An acute bout of resistance exercise (RE) and dietary protein consumption stimulate muscle protein synthesis (MPS). This anabolic effect is believed to be attenuated with resistance exercise training (RET), however, the mechanism for this plateau" is unknown. In addition, the ideal timing for protein consumption to optimize MPS is not well characterized. The central hypothesis of this research is that RE stimulates cumulative (measured over 24-36 h) MPS in rats and humans. Study one determined whether an acute bout of RE in rats enhances MPS when assessed with the traditional flooding dose (~ 25 min) and 2H2O (4 and 24 h measurements); thus a comparison of the two methodologies was made. An acute session of RE did not result in an elevation in MPS when quantified by either the flooding dose or 2H2O over 4 and 24 h (methods compared qualitatively). Therefore, an acute bout of RE in rats does not appear to be anabolic and adaptation resulting from multiple bouts is likely necessary. Study two determined if RET in rats results in attenuation in MPS (plateau effect) 16 h following the final RE session (peak anabolic window) and if it is due to an increase in 4E-BP1 (a key regulator of mRNA translation initiation) activity; or if the timing in anabolism changes, which could be detected with a cumulative assessment (2H2O). MPS at 16 h was unchanged following RE training. Consistent with this finding, there were no differences in 4E-BP1 activity. Conversely, cumulative MPS was significantly increased with RET, suggesting a temporal shift in anabolism. Study three determined if dietary protein consumed immediately following RE augments cumulative (24 h) MPS in young adult human males when energy and macronutrients are controlled. RE and post-RE protein had no effect on mixed MPS; however, myofibrillar MPS was significantly increased with RE suggesting specific changes within a heterogeneous protein pool. Collectively, these are the first studies to assess changes in cumulative MPS with RE in rats and humans. The long term goals of this research are to understand muscle protein anabolism in "free-living" mammals and the mechanisms that regulate this process.

Long duration spaceflight missions out of lower earth orbit, back to the lunar surface, or possibly to Mars highlight the importance of preserving muscle mass and function. Muscle atrophy occurs within days of exposure to microgravity and prevailing thought is that a primary mechanism for muscle atrophy is a reduction in skeletal muscle protein synthesis. This dissertation examines the ability of skeletal muscle to recover muscle protein synthesis with slight perturbation, such as ambulatory reloading during disuse as well as partial loading, similar to body mass seen on the moon or Mars. We use traditional precursor-product labeling to measure protein synthesis, but use a relatively novel tracer, deuterium oxide, in order to make cumulative measures of protein synthesis over 24 h. The overarching goal of this dissertation is to define the response of skeletal muscle protein synthesis to different loading parameters in order to better understand the contribution of protein synthesis to skeletal muscle mass during disuse. In the first study, we demonstrate that muscle atrophy during 5 days of hindlimb unloading is in part due to a decrease in protein synthesis. We also highlight the ability of skeletal muscle to adapt by allowing two 1 h ambulatory reloading sessions on days 2 and 4. Although this countermeasure is able to rescue protein synthesis in soleus and gastrocnemius, it is unable attenuate any losses in muscle mass. In the second study, we compare partial weight loading to traditional hindlimb unloading. Weight bearing of 1/3 or 1/6 body weight is able to attenuate losses in muscle mass seen with unloading. Protein synthesis is maintained after 21 days of the experimental protocol, suggesting that protein synthesis is responsive to load and is likely not the only mechanism for determining muscle mass. In the final study, the effects of < 1 Gy x-ray exposure and partial weight suspension are measured to better understand the complex space environment, which includes a wide variety of radiation. Surprisingly, we found no effects of radiation on muscle protein synthesis in 1 G or partial loading. Targeting only protein synthesis may not be enough of a stimulus as evidenced by the data in this dissertation. Future plans should use a multiple-systems approach to counteract atrophy by increasing protein synthesis to maintain/elevate muscle mass during periods when it is otherwise compromised.

An acute bout of resistance exercise (RE) and dietary protein consumption stimulate muscle protein synthesis (MPS). This anabolic effect is believed to be attenuated with resistance exercise training (RET), however, the mechanism for this plateau" is unknown. In addition, the ideal timing for protein consumption to optimize MPS is not well characterized. The central hypothesis of this research is that RE stimulates cumulative (measured over 24-36 h) MPS in rats and humans. Study one determined whether an acute bout of RE in rats enhances MPS when assessed with the traditional flooding dose (~ 25 min) and 2H2O (4 and 24 h measurements); thus a comparison of the two methodologies was made. An acute session of RE did not result in an elevation in MPS when quantified by either the flooding dose or 2H2O over 4 and 24 h (methods compared qualitatively). Therefore, an acute bout of RE in rats does not appear to be anabolic and adaptation resulting from multiple bouts is likely necessary. Study two determined if RET in rats results in attenuation in MPS (plateau effect) 16 h following the final RE session (peak anabolic window) and if it is due to an increase in 4E-BP1 (a key regulator of mRNA translation initiation) activity; or if the timing in anabolism changes, which could be detected with a cumulative assessment (2H2O). MPS at 16 h was unchanged following RE training. Consistent with this finding, there were no differences in 4E-BP1 activity. Conversely, cumulative MPS was significantly increased with RET, suggesting a temporal shift in anabolism. Study three determined if dietary protein consumed immediately following RE augments cumulative (24 h) MPS in young adult human males when energy and macronutrients are controlled. RE and post-RE protein had no effect on mixed MPS; however, myofibrillar MPS was significantly increased with RE suggesting specific changes within a heterogeneous protein pool. Collectively, these are the first studies to assess changes in cumulative MPS with RE in rats and humans. The long term goals of this research are to understand muscle protein anabolism in "free-living" mammals and the mechanisms that regulate this process.

Ετέρωση, γνωστή επίσης ως υβριδική ευφορία, αναφέρεται στην φαινοτυπική ανωτερότητα ενός ετερόζυγου υβριδίου σε σύγκριση με τους ομόζυγους γονείς του. Πρόσφατες πρόοδοι στην χαρτογράφηση γενετικών τόπων ποσοτικών χαρακτήρων σε Saccharomyces cerevisiae έχουν αποκαλύψει αρκετούς στόχους που συνδέονται με την ετέρωση, όπως το γονίδιο που κωδικοποιεί την ριβοσωματική πρωτεΐνη L39. Για να διερευνήσουμε περαιτέρω τη δυνητική επίδραση της ετέρωσης στην ευκαρυωτική πρωτεϊνική σύνθεση, χρησιμοποιήσαμε τα ομοζυγωτικά γονικά στελέχη 6x6 και BYxBY, σε σύγκριση με τα υβρίδια 6xBY και 6xBY6, και το ημιζυγωτικό στέλεχος Δ6xBY στο οποίο λείπει ένα από τα δύο αλληλόμορφα του γονιδίου. Μετά από πειράματα in vitro, ελέγξαμε την poly(U) εξαρτώμενη δραστηριότητα πολυμερισμού φαινυλαλανίνης, τη μεταφραστική πιστότητα, τη δραστικότητα της πεπτιδυλοτρανσφεράσης και επιπλέον την ευαισθησία έναντι του αντιβιοτικού κυκλοεξιμίδιο. Σύμφωνα με τα στοιχεία μας, τόσο τα υβριδικά στελέχη όσο και το ημιζυγωτικό στέλεχος έδειξαν αυξημένη συχνότητα λάθους, ενώ η δράση της ΡΤάσης παρουσίασε σημαντική αύξηση μόνο στην περίπτωση του υβριδικού στελέχους 6xBY. Επιπλέον, η ανθεκτικότητα έναντι του κυκλοεξιμιδίου ήταν αυξημένη για όλα σε σύγκριση με τα γονικά στελέχη, υποδεικνύοντας ότι η ετέρωση μπορεί να συνδέεται απ 'ευθείας με τον μηχανισμό της πρωτεϊνοσύνθεσης, ανοίγοντας έτσι ένα άλλο νέο παράθυρο για να διερευνηθεί περαιτέρω αυτό το σημαντικό φαινόμενο. / Heterosis, known also as hybrid vigor, refers to the phenotypic superiority of an heterozygous hybrid compared to its homozygous parents. Recent advances in quantitative trait loci (QTL) mapping in Saccharomyces cerevisiae have uncovered several targets associated with heterosis, such as the gene encoding ribosomal protein L39 (RPL39 or spb2)(?). To further explore the potential effect of heterosis on eukaryotic protein synthesis, we used the homogygous parental strains 6x6 and BYxBY, in comparison to hybrids 6xBY and 6xBY6, and the hemizygous strain Δ6xBY lacking one of the two alleles of RPL39 (spb2). Following in vitro experiments, we tested the poly(U) dependent phenylalanine polymerization activity, the translational fidelity, the peptidyl-transferase activity and additionally the susceptibility versus the antibiotic cycloeximide. According to our data, both hybrid and hemizygous strains showed increased error frequency while the PTase activity exhibited significant increase only in the case of the hybrid strain 6xBY. Moreover, the immunity versus the antibiotic cycloeximide was increased for all compared to the parental strains, indicating that heterosis may be directly associated with protein synthesis machinery, thus opening another new window to explore further this important phenomenon.

Η ανάπτυξη των ανθρώπινων δραστηριοτήτων έχει οδηγήσει τα τελευταία χρόνια στην παραγωγή και απελευθέρωση πολλών τοξικών ουσιών στο θαλάσσιο περιβάλλον. Τα δίθυρα μαλάκια, όπως το Mytilus galloprovincialis, μπορούν να συσσωρεύουν ένα μεγάλο εύρος ρυπαντών και να αποκρίνονται με ποικίλες αλλαγές στη φυσιολογία τους, οι οποίες μπορούν να μετρηθούν ως βιοδείκτες έκθεσης ή αποτελέσματος. Το περιβαλλοντικό stress ωθεί πολλούς οργανισμούς στην αρνητική ρύθμιση της πρωτεϊνοσύνθεσης, αποθηκεύοντας ανενεργά ριβοσώματα, τα οποία επανενεργοποιούνται ταχέως όταν οι συνθήκες βελτιωθούν. Έτσι, ένας αποτελεματικός τρόπος εκτίμησης της απόκρισης των οργανισμών στο μολυσματικό stress, είναι η ανάλυση της κατάστασης ριβοσωμικής συσσώρευσης, όπως επίσης και η ικανότητα των ριβοσωμάτων για έναρξη της πρωτεϊνικής σύνθεσης, που αντανακλούν την κατάσταση της μεταφραστικής ενεργότητας. Στην παρούσα μελέτη έγινε εκτίμηση των επιπέδων ρύπανσης του Πατραϊκού Κόλπου, καταποντίζοντας μύδια (M. galloprovincialis) σε δύο σταθμούς στον Πατραϊκό Κόλπο και μία περιοχή αναφοράς. Ο Σταθμός 1 βρίσκεται στις εκβολές του ποταμού Γλαύκου και είναι υπό την επίδραση βιομηχανικών, αγροτικών και αστικών πηγών ρύπανσης. Ο Σταθμός 2 βρίσκεται στον Άγιο Βασίλειο, παρ’ ότι δε φέρει οργανική μόλυνση, έχει αυξημένα επίπεδα βαρέων μετάλλων και κυρίως Cr. Η περιοχή αναφοράς βρίσκεται κοντά στο Γαλαξείδι και δεν επηρεάζεται από πηγές ρύπανσης. Για την εκτίμηση των επιπέδων ρύπανσης στις περιοχές αυτές, εφαρμόστηκε μία σειρά βιοδεικτών που περιλαμβάνει το περιεχόμενο μεταλλοθειονινών, τη σταθερότητα της λυσοσωμικής μεμβράνης και τη συχνότητα μικροπυρήνων. Επίσης, προσδιορίστηκε η συγκέντρωση μετάλλων στο νερό και στους ιστούς των μυδιών, όπως επίσης και το ποσοστό πολυσωμάτων και η ικανοτητα των ριβοσωμάτων για έναρξη της μετάφρασης σε κύτταρα πεπτικού αδένα. Επιπλέον, έγινε στατιστική εκτίμηση της συσχέτισης μεταξύ των επιπέδων ρύπανσης και των εξεταζόμενων βιοδεικτών. Η ανάλυση των βιοδεικτών έδειξε οτι υπάρχει μία διαφοροποίηση στα επίπεδα ρύπανσης ανάμεσα στις διαφορετικές περιοχές του Πατραϊκού Κόλπου, με το μεγαλύτερο βαθμό ρύπανση να εντοπίζεται στο Σταθμό 1. Οι εποχιακές διακυμάνσεις που παρατηρούνται στις τιμές των βιοδεικτών μπορούν να αποδοθούν σε εξωγενείς παράγοντες, όπως τα επίπεδα της ρύπανσης και/ή σε ενδογενείς παράγοντες, όπως ο αναπαραγωγικός κύκλος των μυδιών. Η στατιστική επεξεργασία των αποτελεσμάτων έδειξε οτι υπάρχει σημαντική συσχέτιση (θετική ή αρνητική ανάλογα με το βιοδείκτη) μεταξύ των επιπέδων ρύπανσης και των βιοδεικτών. Το χαμηλότερο πολυσωμικό περιεχόμενο σε συνδυασμό με τη μειωμένη ικανότητα των ριβοσωμάτων για έναρξη της μετάφρασης που παρατηρήθηκε στο Σταθμό 1, καταδεικνύει την επίδραση του περιβαλλοντικού stress στην πρωτεϊνική σύνθεση στην περιοχή αυτή και εισηγείται τη χρήση των διαταραχών της μετάφρασης ως εργαλείο σε μελέτες βιοπαρακολούθησης. Τα παρόντα δεδομένα σε σύγκριση με εκείνα των τελευταίων πέντε ετών αποκαλύπτουν μία προοδευτική μείωση των επιπέδων ρύπανσης του Πατραϊκού Κόλπου, οφειλόμενη πιθανώς στην απομάκρυνση πολλών εργοστασίων από την περιοχή, και στη λειτουργία σταθμού βιολογικού καθαρισμού. / The rapid increase of anthropogenic activities has led to the production and release of several harmful compounds into the marine environment. Bivalve mollusks such as Mytilus galloprovincialis, are able to accumulate in their tissues a wide range of pollutants and respond in a broad range of alterations that can be measured as exposure or effect biomarkers. Such changes are usefull warning tools in marine environment monitoring. Environmental stress forces many organisms to downregulate translation by storing inactive ribosomes, which are rapidly reactivated when conditions improve. Therefore, an effective way to reveal responses to pollution stress is to analyse the aggregation state of ribosomes which reflects the translational efficiency status. Parallel determination of the capability of ribosomes to initiate protein synthesis may help to understand the mechanism of translation downregulation. In the present study, the pollution status of Patraikos Gulf was assessed by caging mussels (M. galloprovincialis) in two stations of the gulf and in a reference area. Station 1 was near the estuaries of Glafkos River, influenced by industrial, agricultural and urban sources. Station 2 located in Agios Vassileios, had no apparent organic pollution, but was enriched in heavy metals, particularly Cr. Reference area was near Galaxidi, far from pollution sources. To verify the degree of pollution in these areas, a battery of biomarkers was assessed, including metallothionein content, lysosomal membrane stability and micronucleus frequency. In addition, metal ion concentrations in the surrounding waters and in mussel tissues as well as polysome content and the efficiency of ribosomes to initiate translation in digestive gland cells were estimated. Furthermore, an effort was made to reveal the correlation between pollution and the employed biomarkers. Biomarker analysis revealed a differentiation in the degree of pollution among different sites of Gulf of Patras. Seasonal variations in the mean values of the biomarkers can be attributed to exogenous factors, like pollution levels, and/or endogenous factors like reproductive cycle of the mussels. Statistical analysis of the data showed a significant correlation (positive or negative, depending on the biomarker) between the pollution levels and the biomarkers.The lower polysome content in combination with the reduced efficiency of the ribosomes to initiate translation observed in Station 1, indicates the effects of pollution stress on protein synthesis in this area and encourages the evaluation of the translational perturbations as a tool in biomonitoring studies. The present data, compared to those collected the past five years, reveal a progressive pollution depression in Patraikos Gulf, propably caused by the removal of many factories previously contaminating this area, and the recent operation of a local sewage cleaning station.

Termination of protein synthesis occurs in response to the translocation of a stop codon (UAA, UAG or UGA) into the A site of the ribosome. Unlike sense codons, stop signals in the mRNA are recognized by two classes of specialized proteins called release factors (RFs): the class I or decoding RF, which recognizes the stop codon and promotes peptidyl-tRNA hydrolysis and class II RF, a G-protein that promotes the dissociation of the decoding RF from the ribosome. The discovery that stop codons are decoded by a protein factor rather than a specific tRNA opened up the possibility that the signal for termination of protein synthesis might extend beyond the stop codon itself. Biochemical and genetic experiments in prokaryotes confirmed that bias in nucleotide usage around stop codons correlates with translation termination efficiency. The objective of the current investigation was to define the eukaryotic termination signal by determining the bias in the nucleotide sequence surrounding eukaryotic stop codons and to identify whether this was a determinant of translation termination efficiency. Bioinformatic analysis of five diverse eukaryotic genomes was undertaken to identify potential eukaryotic translation termination signal elements. Significant nucleotide bias was identified both 5� and 3� of the stop codon in all the genomes investigated. Correlations were identified between nucleotide bias and gene expression levels, and between nucleotide bias and natural recoding sites predicting that nucleotides 5� and 3� of the stop codon affect termination efficiency. These correlations were common to all organisms investigated and suggested the existence of a eukaryotic termination signal. Termination signals identified from the bioinformatic analysis were assayed to determine the efficiency of termination in an in vitro dual luciferase reporter assay. Results indicated that nucleotides both 5� and 3� of the stop codon could significantly alter termination signal efficiency, although readthrough did not vary by greater than 1%. The effect of nucleotides 3� to the stop codon on termination efficiency was investigated further in mammalian cultured cells using the dual luciferase reporter assay. Results showed a significant relationship between the identity of these nucleotides and observed termination efficiencies with nucleotides at positions +4 and +8 giving the strongest correlation. Termination sequence elements of the form UGA CUN NCN mediated up to 5% readthrough in cultured cells. Investigations into the underlying mechanisms that were responsible for the variation in termination efficiency were also undertaken. Co-transfection of specific suppressor tRNAs enhanced but did not change the pattern of observed termination efficiency, indicating that the mechanisms mediated by the termination signal element was not mediated through suppressor tRNA binding. Alignments of 18S rRNA sequences indicated potential extensive interactions between the rRNA and the mRNA termination signal element. Experiments that assessed the effect of eRF1 levels on termination at inefficient termination signals in vitro revealed that increased levels of eRF1 could improve termination efficiency. These results indicate that, as in prokaryotes, specific nucleotides beyond the stop codon modulate translation termination efficiency in eukaryotes, and that the translation termination signal should be considered a sequence element.

Understanding the molecular mechanisms that govern productivity and growth of recombinant host cells is essential to devise informed approaches to increase commercial viability and availability of biopharmaceuticals. This work has focused on the roles of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway in CHO cells, the most widely used expression system in the biopharmaceutical industry. mTORC1 is a master regulator of cell growth, protein synthesis and metabolism in response to availability of nutrients, oxygen and growth factors. Therefore, it was hypothesised that increased activity of mTORC1 enhances growth and productivity of recombinant CHO cells. The study of a recombinant GS-CHO cell line in the serum-free suspension batch culture indicated a gradual decrease in the activity of mTORC1, as defined by the decreased extent of site-specific phosphorylation of two widely ascribed downstream target proteins (ribosomal protein S6 kinase 1 (S6K1) and 4E-BP1, an inhibitor of translation initiation). The decline in the activity of mTORC1 paralleled decreased growth rate, recombinant protein specific productivity and global protein translation. To further clarify the role of the mTOR pathway in cell growth and protein production, cells in batch culture were treated with rapamycin, a specific inhibitor of mTORC1. Treatment with rapamycin stalled the growth of the CHO cell line transiently, but recombinant protein specific productivity, longevity of batch culture, and final antibody titre were greater than control. Rapamycin addition produced discriminating effects on downstream signalling targets, implicating distinct roles for these targets in control of growth and protein synthesis. Engineering the mTORC1 pathway by overexpression of specific components of this pathway (S6K1 and Rheb) generated increased growth and extended viability. Greater proliferation was not associated with improved productivity suggesting highly proliferative phenotypes that prioritise cell growth over synthesis and secretion of recombinant antibody in the recombinant GS-CHO cells examined. Therefore, the engineering of mTORC1 pathway may be beneficial to increase robustness or adaptation to stressed conditions (such as serum- free suspension growth, low nutrition availability and hypoxia).

Intrauterine growth restriction causes impaired growth and development of mammalian fetus, and leads to long-term negative effect on postnatal growth. Among domestic animals, pigs exhibit the most severe naturally occurring IUGR and reduced postnatal muscle growth. The objectives of this research project were to: 1) determine muscle stem cell characteristics in IUGR pigs; 2) determine how intrauterine growth restriction alters protein deposition in skeletal muscle; 3) investigate whether branched-chain amino acids (BCAA) are able to enhance protein synthesis in intrauterine growth restricted (IUGR) pig muscle. Newborn piglets were considered normal body weight (NBWT) or IUGR when birth weight was within ± 0.5 SD and -2 SD of litter average respectively. Muscle satellite cell numbers, believed to be the major nuclei source for postnatal muscle growth, were lower in newborn IUGR pigs which could result in reduced muscle hypertrophy potential. In addition, cultures derived from IUGR muscle satellite cells had a lower fusion percentage. Fewer satellite cells and impaired differentiation ability may contributor to impaired muscle growth in these pigs. Protein synthesis rate was significantly lower in IUGR pig hindquarter in the first hour after feeding, but BCAA supplementation had no effect on protein synthesis in IUGR pigs. Further, eukaryotic translation initiation factor 4E (eIF4E) expression is down regulated in IUGR pig muscle. These results suggest that impaired translation initiation may provide a plausible explanation for the lower protein synthesis rates observed in IUGR pigs. Overall, reduced muscle stem cell number and changes in their activity, as well as impaired translation initiation may be important explanations for compromised postnatal muscle growth in intrauterine growth restricted pigs. / Ph. D.

No description available.

Muscle proteins are in a continuous state of recycling. This process involves a balance between synthesis and breakdown. These opposing processes dictate muscle protein gains and losses. Muscle hypertrophy occurs when synthesis exceeds breakdown. In order for the accretion of new muscle proteins, a chronic state of net positive muscle protein balance (synthesis> breakdown) is required. Resistance exercise is a potent stimulus of protein turnover and the combined effects of exercise and feeding have shown to be necessary for net protein anabolism. Resistance training has been reported to increase muscle strength and induce changes in skeletal muscle morphology. These positive strength adaptations include muscle fibre hypertrophy and a shift in fibre type from IJX to IIA. Previous investigations of resistance training-induced changes in muscle protein synthesis and fibre morphology have utilized cross-sectional or longitudinal, bilateral training designs. Thus, the purpose of this study was to investigate the effects of a progressive eight week unilateral leg resistance training program on skeletal muscle morphology, and resting and exercise-stimulated mixed muscle protein fractional synthesis rate (FSR). Eight young men performed two training sessions each week, and each session consisted of four sets of knee extension (KE) and four sets of leg press (LP) at 80% 1 repetition maximum (1 RM). Needle biopsies from the vastus lateralis muscle of the trained (T) leg were taken before and after training and analyzed for fibre composition, cross-sectional area (CSA), and myosin heavy chain (MHC) content. Muscle protein FSR was determined using a primed constant stable isotope infusion of [13C6]-phenylalanine in both the T and untrained (UT) legs. Training induced type IIX and IIA fibre hypertrophy (P <0.05) with no change for 1ype I fibre CSA. There was no significant change in histochemically determined fibre composition or MHC content. After training, 1RM strength of the T leg significantly increased compared to baseline values (P < 0.01). At rest, FSR was significantly elevated in the T versus the UT leg (P < 0.01). Following an acute bout of resistance exercise, which was performed at the same relative intensity (80% 1 RM) for the T and UT legs, FSR was greater in the UT versus the T leg (P < 0.01). There was a lower exercise-induced increase in muscle FSR in the T versus the UT leg compared to their respective resting values <T: P = 0.08, UT: P < 0.01). These data show that resistance training resulted in significant muscle fibre hypertrophy and elevated rate of muscle protein synthesis at rest. In addition, the acute response to resistance exercise was characterized by an attenuated rise in muscle protein FSR in the T versus the UT leg. We conclude that resistance training markedly attenuates the acute muscle protein synthetic response following resistance exercise, even when loads are matched at the same relative intensity. / Thesis / Master of Science (MS)