The studies reported in this thesis address, firstly, aspects of ribosome recycling in eubacteria, and secondly, a preliminary characterization of an EFG-like locus from Mycobacterium smegmatis. A hitherto unsuspected role of the ribosome recycling factor in governing the fidelity of initiation has been discovered during the course of this work. A summary of the relevant literature is presented in chapter 1. Section I of the ‘General Introduction’ provides a brief review of the current understanding of protein biosynthesis, with a special emphasis on ribosome recycling and the fidelity of translation initiation. Section II provides a brief introduction to mycobacterial translation, and known deviations from the E. coli prototype are highlighted. This is followed by three chapters containing experimental work, as summarized below. (i) Role of elongation factor G in governing specificity of ribosome recycling In eubacteria and the eukaryotic organelles, the post-termination ribosome complexes are recycled by the combined action of ribosome recycling factor (RRF) and elongation factor G (EFG). Earlier studies both from our laboratory and other laboratories have revealed the existence of specific interactions between RRF and EFG that are crucial for ribosome recycling, using ribosomes from E. coli and factors from both E. coli and heterologous sources such as Mycobacterium tuberculosis, Thermus thermophilus etc. In this study, to further understand the mechanism of ribosome recycling, we employed polysomes from both E. coli and M. smegmatis and monitored ribosome recycling in in vitro assays using RRF and EFG from both these sources; in addition, in vivo assays were performed in E. coli using either temperature-sensitive strains or strains carrying a deletion in frr (encoding RRF) or fusA (encoding EFG) genes. It was found that, in E. coli, RRF from Mycobacterium tuberculosis and M. smegmatis function with MtuEFG or MsmEFG but not with EcoEFG. In vitro assays revealed that the mycobacterial EFGs facilitate recycling of both the mycobacterial and E. coli polysomes not only with mycobacterial RRFs but also with EcoRRF. In contrast, although EcoEFG binds to mycobacterial polysomes, carries out GTP hydrolysis and is reported to sustain translocation on mycobacterial ribosomes, its activity in recycling mycobacterial polysomes was undetectable with EcoRRF, as well as with the mycobacterial RRFs. Such an observation allowed us to infer that EFG establishes specific interactions with the ribosome that are crucial for ribosome recycling but not for translocation, suggesting that translocation and ribosome recycling are distinct functions of EFG. In addition, a number of EFG chimeras generated by swapping corresponding domains between Msm- and Eco-EFGs were analyzed for their ability to sustain translocation and/or ribosome recycling in E. coli and M. smegmatis, using a combination of in vivo (for E. coli) and in vitro (for both E. coli and M. smegmatis) approaches. Our observations reveal that a dual set of specific interactions of EFG with RRF and ribosome is essential for ribosome recycling. While the RRF-EFG specific interactions are predominantly localized to the domains IV and V of EFG, the EFG-ribosome specific interactions that are crucial for ribosome recycling are not localized to a specific region of EFG but are found throughout the molecule. Our novel observations also emphasize the importance of using ribosomes from heterologous sources to understand the mechanism of this crucial process. (ii) Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions; however, very little is known about the role of these rRNA modifications in ribosome function. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of the intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. The isolation of a folD122 mutant strain of E. coli with a deficiency in rRNA methylations, as well as the availability of E. coli strains deficient for various individual methyltransferases that modify specific rRNA residues, provided us with a genetic tool to assay the role of rRNA methylations in ribosome recycling. We observed that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, a compromise in the RRF activity was found to afford increased initiation with a mutant tRNAfMet wherein the three consecutive G-C base pairs (29GGG31:39CCC41), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNAMet (29UCA31:39ψGA41). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. In addition, it was also found that IF3 and rRNA methylations, both of which are known to affect fidelity of initiation, exert their effects through distinct mechanisms, despite the proximity of a cluster of methylated rRNA residues to the IF3 binding site on the 30S subunit. (iii) Characterization of the role of EFG2, an EFG-like locus in Mycobacterium smegmatis Several bacteria, including various species of mycobacteria (with the exception of Mycobacterium leprae) contain a second EFG-like locus, denoted as fusA2, which shows considerable homology to fusA (encoding EFG). A comparison of the sequences of EFG and EFG2 from various bacteria reveals that EFG2 contains a GTPase domain and domains with significant homology to EFG domains IV and V, suggesting that it may function as an elongation factor. With the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like protein factors has not been studied so far. Hence, it was of interest to characterize EFG2. In the current study, EFG2 from M. smegmatis was characterized both by in vitro biochemical assays as well as by in vivo experiments targeted to investigate the biological significance of EFG2 in mycobacteria. It was found that, unlike EFG, MsmEFG2 could not sustain either translocation or ribosome recycling in E. coli. Despite the fact that the purified MsmEFG2 could bind guanine nucleotides, it lacked the ribosome-dependent GTPase activity characteristic of EFG and other translation GTPases, suggesting that it was unlikely to function as an elongation factor. However, EFG2 was found to be expressed in stationary phase cultures of M. smegmatis. To understand the biological significance of EFG2, fusA2 was disrupted in M. smegmatis. The viability of the M. smegmatis mc2155 fusA2::kan derivative indicates that MsmfusA2 is a non-essential gene. While disruption of the fusA2 gene (encoding EFG2) in M. smegmatis does not appear to affect its growth and survival in log phase or stationary phase or under hypoxic conditions, preliminary experiments indicate that disruption of fusA2 confers a fitness disadvantage to M. smegmatis when competed against M. smegmatis mc2155 (with wild type fusA2 locus).

Στην παρούσα διατριβή μελετήθηκε ο ρόλος της έλικας h44 του 18S rRNA του Saccharomyces cerevisiae επί διαφόρων παραμέτρων της πρωτεϊνικής σύνθεσης. Η μελέτη διεξήχθη με την βοήθεια των σημειακών μεταλλάξεων A1491G (rdn15) και U1495C (rdnhyg1), οι οποίες εντοπίζονται στην Α-θέση του ριβοσώματος. Η μετάλλαξη rdn15 επιδρά ήπια στον ρυθμό ανάπτυξης των κυττάρων ενώ τα rdn15 ριβοσώματα επιτελούν την πρωτεϊνοσύνθεση με αυξημένη ακρίβεια. Η έλλειψη σοβαρών επιπτώσεων παρουσία της rdn15 φανερώνει ότι το νουκλεοτίδιο 1491 δεν παίζει καθοριστικό ρόλο στην λειτουργία του ριβοσώματος. Τα κύτταρα ζύμης που φέρουν την μετάλλαξη rdnhyg1 αναπτύσσονται βραδύτερα από τα κύτταρα αγρίου τύπου, ενώ τα rdnhyg1 ριβοσώματα πρωτεϊνοσυνθέτουν με ελαφρώς αυξημένη συχνότητα λάθους. Η μετάλλαξη αυξάνει επίσης την συγγένεια της Α-θέσης του ριβοσώματος για το αμινοακυλο-tRNA και επιδρά αρνητικά στο στάδιο της μετατόπισης, χωρίς να επηρεάζει την ενεργότητα πεπτιδυλοτρανσφεράσης. Η επίδραση της μετάλλαξης rdnhyg1 επί διαφόρων παραμέτρων της πρωτεϊνοσύνθεσης δικαιολογεί την συντήρηση της U1495 κατά την εξέλιξη. Η μετάλλαξη sup45-R2ts εντοπίζεται στο γονίδιο που κωδικοποιεί τον παράγοντα τερματισμού eRF1 και οδηγεί στην αντικατάσταση της προλίνης 86 από αλανίνη. Η μετάλλαξη δεν επηρεάζει τις περισσότερες από τις λειτουργίες του ριβοσώματος που εξετάστηκαν, αλλά μειώνει την μεταφραστική πιστότητα. Σε κύτταρα που φέρουν ταυτόχρονα την μετάλλαξη sup45-R2ts και την ριβοσωματική μετάλλαξη rdn15, η συχνότητα λάθους αυξάνεται σε βαθμό μεγαλύτερο από την αθροιστική επίδραση των δύο επιμέρους μεταλλάξεων, επιβεβαιώνοντας μια ιδιαίτερη αλληλεπίδραση του μεταλλαγμένου παράγοντα eRF1 με τα rdn15 ριβοσώματα, που, όπως προκύπτει, αντιστρέφει τον υπερακριβή χαρακτήρα των μεταλλαγμένων ριβοσωμάτων. Όταν η μετάλλαξη sup45-R2ts συνυπάρχει με την ριβοσωματική μετάλλαξη rdnhyg1 η συχνότητα λάθους δεν επηρεάζεται σημαντικά. Η rdnhyg1 φαίνεται να ελαχιστοποιεί την επίδραση του μεταλλαγμένου παράγοντα eRF1 ενισχύοντας την δράση GTPάσης του eRF3. Τα παραπάνω αποτελέσματα φανερώνουν επιπλέον ότι η sup45 δύναται να μεταβάλει τις ιδιότητας ορισμένων μεταλλάξεων κατά την ριβοσωματική λειτουργία. Το στέλεχος που φέρει την μετάλλαξη rdn15 είναι πολύ ευαίσθητο έναντι της παρομομυκίνης αλλά και έναντι της τομπραμυκίνης, αν και σε μικρότερο βαθμό. Τα αποτελέσματα αυτά αποδίδονται στην ικανότητα της μετάλλαξης να αυξάνει την συγγένεια της Α-θέσης του ριβοσώματος για τα εν λόγω αντιβιοτικά. Αντίθετα, η μετάλλαξη rdn15 προσδίδει ανθεκτικότητα στην υγρομυκίνη, φανερώνοντας ότι ο τρόπος πρόσδεσης και δράσης του συγκεκριμένου αμινογλυκοζίτη διαφοροποιείται. Το στέλεχος που φέρει την μετάλλαξη rdnhyg1 είναι ανθεκτικό και στα τρία αντιβιοτικά σε σύγκριση με το αγρίου τύπου, φανερώνοντας ότι η U1495 είναι καθοριστική για την πρόσδεση των αμινογλυκοζιτών στο ριβόσωμα. Τα κύτταρα που φέρουν την εξωριβοσωματική μετάλλαξη sup45-R2ts είναι πιο ευαίσθητα από τα αντίστοιχα αγρίου τύπου έναντι και των τριών αμινογλυκοζιτών. Ωστόσο η μετάλλαξη sup45-R2ts, δεν επηρεάζει την ικανότητα των αντιβιοτικών αυτών να προσδένονται στα αγρίου τύπου και μεταλλαγμένα ριβοσώματα και να επάγουν άμεσα τα μεταφραστικά λάθη. Η μελέτη επίδρασης των αμινγλυκοζιτών επιβεβαίωσε ότι η παρομομυκίνη και η υγρομυκίνη αναστέλλουν την ανάπτυξη των κυττάρων ζύμης, ενώ η τομπραμυκίνη δεν έχει καμία επίδραση. Το γεγονός αυτό συνδυάζεται με την αδυναμία της τομπραμυκίνης να αναστείλει την πρόσδεση του υποστρώματος στην Α-θέση των ριβοσωμάτων. Ωστόσο η τομπραμυκίνη, όπως η παρομομυκίνη και η υγρομυκίνη, είναι ικανή να αυξήσει την συχνότητα λάθους και την σύνθεση πολυφαινυλαλανίνης. / In present study, we investigated the role of helix h44 of 18S rRNA of Saccharomyces cerevisiae on several parameters of protein synthesis. For this purpose we employed mutations A1491G (rdn15) and U1495C (rdnhyg1) which are located in the A-site of the ribosome. The rdn15 mutation slightly delays cell growth, while rdn15 ribosomes translate proteins with higher fidelity. The lack of severe impairment of ribosomal function by mutation rdn15 indicates that the nature of nucleotide 1491 is not essential for ribosomal function. Yeast cells carrying the rdnhyg1 mutation grow slower than wild-type cells, while their ribosomes possess a slightly increased error rate. This mutation also increases the affinity of the A-site for aminoacyl-tRNA and renders ribosomes less efficient in translocation without affecting peptidyltransferase activity. The effect of mutation rdnhyg1 on several parameters of protein synthesis explains why U1495 is evolutionarily conserved. Mutation sup45-R2ts is located in the gene encoding eukaryotic Release Factor 1 (eRF1) and results in the substitution of proline 86 by alanine. This mutation leaves unaffected most ribosomal functions but it decreases translational fidelity. When ribosomal mutation rdn15 is introduced in cells already carrying sup45-R2ts mutation, the error frequency is increased to a degree which is higher than the additive effect of the two mutations, testifying to a previously reported special interaction of eRF1 with rdn15 ribosomes, which in this case reverses the hyperaccurate character of rdn15 ribosomes. When mutation sup45-R2ts is expressed in cells also carrying ribosomal mutation rdnhyg1, the error frequency is not significantly altered. Mutation rdnhyg1 seems to minimize the effect of the mutant factor eRF1 on ribosomal function by enhancing GTPase activity of eRF3. The results obtained with rdn15 and rdnhyg1 alone or in combination with sup45-R2ts show for the first time that the presence of sup45 may result in significant changes in the properties of the mutations under study. The strain carrying mutation rdn15 exhibits extremely high sensitivity toward paromomycin and also increases sensitivity of yeast ribosomes to tobramycin but to a lesser degree. These results demonstrate the ability of this mutation to increase affinity of the A-site for aminoglycosides. In contrast, mutation rdn15 causes resistance to hygromycin, revealing that binding and possibly action of hygromycin is differentiated from the other two aminoglycosides. The strain carrying mutation rdnhyg1 is resistant to all three antibiotics tested compared to wild type, indicating that U1495 participates in aminoglycoside binding to the ribosome. Cells carrying the extraribosomal mutation sup45-R2ts are more sensitive toward all three antibiotics compared to their wild type cells. Nevertheless, mutation sup45-R2ts does not affect the ability of these antibiotics to bind to the ribosome and directly induce translational errors. The study of amingolycoside action confirmed that paromomycin and hygromycin inhibit cells growth, while no such effect is observed during cell growth in the presence of tobramycin. This fact is combined with the inability of tobramycin to inhibit substrate binding to the ribosomal A-site Nevertheless, it was shown that tobramycin, like paromomycin and hygromycin, is effective both in inducing translational errors and increasing polyphenylalanine synthesis in wild-type and mutant ribosomes.

Ο σκοπός της παρούσας διατριβής εστιάζεται στη διερεύνηση της λειτουργίας και της δομής του ευκαρυωτικού ριβοσώματος. Συγκεκριμένα, ερευνά την επίδραση ορισμένων συστατικών του ριβοσωματικού RNA (rRNA) της μεγάλης και της μικρής υπομονάδας του ριβοσώματος αλλά και ορισμένων εξωριβοσωματικών συστατικών επί της πιστής αποκωδικοποίησης της γενετικής πληροφορίας, επί της ικανότητας κατάλυσης του σχηματισμού πεπτιδικού δεσμού, αλλά και επί της μετατόπισης του πεπτιδυλο-tRNA από την Α στην Ρ ριβοσωματική περιοχή. Τέλος, διερευνήθηκε για πρώτη φορά σε ευκαρυωτικά κύτταρα, η πιθανότητα συσχέτισης της πιστότητας με την οποία επιτελούν τα κύτταρα τη μετάφραση με την οξειδωτική τους κατάσταση. Η μεθοδολογία που αναπτύχθηκε περιλαμβάνει α) τον προσδιορισμό της συχνότητας λάθους (E.F) in vitro, η οποία αποτελεί μέτρο της πιστότητας της μετάφρασης, β) τον προσδιορισμό της ταχύτητας κατάλυσης του σχηματισμού πεπτιδικού δεσμού που αποτελεί μέτρο της ενεργότητας της ριβοσωματικής πεπτιδυλοτρανσφεράσης και γ) την εξάρτηση του σταδίου μετατόπισης από την συγκέντρωση των διαλυτών πρωτεϊνικών παραγόντων και του κυκλοεξιμιδίου. Επιπλέον, διερευνήθηκε η επίδραση ορισμένων αντιβιοτικών, κυρίως της παρομομυκίνης και του κυκλοεξιμιδίου, in vivo και in vitro. Επίσης, προσδιορίστηκε η ικανότητα συγκρότησης των ριβοσωματικών υπομονάδων, των ακέραιων ριβοσωμάτων και των πολυσωμάτων. Τέλος, προσδιορίστηκαν χαρακτηριστικοί δείκτες της οξειδωτικής κατάστασης του κυττάρου. Παρά το γεγονός ότι συστατικά του rRNA είναι κυρίως υπεύθυνα για τη ριβοσωματική λειτουργία, τα αποτελέσματα της πρώτης ενότητας οδηγούν στο συμπέρασμα ότι η πιστότητα της μετάφρασης, η καταλυτική ενεργότητα αλλά και το στάδιο μετατόπισης της μετάφρασης καθορίζονται ως ένα βαθμό και από εξωριβοσωματικά συστατικά όπως είναι η φωσφατάση σερίνης/θρεονίνης SAL6 και το προϊόν του γονιδίου ASU9. Τα συμπεράσματα της δεύτερης θεματικής ενότητας δίνουν έμφαση στην συνεχή ενδοεπικοινωνία που υφίσταται μεταξύ των δυο ριβοσωματικών υπομονάδων. Πράγματι, ορισμένες μεταλλάξεις στο rRNA της μικρής υπομονάδας του ριβοσώματος επηρεάζουν εκτός από την κύρια λειτουργία αυτής, δηλαδή την αποκωδικοποίηση, και την ταχύτητα σχηματισμού πεπτιδικών δεσμών. Αντίστροφα, ορισμένες μεταλλάξεις στο rRNA της μεγάλης υπομονάδας του ριβοσώματος επηρεάζουν εκτός από την κύρια ενεργότητα αυτής, δηλαδή την ενεργότητα πεπτιδυλοτρανσφεράσης, και την πιστότητα της αποκωδικοποίησης. Ορισμένες εξ αυτών των μεταλλάξεων επηρεάζουν επίσης και το στάδιο της μετατόπισης. Στην τρίτη ενότητα αποδεικνύουμε ότι οι επιρρεπείς σε λάθη μεταλλάξεις παρουσιάζουν χαμηλότερο οξειδωτικό στρες ενώ οι υπερακριβείς μεταλλάξεις παρουσιάζουν υψηλότερο οξειδωτικό στρες. Μια ελκυστική ερμηνεία των αποτελεσμάτων είναι ότι όσο πιο υπερακριβές είναι ένα κύτταρο κατά τη μετάφραση τόσο περισσότερο είναι το ποσό της ενέργειας που πρέπει να καταναλώσει ώστε να εξασφαλίσει την αποφυγή λαθών κατά τη πρωτεϊνοσύνθεση, ελαττώνοντας κατά αυτό τον τρόπο το ποσό ενέργειας με το οποίο θα αντιμετωπίζονταν οι ελεύθερες ρίζες. / The aim of the present diatribe focuses on the function and the structure of the eukaryotic ribosome. Specifically, the influence of several ribosomal RNA (rRNA) residues from the small and the large ribosomal subunit as well as the influence of several extra-ribosomal elements on the accurate decoding of the genetic information, on the catalysis of peptide bond formation and on the translocation of peptidyl-tRNA from the A to the P ribosomal site, is investigated. In the last part, the possible correlation between translational fidelity and the cell’s oxidative status is determined for the first time in eukaryotic cells. The methodology that was applied includes a) the error frequency (E.F) determination that measures translational fidelity, b) the determination of the catalytic rate constant for peptide bond formation that reflects the ribosomal peptidyltransferase activity and c) the dependence of the translocation step on soluble protein factors concentration and on cycloheximide concentration. Moreover, we studied the effects of the antibiotics paromomycin and cycloheximide in vivo and in vitro. The assembly of ribosomal subunits, of ribosomes and of polysomes was also investigated. Finally, typical markers of the cell’s oxidative status were determined. Despite the fact that rRNA residues are mainly responsible for ribosomal function, the results from the first part of the thesis lead to the conclusion that the translational fidelity, the catalytic activity and the translocation step of translation are determined up to a certain level by extra-ribosomal elements such as the serine/threonine phosphatase SAL6 as well as the ASU9 gene product. The conclusions drawn from the second part of the diatribe point to the constant intercommunication between the two ribosomal subunits. Indeed, several mutations in the small ribosomal subunit rRNA not only affect its major function, i.e. the decoding process, but they also affect the rate of peptide bond formation. Reversely, several mutations in the large ribosomal subunit rRNA not only affect its major activity, i.e. the peptidyltransferase activity, but they also affect the accuracy of decoding. Some of these mutations influence also the translocation step of protein synthesis. In the third part, we prove that error-prone mutations display lower oxidative stress whereas the hyperaccurate mutations display higher oxidative stress. An attractive interpretation of these results is that a cell might spend more energy in order to achieve hyperaccuracy during translation thus reducing the amount of energy left in order to combat free radicals.

No description available.

Interferons (IFNs) are a class of structurally related proteins first discovered to be produced by virus-infected cells. By now, several other inducing agents have been described. IFNs exert multiple effects on cells exemplified by the establishment of an antiviral state, inhibition of cell proliferation and alteration of different immune reactions. In the present thesis the inhibition of cellular growth concentrated on effects in the early cell cycle have been studied. The human glioma cell line 251 MG was found to be blocked in the S phase of the cell cycle upon addition of IFN both to exponentially growing and growth-factor depleted, synchronized cells. Thymidine kinase and DNA-polymerase activities were reduced in parallel with the S phase effect. 2-5 oligo Anucleotides transfected into glioma cells lead to inhibition of cell growth, exponentially growing cells being blocked in the S phase as during IFN treatment. In contrast, synchronized, restimulated cells were blocked in the cellcycle phase where they resided at the time of transfection. As 2-5 oligo A synthetase activity was induced in the middle of the Gl phase, these results might indicate that the kinetics of expression of oligonucleotides after IFN additiondetermines the type of cell cycle block obtained in differenttumor cells. IFN inhibited preferentially proteins originating from newly synthesized mRNA in Sw 3T3 cells, c-mvc did not seem to be included among these proteins. In both cell systems c-myc expression was unaltered after IFN treatment. In clone T1 selected from the the Sw 3T3 cell line , c-mvc expression was uncoupled to growth and seemed to be growth factor independent. The change in c-myc expression in clone T1 compared to SW 3T3 cells did not render the cells sensitive to IFN. Hence, c-myc regulation does not seem to be the mechanism by which IFN regulates cell growth in this system. The proliferation marker KI-67 antigen was shown not to be causatively involved in growth inhibition of IFN. The reduced levels of the antigen was proposed to be a secondary effect caused by the G0/G1 arrest. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1991, härtill 6 uppsatser</p> / digitalisering@umu

Critical illness associated muscle weakness and muscle dysfunction in intensive care unit (ICU) patients lead to severe morbidity and mortality as well as significant adverse effect on quality of life. Immobilization, mechanical ventilation, neuromuscular blocking agents, corticosteroids, and sepsis have been implicated as important risk factors, but the underlying molecular and cellular mechanisms remain unclear.  A unique porcine ICU model was employed to investigate the effect of these risk factors on the expression profiles, gene expression and contractile properties of limb and diaphragm muscle, in the early phase of ICU stay. This project has focused on unraveling the underlying molecular and cellular pathways or networks in response to ICU and critical illness interventions. Upregulation of heat shock proteins indicated to play a protective role despite number of differentially transcribed gene groups that would otherwise have a negative effect on muscle fiber structure and function in response to immobilization and mechanical ventilation.  Mechanical ventilation appears to play a critical role in development of diaphragmatic dysfunction. Impaired autophagy, chaperone expression and protein synthesis are indicated to play a pivotal role in exacerbating muscle weakness in response to the combined effect of risk factors in ICU. These results may be of therapeutic importance in alleviating critical illness associated muscle weakness.

Les fibres amyloïdes sont souvent à l’origine de nombreuses maladies dégénératives telles que la maladie d’Alzheimer ou la maladie de Parkinson. La formation de ces plaques insolubles est due à une agrégation anormale de protéines. Les études structurales et biologiques des amyloïdes sont hautement complexes du fait de leur organisation sous forme de superstructures unidirectionnelles composées d’une infinité d’unités peptidiques ou protéiques, mais aussi à cause de leur hétérogénéité conformationnelle et polymorphique. Au cours de ces différents travaux de thèse en collaboration avec différents laboratoires d’analyses structurales, nous avons développé plusieurs outils de synthèse tant pour la formation de différents polymorphes de fibres amyloïdes que pour la formation d’espèces oligomériques de tailles conséquentes qui sont un challenge du point de vue synthétique et méthodologique mais aussi pour leur caractérisation. Ces différentes avancées permettront de mieux comprendre les mécanismes de formation de fibres amyloïdes et de préparer des échantillons homogènes pour les analyses structurales et biologiques. L’étude de modifications chimiques telles que la N-méthylation ou les polypeptides D est également un enjeu important pour l’élucidation des interactions protéine-protéine vis-à-vis des structures amyloïdogéniques et ainsi permettre l’élaboration de nouveaux composés inhibant la formation de plaques amyloïdes. / Amyloid fibrils are associated with many human disorders including Alzheimer’s or Parkinson’s diseases. The formation of insoluble plaques is the result of protein misfolding and aggregation due to abnormal conformational isomerization of the involved protein. The structural and biological studies of amyloids are highly complex. In this thesis, we report on the development of different synthetic methodologies for the preparation of distinct amyloid fibril polymorphs as homogeneous samples for structural and biological studies. We also synthesized covalently-tethered oligomers composed of nine copies of an amyloidogenic peptide segment, where we were able to control the self-assembly of the structure by insertion of N-methylated amino-acids and to obtain monomeric oligomers mimicking a cross section of an amyloid fibril. We also report on the chiral recognition of L-peptides and L-proteins towards corresponding D-enantiomers during amyloid formation. Moreover, we studied various N-methylated peptide analogues to suppress amyloid growth. Overall, the results obtained in this thesis pave the way towards rational design of peptide-based inhibitors and diagnostics against amyloid propagation.

Neurons connect to their remote targets via axons, which usually survive for the lifetime of an organism. Spatiotemporal regulation of the axonal proteome by local protein synthesis (LPS) plays a critical role in neuronal wiring and axon survival, raising the intriguing possibility that some neurological disorders involve LPS dysfunction. To visualise LPS in situ, I optimised multiple imaging techniques to investigate Netrin-1-induced translation in cultured retinal axons. Total axonal protein synthesis measured by metabolic and puromycin labelling indicates axons experience stage-dependent alterations in translation rate upon Netrin-1 stimulation. Remarkably, Netrin-1 triggers a burst of β-actin synthesis starting within 20 seconds of cue application at multiple non-repetitive sites visualised by single molecule translation imaging, an approach that allows direct visualisation of translation dynamics in response to external stimuli. Further studies have shown that local translation can occur on Rab7a-associated late endosomes, where mRNA recruitment and translation are coordinately regulated. Notably, mRNAs encoding mitochondria-related proteins are found translating on late endosomes docking in the vicinity of mitochondria, suggesting late endosomes act as ‘platforms’ for the localised synthesis of mitochondrial proteins necessary for maintaining mitochondrial integrity. Moreover, this process is affected in axons expressing the Charcot-Marie-Tooth disease type 2B (CMT2B)-related Rab7a mutants, leading to abnormal mitochondrial biogenesis and activity and compromised axon survival. Finally, attenuated de novo protein synthesis is observed in axons expressing amyotrophic lateral sclerosis (ALS)-associated fused in sarcoma (FUS) mutants and hypomethylated wild-type FUS. Live imaging reveals mislocalised mutant or hypomethylated FUS granules are transported along axons and accumulate at growth cones, possibly irreversibly trapping RNA molecules, resulting in reduced distance travelled by RNA granules in axons. Furthermore, mutant FUS expression results in defective retinal projections in vivo, highlighting the importance of RNA metabolism and local translation in axonal homeostatic mechanisms. In conclusion, aberrant translational activity in axons leads to prominent axonopathy, which recapitulates features of early stages of neurological diseases, providing the basis for novel therapeutic strategies.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Eight Texel x Polwarth crossbred wethers (31.1±3.8 kg BW), four with duodenal cannulae were used in a replicated 4x4 Latin square design to evaluate effects of canola meal (44.4% CP, 29.5% NDF and 3.2% EE; DM basis) on intake, whole-tract digestibility, microbial protein synthesis and nitrogen retention. The basal diet consisted of ad libitum access to sudangrass (10% refusals). Treatments were sudangrass only (control), or supplemented with 5, 10, or 15 g/kg BW of concentrate, offered twice daily at 0800 and 1700h. Concentrate was 90% canola meal and 10% finely ground corn. Wethers were adapted to diets for 10 d followed by a 5-d collection period. Forage DMI decreased linearly (P<0.001) as supplement intake increased, and was 26.1% lower in supplemented animals in relation to the control group. Total DMI, which included forage and supplement, increased 30.6% and digestible OM intake increased 41% with supplementation. Supplementation did not affect DM and OM digestibility, while depressed NDF digestibility and improves N digestibility. Microbial protein synthesis and microbial efficiency were not affected by supplementation. Nitrogen retention was markedly higher in supplemented animals (236% higher), and was due mainly to the higher duodenal flow of amino acids. Supplementation with canola meal improves total nutrient supply, however, exerted a negative effect on forage intake and fiber digestibility in wethers. / Este estudo foi conduzido com o objetivo de avaliar a inclusão de farelo de canola na dieta de ovinos e seus efeitos sobre o consumo de volumoso, digestibilidade, síntese protéica microbiana ruminal e retenção de nitrogênio. Oito ovinos machos cruzados Texel x Polwarth (31,1±3,8 kg PV), quatro destes implantados com cânula duodenal foram utilizados em um delineamento quadrado Latino 4x4 duplo com períodos de 15 dias, sendo 10 dias adaptação e cindo dias coleta de dados. A dieta experimental basal foi composta de capim Sudão (ad libitum, 10% de sobras) e os tratamentos foram: capim Sudão (controle), ou suplementado com 5, 10 ou 15 g/kg PV de concentrado, ofertados duas vezes ao dia às 08:00 e 17:00h. O concentrado foi 90% farelo de canola e 10% de milho moído. O consumo de MS da forragem decresceu linearmente (P<0,001) com o aumento no consumo de suplemento e foi em média 26,1% menor em relação ao grupo controle. O consumo total de MS que inclui forragem e suplemento aumentou 30,6% e o consumo de MOD aumentou 41% nos animais suplementados. Não houve efeito da suplementação sobre a digestibilidade da MS e da MO, todavia, houve redução na digestibilidade da FDN e aumento na digestibilidade do N. A síntese e a eficiência de síntese protéica microbiana ruminal não foram afetadas (P>0,05) pela adição do suplemento à dieta. A retenção de N foi substancialmente maior(P<0,001) nos animais recebendo o farelo de canola (236%), e foi devida principalmente à maior oferta de aminoácidos. A suplementação aumentou a oferta total de nutrientes e a retenção de nitrogênio em ovinos, todavia, exerceu um efeito negativo sobre o consumo de forragem e a digestibilidade da fibra.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-07-19T20:36:06Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) katharine_kelly_azevedo.pdf: 1197322 bytes, checksum: 47b2dff938cb953a1d3c90817b658008 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-08-22T12:59:16Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) katharine_kelly_azevedo.pdf: 1197322 bytes, checksum: 47b2dff938cb953a1d3c90817b658008 (MD5) / Made available in DSpace on 2018-08-22T12:59:16Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) katharine_kelly_azevedo.pdf: 1197322 bytes, checksum: 47b2dff938cb953a1d3c90817b658008 (MD5) Previous issue date: 2018 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Objetivou-se avaliar o efeito de n?veis crescentes de substitui??o da PB do concentrado (0, 25, 50 e 75% com base na MS) pela PB do farelo de crambe (FC) em dietas para ovinos, sobre o consumo e digestibilidade de nutrientes, par?metros ruminais, N ureico no plasma sangu?neo (NUP), excre??o urin?ria de N ureico (EUNU), balan?o de N, fluxo intestinal de N microbiano (NMIC) e efici?ncia de s?ntese de prote?na microbiana (EFIM). Foram utilizados quatro ovinos fistulados no r?men, SRD, machos, castrados, alojados em gaiolas metab?licas, com idade m?dia inicial de 18 meses e peso vivo m?dio inicial de 50 kg, distribu?dos em delineamento quadrado latino 4 x 4 (4 tratamentos e 4 per?odos). Cada per?odo foi composto de 14 dias, sendo sete dias destinados ? adapta??o dos animais ? dieta e ?s condi??es experimentais e sete dias para as coletas. As dietas foram compostas por 50% de volumoso (silagem de milho) e 50% de concentrado (%MS). Os resultados foram submetidos ? an?lise de vari?ncia e estudo de regress?o a 5% de signific?ncia, utilizando-se o programa estat?stico SAS. Foi verificado efeito linear crescente para o consumo de extrato et?reo e linear decrescente para o consumo de carboidratos n?o fibrosos corrigidos para cinzas e prote?na. Com o aumento dos n?veis de FC nas dietas observou-se redu??o na digestibilidade de todos os nutrientes avaliados, exceto para PB e EE. N?o houve efeito para o pH do l?quido ruminal, por?m para os valores de N amoniacal no l?quido ruminal foi observado efeito linear decrescente com a inclus?o do FC na dieta. Tamb?m n?o foi observado efeito das dietas para o balan?o de N e EUNU. Contudo, para a concentra??o de NUP houve efeito linear decrescente. O NMIC e EFIM apresentaram efeito linear crescente com a inclus?o do FC. De acordo com os resultados alcan?ados no presente estudo, o FC possui potencial como alimento proteico alternativo na dieta de ovinos, pois assegura consumo e utiliza??o do N semelhante a alimentos convencionais e contribui pra melhor s?ntese de prote?na microbiana. Apesar da redu??o da digestibilidade dos nutrientes com a inclus?o do FC ?s dietas, o consumo de NDT n?o foi prejudicado. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Zootecnia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The objective of this study was to evaluate the effect of increasing levels of CP replacement of concentrate (0, 25, 50 and 75% based on DM) for crambe meal (CM) CP in sheep diets regarding on nutrient intake, digestibility, ruminal parameters, blood plasma urea nitrogen (NUP), urinary urea nitrogen excretion (EUNU), N balance, intestinal flow of microbial nitrogen (NMIC), and efficiency of microbial protein synthesis (EFIM). Four rumen fistulated male sheep of undefined breed, castrated, housed in metabolic cages, with initial mean age of 18 months and initial mean body weight (BW) of 50 kg, were distributed in a 4 x 4 Latin square design (4 treatments and 4 periods). Each period was composed of 14 days, seven days for the adaptation of the animals to the diet and the experimental conditions and seven days sampling. The diets were composed of 50% of roughage (corn silage) and 50% of concentrate (% MS). The results were submitted to analysis of variance and regression study at 5% of significance using the SAS statistical program. It was verified crescent linear effect for the intake of ethereal extract and linear effect decreasing for the intake of non-fibrous carbohydrates corrected for ashes and protein. It was observed a reduction of the digestibility of all the nutrients with increase of CM levels in the diets, except for PB and EE. There was no effect on the ruminal fluid pH, but it was observed linear decreasing effect for the values of ammoniacal nitrogen in the rumen according to the inclusion of the CM in the diets. It was not observed effect of diets on N balance and EUNU. However, there was a linear decreasing effect for NUP. The was in increasing linear effect on NMIC and the EFIM with the inclusion of CM. According to the results obtained in the present study, the CM has potential as an alternative protein food in the diets of sheep, because the intake and use of N is similar to conventional foods and improves the microbial protein synthesis. Even with the reduction of nutrient digestibility with the inclusion of CM in diets, the intake of TDN was not affected.