμους δίσκους. Οι παρατηρήσεις αυτές υπογραμμίζουν το βαθμό στον οποίο η δεσμίνη συμμετέχει στην εξέλιξη του ΜΗCsTNF φαινοτύπου. Παραδόξως, στο μυοκάρδιο των ΜΗCsTNF des-/- ποντικιών δεν παρατηρούνται τα μορφολογικά χαρακτηριστικά που προκαλεί η έλλειψη της δεσμίνης, ενώ διατηρούνται αυτά της υπερέκφρασης του TNF-α, υποδηλώνοντας ενδεχόμενη προστατευτική δράση του TNF-α για τα des-/- καρδιομυοκύτταρα. Η διερεύνηση του μηχανισμού της πέψης της δεσμίνης από την κασπάση-6, αλλά και της απομάκρυνσής της από τους εμβόλιμους δίσκους, με τις συνακόλουθες συνέπειες, αποτέλεσε τον επόμενο στόχο αυτής της εργασίας. Για το σκοπό αυτό, δημιουργήθηκαν διαγονιδιακά ποντίκια που εκφράζουν τη μεταλλαγμένη δεσμίνη D263E, η οποία στο 263 αμινοξύ φέρει αντί για ασπαρτικό, γλουταμινικό οξύ. Η D263E δεσμίνη δεν πέπτεται από τις κασπάσες στο ΜΗCsTNF μυοκάρδιο, διατηρεί σε μεγάλο βαθμό την παρουσία της στους εμβόλιμους δίσκους και φαίνεται ότι συγκρατεί και τις υπόλοιπες πρωτεΐνες της “area composita” στη δομή αυτή. Επιπλέον, τα συσσωματώματα της ουμπικουϊτίνης μειώνονται σημαντικά και χάνεται ο συνεντοπισμός με τη δεσμίνη σε όσα από αυτά σχηματίζονται. Τέλος, η παρουσία της D263E δεσμίνης μείωσε τον αριθμό των αποπτωτικών καρδιομυοκυττάρων στο ΜΗCsTNF μυοκάρδιο και βελτίωσε σημαντικά την καρδιακή λειτουργία. Συνοψίζοντας, τα αποτελέσματα αυτής της εργασίας υπογραμμίζουν ότι η δεσμίνη είναι ένα μόριο με σημαντικό ρόλο στην εξέλιξη της μυοκαρδιοπάθειας και της καρδιακής ανεπάρκειας, στο μοντέλο που προκαλείται από την υπερέκφραση του TNF-α. Η συμμετοχή της στο μονοπάτι της απόπτωσης ως υπόστρωμα πέψης για τις κασπάσες και οι επακόλουθες συνέπειες, αποτελούν βάση μελέτης του ρόλου των ενδιαμέσων ινιδίων και για άλλες εκφυλιστικές νόσους. / Desmin, the muscle specific intermediate filament protein, forms a three-dimensional scaffold which links the contractile apparatus to the plasma membrane intercalated disks (IDs), the nucleus and also other membranous cellular organelles. The main goal of this study was to assess the mechanism of cardioprotection by the desmin cytoskeleton in vivo and its participation in the apoptotic pathway. Disruption of the cytoskeleton is a key event in apoptotic cell death pathways. Both actin microfilaments and intermediate filaments -including the nuclear lamins- are substrates for cleavage by different members of the caspase family. Desmin is known to be specifically cleaved in vitro, by caspase-6, at the 263 aspartic acid residue. The amino-terminal product of desmin cleavage is completely unable to assemble into filaments and forms intracellular aggregates. The in vivo model used for the induction of apoptosis was transgenic mice that overexpress specifically in the heart the Tumor Necrosis Factor-α (ΤΝF-α). ΤΝF-α has been described to induce several cell death pathways that converge on activation of different members of the caspase proteolytic enzyme family. The myocardium of mice that overexpress ΤΝF-α (ΜΗCsTNF) is characterized by concentric hypertrophy of cardiomyocytes followed by chamber dilation. 25% of ΜΗCsTNF mice die by heart failure before the age of sixth month. We examined the subcellular localization of desmin in the myocardium of ΜΗCsTNF mice. Desmin is absent from the IDs of ΜΗCsTNF myocardium whereas it preserves, in part, its presence at Z-disks. In order to delineate whether the relocalization of desmin influences the position of other ID proteins, we examined the subcellular localization of desmoplakin, β-catenin and connexin 43, which are characteristic proteins of desmosomes, adherent junctions and gap junctions respectively. At early stages, these proteins localize almost normally in MHCsTNF cardiomyocytes, however, in the 3-month old MHCsTNF mice, all studied proteins were primarily localized at the lateral, non-junctional, side of the cardiomyocytes. Ultrastructural study of the MHCsTNF myocardium revealed that the overall ID architecture is altered. Additionally, we show that desmin is cleaved in vivo and accumulates into cytoplasmic aggregates. The presence of desmin positive aggregates has been correlated with several human inherited myopathies called Desmin Related Myopathies (DRM). Mutations within its gene or in the genes of other desmin associated cytoskeletal proteins, result in accumulation of aggregates in the cytoplasm and have been linked to Dilated Cardiomyopathy (DCM) and heart failure. In the MHCsTNF myocardium we observe two distinct kinds of aggregates that are positive for desmin. The first ones are diffused in the cytoplasm and desmin colocalizes with HSP25, while in the second category the aggregates tend to be restricted near the nucleus and desmin colocalizes with ubiquitin. Ultrastructural study of the aggregates reveals that they are variable in size and composition, consisting of vesicles, mitochondria, membranous whorls and, occasionally, clumps of electron dense material, suggesting that, in addition to other functions such as proteasome activity, they may interfere with the sarcomere contraction thereby leading to impairment of cardiac function. In order to study the importance of desmin in the development of the ΜΗCsTNF pathology, we crossed ΜΗCsTNF mice with desmin null mice (des-/-). In desmin-deficient mice transient cardiomyocyte hypertrophy is followed by development of a DCM that is characterized by mitochondrial functional, ultrastructural and other defects, extensive cell death, fibrotic lesions and calcium deposition at the external surface of the heart. ΜΗCsTNF des-/- mice showed significant decrease in both kind of aggregates, whereas desmoplakin and β-catenin -proteins that belong to the uniform cardiomyocyte junctional structure “area composita”- preserve at high degree their presence at IDs. These observations underline the level at which desmin participates in the progress of ΜΗCsTNF phenotype. Surprisingly, the myocardium of ΜΗCsTNF des-/- mice eliminated the morphological defects caused by the absence of desmin while retained those caused by TNF-α overexpression, suggesting a possible counteracting action of TNF-α in the des-/- cardiomyocytes. To investigate the mechanism and the importance of desmin cleavage by caspase-6 and its withdrawal from IDs with the concomitant consequences, we generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E), harboring a substitution of the 263 aspartic acid (D) with a glutamic acid (E), which renders desmin resistant to caspase mediated cleavage. We showed that D263E desmin is indeed resistant to caspase cleavage in the ΜΗCsTNF myocardium, largely retains its proper ID localization and allows the same for the other proteins of the area composita. Additionally, the ubiquitin positive aggregates are diminished and the few generated do not colocalize with desmin. Importantly, D263E desmin expression attenuated cardiomyocyte apoptosis, prevented left ventricular wall thinning and improved function of MHCsTNF hearts. To summarize, the data presented in this study underline that desmin is a target and mediator of ΤΝF-α induced cardiomyopathy and heart failure. Its contribution in the pathway of apoptosis as a substrate for caspase cleavage and the subsequent participation in the formation of aggregates and the alteration of the ID architecture is the baseline for the study of the role of Intermediate Filaments in degenerative diseases.

Το επιστημονικό ενδιαφέρον της παρούσας διδακτορικής διατριβής εστιάζεται στο ρόλο του κυτταροσκελετού της δεσμίνης (desmin), της μυοειδικής πρωτεΐνης, μέλος της οικογένειας των ενδιάμεσων ινιδίων, στη διατήρηση και τη λειτουργία του καρδιακού μυός. H καταστολή της έκφρασης της δεσμίνης σε μύες με τη χρησιμοποίηση της γονιδιακής στόχευσης, έδειξε πως η έκφραση της δεσμίνης είναι απαραίτητη για τη διατήρηση της λειτουργικής και δομικής ακεραιότητας του καρδιακού μυός. Μύες που δεν εκφράζουν δεσμίνη αναπτύσσουν διατατική καρδιομυοπάθεια, που χαρακτηρίζεται από ανωμαλίες σε υποκυτταρικά οργανίδια, όπως είναι τα μιτοχόνδρια, εκτεταμένη ίνωση και εναπόθεση ασβεστίου, με επακόλουθο τον κυτταρικό θάνατο, και τελικά την καρδιακή ανεπάρκεια. Πιο συγκεκριμένα σε μια προσπάθεια διερεύνησης της βιολογικής δράσης της δεσμίνης, μελετήθηκαν οι πρωτεϊνικές αλληλεπιδράσεις αυτού του μορίου. Η ανεύρεση μορίων με τα οποία η δεσμίνη αλληλεπιδρά μπορεί να δώσει σημαντικές πληροφορίες για τη βιολογικής της δράση και να βοηθήσει στην κατανόηση της παθολογίας που παρατηρείται σε επίμυς όπου έχει κατασταλεί η έκφρασή της (διαγονιδιακό μοντέλο διατατικής καρδιομυοπάθειας που έχει δημιουργηθεί από το εργαστήριό μας, des-/-) ή σε ασθενείς με καρδιομυοπάθειες εξαιτίας μεταλλαγών στο μόριο της δεσμίνης. Για το σκοπό αυτό εφαρμόστηκε η μέθοδος του διυβριδικού συστήματος ζύμης (Yeast two-hybrid), όπου χρησιμοποιήθηκε τμήμα του μορίου της δεσμίνης (το αμινο-τελικό άκρο), με το οποίο ‘σαρώθηκε’ (screening) μια cDNA καρδιακή βιβλιοθήκη, και βρέθηκε ένας αριθμός δυνητικών αλληλεπιδράσεων. Η σάρωση αποκάλυψε την αλληλεπίδραση της δεσμίνης με ένα νέο μόριο, τη Myospryn, μια μυοειδική πρωτεΐνη 413 kDa, μέλος της οικογένειας των TRIM-like πρωτεϊνών. Η πρωτεΐνη myospryn βρέθηκε αρχικά να αλληλεπιδρά με την πρωτεΐνη dysbindin, η οποία εμπλέκεται σε διαδικασίες διαμετακίνησης πρωτεϊνών, κυστιδίων και βιοσύνθεσης οργανιδίων, και αποτελεί συστατικό ενός πρωτεϊνικού συμπλόκου υπεύθυνου για τη βιογένεση λυσοσωμάτων και λυσοσωματικά συσχετιζόμενων οργανιδίων, γνωστό ως Biogenesis of Lysosome Related Organelles (BLOC-1). Η πρόσδεση της δεσμίνης με τη myospryn επαληθεύτηκε με in vitro μεθόδους (GST pull down assay, Co-immunoprecipitation). H ανάλυση ανοσοαποτύπωσης (western blot) απεκάλυψε ότι το ανοσολογικό σύμπλοκο που κατακρημνίζεται με χρήση αντισωμάτων δεσμίνης περιέχει, εκτός της myospryn, και δυο τουλάχιστον υπομονάδες του πρωτεϊνικού συμπλόκου BLOC-1, dysbindin και pallidin. Πειράματα με το δι-υβριδικό σύστημα ζύμης απεκάλυψαν την ακριβή περιοχή αλληλεπίδρασης των δύο πρωτεϊνών, και η οποία περιλαμβάνει τα αα58-103 του αμινο-τελικού άκρου της δεσμίνης και τα τελευταία 24αα του καρβόξυ-τελικού άκρου του μορίου της myospryn, που αντιστοιχούν στο SPRY τμήμα του TRIM-like μοτίβου της οικογένειας των πρωτεϊνών στην οποία ανήκει η myospryn. Πειράματα ανοσοφθορισμού σε απομονωμένα καρδιομυοκύτταρα απεκάλυψαν ότι η myospryn τοπολογείται στην περιφέρεια του πυρήνα, σε στενή γειτνίαση με τις μεμβράνες του ενδοπλασματικού δικτύου, και στο σημείο αυτό αλληλο-επικαλύπτεται με τη δεσμίνη, η οποία σχηματίζει ένα δίκτυο ινιδίων που διατρέχει όλο το μήκος του κυττάρου. Στον ενήλικο καρδιακό ιστό, η myospryn βρίσκεται κυρίως στους εμβόλιμους δίσκους, καθώς και στο επίπεδο της πλασματικής μεμβράνης, του σαρκολείματος, όπου και αλληλοεπικαλύπτεται με τη δεσμίνη. Η αλληλο-επικάλυψη των δυο πρωτεϊνών είναι έντονη στο επίπεδο των εμβόλιμων δίσκων. Δείχθηκε πως η myospryn άλληλο-επικαλύπτεται με τα λυσοσώματα καθώς και ότι η δεσμίνη είναι απαραίτητη τόσο για τη σωστή τοπολόγηση της myospryn, όσο και για αυτήν των λυσοσωμάτων. Τέλος, με πειράματα καταστολής τηw έκφρασης της myospryn, αποκαλύφθηκε πως η παρουσία της myospryn είναι απαραίτητη προκειμένου η δεσμίνη να μπορεί να ανοσοκατακρημνίσει τις υπομονάδες του BLOC-1, dysbindin και pallidin Τέλος, στην προσπάθεια χαρακτηρισμού του λειτουργικού ρόλου της myospryn συμπεριλήφθηκε η χρησιμοποίηση του μοντέλου του D. rerio (zebrafish). Χρησιμοποιώντας την τεχνολογία των antisense morpholinos, ήταν δυνατή η παρεμπόδιση της έκφρασης της myospryn και η μελέτη των επιπτώσεων στην ανάπτυξη και λειτουργία του καρδιακού ιστού. Η καταστολή της έκφρασης της myospryn είχε ως αποτέλεσμα σοβαρές δομικές και λειτουργικές ανωμαλίες στο καρδιακό σύστημα ατόμων Danio rerio, καταδεικνύοντας την σπουδαιότητα αυτού του μορίου στην ανάπτυξη του καρδιακού συστήματος του D.rerio. Συμπερασματικά η παρούσα διδακτορική διατριβή διαπραγματεύεται τις πρωτεϊνικές αλληλεπιδράσεις της μυοειδικής πρωτεΐνης δεσμίνης, αποκαλύπτοντας νέους ρυθμιστικούς ρόλους για το μόριο αυτό. / The scientific interest of this PhD thesis is mainly the role of desmin cytoskeleton (the muscle-specific intermediate filament) in myocyte maintenance and function. The ablation of desmin expression in mice by gene targeting demonstrated that desmin expression is crucial for maintanance of healthy muscle. Mice null for desmin develop dilated cardiomyopathy characterized by mitochondrial abnormalities, extensive cardiomyocyte death, fibrosis, calcification and eventually heart failure. Study of desmin protein interactions could facilitate our understanding of the molecular mechanisms responsible for the observed pathology. To that end, we have performed a yeast two-hybrid screening of a cardiac cDNA library We showed that the desmin amino-terminal domain [Naa(1 -103)] binds to a 413kDa TRIM-like protein, myospryn, originally identified as the muscle-specific partner of dysbindin, a component of the Biogenesis of Lysosome Related Organelles Complex 1 (BLOC-1). Binding of desmin with myospryn was confirmed with GST pull down assays and co-immunoprecipitation experiments. Western blot analysis revealed that the complex immunoprecipitated by desmin antibodies, in addition to myospryn contained the BLOC-1 components dysbindin and pallidin. Deletion analysis revealed that only the [Naa(1 -103)] fragment of desmin binds to myospryn carboxyl terminus and that this association takes place through the 24aa long C-terminal end of the SPRY domain of myospryn. Using an antibody against the COOH terminus of myospryn, we demonstrated that myospryn co-localizes with desmin at the periphery of the nucleus, in close proximity to the endoplasmic reticulum, of mouse neonatal cardiomyocytes. In adult heart muscle, the two proteins co localize, predominantly, at intercalated discs and costameres. We also showed that myospryn colocalizes with lysosomes. Using desmin null hearts, we demonstrated that desmin is required for both the proper perinuclear localization of myospryn, as well as the proper positioning of lysosomes, thus suggesting a potential role of desmin IFs in lysosomes and lysosome-related organelle biogenesis and/or positioning. siRNAs experiments for myospryn knock down revealed that when myospryn is absent, desmin is not able to immunoprecipitate BLOC-1 subunits. Additionally, the in vivo role of myospryn was examined using the model of D. rerio. Using a splice morpholino for knocking down myospryn transcript, we examined the effects on zebrafish heart development and function caused by the absence of myospryn, where it was shown that myospryn is essential for the early heart development in D.rerio. In summary, this work reveals the association of desmin with a novel molecule, myospryn, and it sheds light on novel regulatory role for this member of intermediate filament family.

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.

Biochemical changes in muscle postmortem have been associated with initial beef tenderness early postmortem, and with improvements in tenderness during postmortem storage, defined as meat aging. Differences in the initial contractile state of the sarcomere, the ionic environment of the sarcoplasm including pH, the activity of neutral proteolytic enzymes, and collagen content and solubility have been associated with beef tenderness. In Phase I, steaks from four genetic lines of steers and heifers were used to understand the biochemical differences between tough and tender steaks. The most tender (< 30 N Warner Bratzler shear force (WBS)) and toughest Longissimus steaks (< 30 N WBS) from Angus, Braford, Brangus, and Simbrah heifers and steers were used. For Phase II, samples were obtained from a subset of Santa Cruz yearling heifers selected based of genotypes for tenderness (tough and tender) using a commercial genetic marker. Within genotype for tenderness, each animal was randomly assigned to one of four growth enhancement treatments. The most tender (< 30 N WBS) and toughest Longissimus steaks (< 30 N WBS) were selected for use in this study. In Phase I, tough steaks after 3, 10, and 17d postmortem had higher (P < 0.0005) WBS values than tender steaks. Tender steaks came from carcass with slightly higher (P = 0.008) marbling score and (P = 0.01) Quality grade. Sarcomere length, total and soluble collagen, potassium concentration, and m and µcalpain did not differ (P > 0.05) between tough and tender steaks. Sodium concentration at 10 d was higher (P = 0.03) in tough steaks, but only account for 0.05% of the variation in WBS at 3d. Tender steaks had less (P = 0.04) intact desmin at 24h, but intact desmin was not correlated (P > 0.05) with WBS. In Phase II, tough steaks after 3, 10, and 17d postmortem had higher (P < 0.0001) WBS values than tender steaks. Tender steaks came from carcass with slightly higher (P < 0.03) marbling score and (P = 0.02) Quality grade. Tender teaks were slightly lighter (P = 0.02), with more red (P = 0.02) and yellow (P = 0.007) color, and had slightly lower (P = 0.02) pH, compared with tough steaks. Sarcomere length, total and soluble collagen, sodium and potassium concentration, and m and µcalpain did not differ (P > 0.05) between tough and tender steaks. Tender steaks had less (P < 0.0001) intact desmin at 17d postmortem than tough steaks. Intact desmin at 17d was responsible for 4%, 47%, and 30% of WBS variation after 3, 10, and 17d postmortem, respectively. The slight difference in marbling and quality grade did not account for a significant amount of variation in WBS. However, meat color and pH accounted for variation in shear WBS. Calcium flux may have influenced meat tenderness by activation of calpains and may have altered protein to protein interactions. Results suggested that marbling, µ calpain activity, and desmin degradation, and to a lesser extent pH and meat color contributed to meat tenderness.

The occurrence of clinically diagnosed and/or ruptured abdominal aortic aneurysms (AAAs) in the families of 220 patients with AAAs, treated at the Surgical Clinic, University Hospital of Umeå in the northern part of Sweden during the years 1965-82, was studied. A questionnaire concerning the blood relatives was answered by 87/89 patients. 16/87 patients (18%) had blood relatives with AAAs. In 14 families one blood relative was affected, and in 2 families two blood relatives were affected. First degree relatives were affected in 9/87 cases (10%), and second degree relatives in 7/87 cases (8%). 9/468 (1.9%) of the patients' brothers and sisters but only five of all their cousins had AAAs, and 7/204 (3.4%) of the dead brothers and sisters had died of ruptured AAAs. Concerning the patients who were not included in the letter survey at least 14/133had blood relatives with AAAs. However, the great majority of these patients were dead when the study was performed and could not be asked aboutthe occurrence of AAAs in their families. The patients with AAAs had significantly higher serum concentrations of triglyceride and (YLDL + LDL)-cholesterol and a significantly lower serum concentration of HDL-cholesterol than randomly selected healthy controls of the same sex and age as the patients. We also compared the distributions of genetic markers (HLA antigens, the blood group systems ABO, Rh, MNSs, P, Kell, Lewis and Duffy and the serum protein group systems haptoglobin, transferrin, group-specific component, complement C3, properdin factor and alpha-1-antitrypsin) in patients with AAAs with the distributions in controls and in some cases with the expected distributions according to the Hardy-Weinberg law. A significantly decreased frequency of Rh-negative individuals, and significantly increased frequencies of Kell-positi ve individuals, of MN heterozygotes and of heterozygotes concerning haptoglobin type was found. Furthermore, the aneurysm walls of patients with and without AAAs in the family were compared concerning the morphology, but no differences were found. We also studied the occurrence of collagen types I and III in the aneurysm walls, and the occurrence of vimentin and desmin in the smooth muscle cells of the aneurysm walls, but all these components were present in the aneurysm walls of both the patients with and those without AAAs in the family. To summarize the results, there seems to be an increased frequency of AAAs, and especially of ruptured AAAs, among the brothers and sisters of patients with AAAs. Elevated serum concentrations of triglyceride and (VLDL + LDL)- cholesterol and a lowered serum concentration of HDL-cholesterol seems to be common in patients with AAAs. There seems to be a hereditary predisposition to the development of AAAs, because we found associations with four different genetic markers (Rh, MN, Kell, haptoglobin group). However, there is probably no specific "familial" type of AAAs, because we found no differences between the patients with and those without AAAs in the family.Key words: / <p>S. 1-42: sammanfattning, s. 43-103: 5 uppsatser</p> / digitalisering@umu

The muscle fibre cytoskeleton of skeletal and heart muscle cells is composed mainly of intermediate filaments (IFs), that surround the myofibrils and connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. Desmin is the first muscle specific IF protein to be produced in developing muscles and is the main IF protein in mature muscles. In skeletal muscle, desmin is particularly abundant at myotendinous and neuromuscular junctions. In the heart an increased amount of desmin is found at intercalated discs and in Purkinje fibres of the conduction system. Interactions between the IFs themselves, and between IFs and other structures such as Z-discs and the sarcolemma, are mediated by intermediate filament associated proteins (IFAPs). A transgenic mice model, which lacks the desmin gene have been developed to study the function of desmin. In these mice, morphological abnormalities are observed in both heart and skeletal muscles. Similar defects have been observed in human myopathies, caused by different mutations in the desmin gene. In the present thesis, skeletal and heart muscles of both wild type and desmin knock-out (K/O) mice have been investigated. Furthermore the cytoskeletal organisation in skeletal muscles from human controls and from a patient with desmin myopathy was examined. In the desmin K/O mice, no morphological alterations were observed during embryogenesis. These mice postnatally developed a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. Ruptures of the sarcolemma appear to be the primary event leading to muscle degeneration and fibrosis both in cardiac and affected skeletal muscles. In the heart the muscle degeneration gave rise to calcifications, whereas in skeletal muscles regeneration of affected muscle was seen. In mature wild type mice, the IF proteins synemin and paranemin, and the IFAP plectin were present together with desmin at the myofibrillar Z-discs, the sarcolemma, the neuromuscular junctions and the myotendinous junctions. Nestin was only found in these junctional regions. In desmin K/O mice, all four proteins were detected at neuromuscular and myotendinous junctions. The normal network of synemin and paranemin were not observed, whereas the distribution of plectin was preserved. In normal human muscles, synemin, paranemin, plectin and αB-crystallin were colocalised with desmin in between the myofibrils, at the sarcolemma and at myotendinous and neuromuscular junctions. In the human desmin myopathy, the distribution of desmin varied considerably. A normal pattern was seen in some fibres areas, whereas other regions either contained large subsarcolemmal and intermyofibrillar accumulations of desmin or totally lacked desmin. Nestin, synemin, paranemin, plectin and αB-crystallin also exhibited an abnormal distribution. They were often aggregated in the areas that contained accumulations of desmin. In cultured satellite cells from the patient, a normal network of desmin was present in early passages, whereas aggragates of desmin occurred upon further culturing. In the latter, also the nestin network was disrupted, whereas vimentin showed a normal pattern. αB-crystallin was only present in cells with a disrupted desmin network. Plectin was present in a subset of cells, irrespective of whether desmin was aggregated or showed a normal network. From the present study it can be concluded that an intact desmin network is needed to maintain the integrity of muscle fibres. Desmin may be an important component in the assembly of proteins, which connect the extrasarcomeric cytoskeleton with the extracellular matrix.

Modelos de desusos do músculo esquelético como imobilização gessada, suspensão são frequentemente utilizados em grupos de pesquisas experimentais. Esse tempo de desuso por período prolongado pode determinar alterações significativas na citoarquitetura muscular. Este estudo teve como objetivo avaliar os aspectos morfológicos do músculo sóleo de ratas em desenvolvimento pós-natal que tiveram seus membros posteriores direitos imobilizados, e posteriormente foram submetidas ao protocolo passivo de alongamento (alongamento manual passivo intermitente), por um período de sete dias. Utilizou-se 20 ratas da raça Wistar (Rattus Norvegicus Albinus) com 21 dias de idade, dividas em cinco grupos: Grupo Controle 21 dias (GC21- Animais com 21 dias), Grupo Imobilizado (GI- Animais de 21 dias que foram imobilizados por 7 dias), Grupo Imobilizado e Alongado (GIA- Animais de 21 dias que foram imobilizados por 7 e reabilitados pelo alongamento durante 3 dias) e Grupo Alongado (GA- Animais de 21 dias não imobilizados por 7 dias e posteriormente alongamentos durante 3 dias), Grupo Controle 30 (GC30 - Animais com 30 dias). Fragmentos dos músculo sóleo foi processado sob diferentes métodos histoquímicos, coloração hematoxilina-eosina e picro-sirius. As variáveis foram avaliadas inter- e intra-grupos através de técnicas estatísticas como: Teste de Kruskall Wallis e pós teste de Dunn. Conclusão: Os resultados indicaram que o músculo sóleo de ratas bebês sofreram modificações citoarquiteturais significativas quando o alongamento manual intermitente foi usado como recurso terapêutico após 7 dias de desuso do segmento posterior direito (imobilização em flexão plantar). Nos músculos imobilizados, ambas as proteínas (desmina e vimentina) tiveram seus conteúdos reduzidos em relação aos valores controle (21 ou 30 dias), indicando balanço negativo para o tecido pós-desuso. A quantidade dessas proteínas não foi modificada nos animais submetidos somente ao procedimento de alongamento intermitente. Os animais que sofreram imobilização e que foram reabilitados, a quantidade de desmina não aumentou significativamente, não atingindo valores similares ao grupo controle 30 dias. Esses dados sugerem que os filamentos de desmina necessitam de tempo superior a 3 dias de reabilitação por alongamento intermitente para restabelecerem a arquitetura intersticial das fibras e consequentemente favorecerem a transdução mecânica de sinais entre os meios intra e extracelular. Porém, o efeito citoarquitetural do alongamento sobre os filamentos intermediários deve ser acompanhado longitudinalmente e confirmados em adicionais estudos bioquímicos e moleculares. / Disuses models of skeletal muscle as immobilization , suspension are often used in experimental research groups. This time of disuse for a prolonged period can cause significant changes in muscle cytoarchitecture .This study aimed to evaluate the morphology of the soleus muscle of rats in postnatal development that had its members later immobilized rights, and subsequently were subjected to passive stretching protocol (passive manual stretching flashes), for a period of seven days. We used 20 Wistar rats (Rattus Norvegicus Albinos) race with 21 days of age , divided into five groups: Control Group (CG21- Animal 21 days ), Immobilized Group (IG- Animal 21 days that were immobilized for 7 days ) , Immobilized and Stretched Group (ISG - Animal 21 days that were immobilized for 7 and rehabilitated by stretching for 3 days) and Stretched Group (SG -Animal 21 days not immobilized for 7 days and subsequently stretching for 3 days), Control Group (CG30 - Animals 30 days).Fragments of the soleus muscle was processed under different histochemical methods, hematoxylin - eosin staining and picro-sirius. Variables were evaluated inter - and intra - groups through statistical techniques such as Kruskall Wallis and Dunn\'s post test . Conclusion: The results indicated that the soleus muscle of rats were babies citoarquiteturais significant changes when the manual stretching intermittently been used as a therapeutic resource after 7 days of disuse of the right posterior segment ( immobilization in plantar flexion). The immobilized muscles , both proteins (desmin and vimentin ) content had decreased compared to control values (21 or 30 days), indicating negative after- tissue balance disuse. The amount of these proteins was not modified in animals subjected only to intermittent stretching procedure. The animals underwent immobilization have been rehabilitated and that the amount of desmin was not significantly increased, not reaching values similar to the control group 30 days. These data suggest that desmin filaments need to 3 days longer than the time for rehabilitation to restore the intermittent stretching interstitial fiber architecture and hence favor the mechanical transduction of signals between intra-and extracellular media. However, the effect of stretching on cytoarchitectural intermediate filaments should be followed longitudinally and confirmed in additional biochemical and molecular studies .

O objetivo deste trabalho foi quantificar as concentrações de heat shock protein (HSP) 27 e 70 no músculo Longissimus thoracis bovino e avaliar o perfil proteico das amostras no decorrer do processo de maturação, bem como, a relação com as características de qualidade da carne. Foram utilizados 191 animais ½ Simental Sul Africano x ½ Nelore (½ S x ½ N), castrados e terminados em confinamento. Para qualidade da carne mensurou-se os valores de pH, cor (L*, a* e b*), perda de água por cocção (PAC) e força de cisalhamento (FC) em dois tempos de maturação (um e 14 dias). Para a quantificação de HSP 27 e 70 foram selecionadas 40 amostras em função dos valores de FC, e separadas em dois grupos (mais e menos macia) com 20 amostras cada, dentro de cada tempo de maturação (48h e 14 dias). Para realização da análise proteômica foram selecionados três animais de cada grupo e tempo de maturação. A concentração de HSP 27 apresentou diferença significativa (P &lt; 0,05) entre os tempos de maturação dentro dos dois grupos estudados. Já para a HSP 70 verificou-se diferença significativa (P &lt; 0,05) apenas para as amostras do grupo mais macia. A análise de correlação mostrou que as HSP 27 e 70 não interferiram nas características de qualidade da carne. A análise proteômica identificou seis proteínas diferencialmente abundantes (P &lt; 0,05) e o enriquecimento funcional demonstrou que essas proteínas estão relacionadas com processos biológicos e componentes celulares. A desmina e a glicerol-3-fosfato desidrogenase demonstraram correlação com a FC e coloração da carne, respectivamente. Os resultados indicam que as HSP 27 e 70 não podem ser apontadas como biomarcadores eficientes para qualidade de carne em animais ½ S x ½ N. Já a glicerol-3-fosfato desidrogenase demonstrou forte potencial em predizer a estabilidade da cor da carne. Desmina pode ser apontada como um importante biomarcador candidato associado à maciez da carne em animais ½ S x ½ N. / The aim of this work was to quantify the concentrations of heat shock protein (HSP) 27 and 70 in Longissimus thoracis muscle and evaluate the protein profile of the samples during the aging process and its relationship with meat quality characteristics. A total of 191 steers ½ Simmental South African x ½ Nellore (½ S x ½ N) feedlot-finished, were used in this work. For meat quality, pH values, color values (L *, a * and b *), cooking loss (CL) and shear force (SF) were measured in two aging times (one and 14 days). For quantification of HSP 27 and 70, 40 samples were selected according to the SF values and separated into two groups (more and less tender) with 20 samples each, according to the aging time (one and 14 days). To perform the proteomic analysis were selected three animals from each group and aging time. The concentration of HSP 27 showed a significant difference (P &lt; 0.05) between aging times within both groups. As for the HSP 70, there was a significant difference (P &lt; 0.05) for the tenderness samples. Correlation analysis showed that the HSP 27 and 70 didn\'t affect the meat quality traits. The proteomic analysis identified six proteins differentially expressed (P &lt; 0.05) and functional enrichment analysis demonstrated that these proteins are associated with biological processes and cellular components. Desmin and glycerol-3-phosphate dehydrogenase showed correlation with meat traits of SF and color, respectively. The results indicate that HSP 27 and 70 could not be identified as effective biomarkers for meat quality in ½ S x ½ N. However, glycerol-3-phosphate dehydrogenase showed a strong potential in predicting meat color stability. Desmin can be shown as a candidate biomarker associated with meat tenderness in ½ S x ½ N.

O objetivo deste trabalho foi quantificar as concentrações de heat shock protein (HSP) 27 e 70 no músculo Longissimus thoracis bovino e avaliar o perfil proteico das amostras no decorrer do processo de maturação, bem como, a relação com as características de qualidade da carne. Foram utilizados 191 animais ½ Simental Sul Africano x ½ Nelore (½ S x ½ N), castrados e terminados em confinamento. Para qualidade da carne mensurou-se os valores de pH, cor (L*, a* e b*), perda de água por cocção (PAC) e força de cisalhamento (FC) em dois tempos de maturação (um e 14 dias). Para a quantificação de HSP 27 e 70 foram selecionadas 40 amostras em função dos valores de FC, e separadas em dois grupos (mais e menos macia) com 20 amostras cada, dentro de cada tempo de maturação (48h e 14 dias). Para realização da análise proteômica foram selecionados três animais de cada grupo e tempo de maturação. A concentração de HSP 27 apresentou diferença significativa (P &lt; 0,05) entre os tempos de maturação dentro dos dois grupos estudados. Já para a HSP 70 verificou-se diferença significativa (P &lt; 0,05) apenas para as amostras do grupo mais macia. A análise de correlação mostrou que as HSP 27 e 70 não interferiram nas características de qualidade da carne. A análise proteômica identificou seis proteínas diferencialmente abundantes (P &lt; 0,05) e o enriquecimento funcional demonstrou que essas proteínas estão relacionadas com processos biológicos e componentes celulares. A desmina e a glicerol-3-fosfato desidrogenase demonstraram correlação com a FC e coloração da carne, respectivamente. Os resultados indicam que as HSP 27 e 70 não podem ser apontadas como biomarcadores eficientes para qualidade de carne em animais ½ S x ½ N. Já a glicerol-3-fosfato desidrogenase demonstrou forte potencial em predizer a estabilidade da cor da carne. Desmina pode ser apontada como um importante biomarcador candidato associado à maciez da carne em animais ½ S x ½ N. / The aim of this work was to quantify the concentrations of heat shock protein (HSP) 27 and 70 in Longissimus thoracis muscle and evaluate the protein profile of the samples during the aging process and its relationship with meat quality characteristics. A total of 191 steers ½ Simmental South African x ½ Nellore (½ S x ½ N) feedlot-finished, were used in this work. For meat quality, pH values, color values (L *, a * and b *), cooking loss (CL) and shear force (SF) were measured in two aging times (one and 14 days). For quantification of HSP 27 and 70, 40 samples were selected according to the SF values and separated into two groups (more and less tender) with 20 samples each, according to the aging time (one and 14 days). To perform the proteomic analysis were selected three animals from each group and aging time. The concentration of HSP 27 showed a significant difference (P &lt; 0.05) between aging times within both groups. As for the HSP 70, there was a significant difference (P &lt; 0.05) for the tenderness samples. Correlation analysis showed that the HSP 27 and 70 didn\'t affect the meat quality traits. The proteomic analysis identified six proteins differentially expressed (P &lt; 0.05) and functional enrichment analysis demonstrated that these proteins are associated with biological processes and cellular components. Desmin and glycerol-3-phosphate dehydrogenase showed correlation with meat traits of SF and color, respectively. The results indicate that HSP 27 and 70 could not be identified as effective biomarkers for meat quality in ½ S x ½ N. However, glycerol-3-phosphate dehydrogenase showed a strong potential in predicting meat color stability. Desmin can be shown as a candidate biomarker associated with meat tenderness in ½ S x ½ N.

No description available.