<p>There is a prevalent belief that acute hormone responses to resistance exercise mediate adaptations in skeletal muscle hypertrophy; however, there is little supporting evidence. We conducted studies to examine the relationship between acute hormonal increases after resistance exercises and subsequent changes in muscle anabolism.</p> <p>We tested the hypothesis that exercise-induced responses of anabolic hormones—growth hormone (GH) and testosterone—would enhance rates of myofibrillar protein synthesis (MPS) after an acute bout of resistance exercise, and would augment muscle hypertrophy after training. We concluded, however, that resistance exercise-induced increases in putative anabolic hormones do not enhance MPS or hypertrophy.</p> <p>We also examined whether rates of MPS would be attenuated in women (compared with men) after resistance exercise, due to their lack of post-exercise testosteronemia. We reported similar increases in MPS in men and women; post-exercise testosterone responses in women, which were 45-fold lower than men, did not attenuate elevations in MPS.</p> <p>Collectively, our work leads to the conclusion that the acute rise in hormones such as testosterone and GH has very little bearing on MPS and hypertrophy responses to resistance exercise. Instead, the rise in these hormones appears to be a non-specific response to exercise stress rather than a response that is important for muscle anabolism. Contrary to widely used principles, our data suggests that exercise programs should not be designed based on nuances in the post-exercise hormonal milieu. Alternatively, understanding local mechanotransduction, which is directly linked to muscle fibre loading, will reveal the processes that drive human exercise-mediated muscle hypertrophy.</p> / Doctor of Philosophy (PhD)

<p>Amino acids are key nutritional stimuli that are both substrate for muscle protein synthesis (MPS), and signaling molecules that regulate the translational machinery. There is a dose-dependent relationship between protein intake and MPS that differs between young and elderly subjects. The current thesis contains results from three separate studies that were conducted to examine to potential to enhance smaller doses of protein, known to be suboptimal in their capacity to stimulate MPS, through supplementation with specific amino acids, namely leucine and citrulline.</p> <p>The first two studies examined the potential to enhance the muscle protein synthetic capacity of a smaller, suboptimal dose of whey protein with leucine. In study one, we concluded that leucine supplementation of a suboptimal dose of protein could render it as effective at enhancing rates of MPS as ~four times as much protein (25 g) under resting conditions, but not following resistance exercise. In study two, we examined the potential of leucine and branched-chain amino acids to enhance the MPS response of a suboptimal dose of protein within the context of mixed macronutrient ingestion. We concluded that supplementation with a relatively high dose of leucine could render it as effective at enhancing MPS rates as ~four times as much protein (25 g) under both resting and post-exercise conditions.</p> <p>In study three, we examined the potential of citrulline supplementation to enhance blood flow, microvascular circulation, and MPS in response to a suboptimal dose of whey protein in elderly subjects. We concluded that supplementation of a suboptimal dose of protein with citrulline did not augment bulk blood flow or muscle microvascular circulation. The major findings from the works presented in this thesis is that smaller doses of protein that normally elicit a suboptimal increase in MPS can be made more anabolic when supplemented with specific amino acids.</p> / Doctor of Philosophy (PhD)

The neonatal period in mammals is characterized by high rates of growth, attributed to rapid myonuclear accretion and protein deposition in muscle. Low-birth-weight (LBWT) neonates experience restricted muscle development, which leads to impaired postnatal growth and metabolic disorders later in life. The overall hypothesis of this dissertation was that dysfunction of myogenic satellite cells and aberrant regulation of protein synthesis and degradation signaling predispose LBWT neonatal pigs to slower postnatal growth. We sought to determine the proliferation and differentiation of satellite cells (SCs) derived from skeletal muscle of LBWT neonatal pigs and to elucidate the cellular mechanisms that regulate protein synthesis and degradation in LBWT pig muscles. Newborn pigs were considered as normal-birth-weight (NBWT) or LBWT when weight at birth was within 0.5 SD and below 2 SD of litter average respectively. SCs isolated from longissimus dorsi (LD) muscle of NBWT and LBWT neonatal pigs displayed similar proliferation rates. Fusion was modestly diminished in SCs from muscle of LBWT pigs compared with their NBWT siblings, suggesting SCs were not intrinsically different between the two groups and were unlikely a major contributor to the impaired muscle growth of LBWT pigs. Plasma and muscle insulin-like growth factor (IGF)-I was diminished in LBWT compared with NBWT pigs. In addition, reduced activation of key components of IGF-I downstream signaling pathway in LBWT pigs muscle may lead to diminished translation initiation signaling and thus decreased protein synthesis in these animals. However, IGF-I receptor expression and myostatin signaling inversely correlated to LBWT, indicating they may participate in compensatory responses for the reduction in protein synthesis signaling. Expression of eukaryotic initiation factor (eIF) 4F complex subunits, eIF4E, eIF4G, and eIF4A was reduced in LBWT compared with NBWT pigs. This would suggest that diminished translation initiation signaling in skeletal muscle of LBWT pigs is the main factor that predisposes LBWT pigs to slower growth rates in the neonatal period. In contrast, changes in protein degradation signaling do not appear to affect protein turnover in LBWT neonatal pigs. / PHD

This thesis reports the viability of E. coli cell extracts prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples and expression of proteins containing unnatural amino acids (UAAs) at higher level using PCR amplified linear DNA templates (LETs) in cell-free protein synthesis (CFPS) system. E. coli-based cell extracts are a vital component of inexpensive and high-yielding CFPS reactions. However, effective preparation of E. coli cell extract is limited to high-pressure homogenizers (French press style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. This work specifically assessed the following capital cost lysis techniques: (1) sonication, (2) bead vortex mixing, (3) freeze-thaw cycling, and (4) lysozyme incubation to prepare E. coli cell extract for CFPS. In this work, simple shake flask fermentation with a commercially available E. coli strain was used. Additionally, the RNA polymerase was over expressed in the E. coli cells prior to lysis which eliminated the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high yielding E. coli-based cell extract was prepared using equipment requiring reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing. LETs are an attractive alternative to plasmids for site-specific incorporation of unnatural amino acids in proteins in the CFPS system because of their short preparation time and ease of production. However, major limitations associated with LETs are: (1) their degradation by RecBCD enzyme present in the cell-extract used for CFPS and (2) high CFPS energy costs. In this work, we report the optimization of LET-based CFPS for improved protein yield by inhibiting the RecBCD enzyme with small inhibitor molecules resulting in three fold increment in yield of protein containing UAA. We also assessed alternative energy sources such as glucose, fructose-1,6-bisphospate, creatine phosphate/creatine kinase, and high glutamate salt for cost reduction. This work could be important for high-throughput applications based on linear expression templates. This work demonstrates simple E. coli extract preparation and improved yield with linear expression templates for further advancements of cell-free protein synthesis system.

A arginina é um aminoácido condicionalmente essencial que participa de inúmeras reações metabólicas no organismo como, por exemplo, o ciclo da uréia, a síntese de creatina e a geração de óxido nítrico (NO). Além dessas funções a arginina é associada, com a sensibilidade à insulina, a secreção de GH e mais recentemente com a síntese protéica muscular. O objetivo deste trabalho foi investigar o efeito da suplementação via oral crônica de L-arginina sobre a síntese protéica muscular, pela via da mTOR, a fim de contribuir com as novas discussões científicas acerca deste aminoácido de ampla atuação. Métodos: Foram utilizados ratos wistar machos adultos com cerca de 200g de peso corporal divididos em quatro grupos de quatorze animais denominados na seguinte forma: Arginina Treinado (AT), Arginina Sedentário (AS), Dieta-Controle Treinado (CT) e Dieta-Controle Sedentário (CS). Ambas as dietas foram elaboradas com base das recomendações da AIN-93, sendo que a dieta enriquecida com arginina recebeu acréscimo de 2% deste aminoácido e a dieta controle recebeu um mix de aminoácidos não essenciais para garantir que ambas fossem isonitrogenadas e isocalóricas e as proporções de aminoácidos presente nas rações foi conferida por aminograma. O treinamento dos animais consistiu em exercício anaeróbio com sessões que eram compostas de 4 séries de 10 saltos com um minuto de descanso entre estas em tanque de água. Os saltos eram desempenhados com carga de 50% do peso corporal acoplado ao tórax dos animais na freqüência de 5 dias por semana por 6 semanas. A evolução da massa corporal dos animais bem como o consumo de ração foram avaliadas três vezes por semana e estimada uma média semanal. Foram realizados testes de tolerância oral à glicose (OGTT) e tolerância à insulina (ITT) no início e ao final do experimento em todos os animais para avaliar alterações na sensibilidade à insulina. Após 72hs da última sessão de treinamento os animais foram anestesiados para infusão de insulina, coleta dos músculos gastrocnêmio e plantar, fígado, sangue e eutanasiados conforme protocolo aprovado pelo CEA-USP. As análises bioquímicas foram determinações séricas de insulina, GH, IGF-1 e a proteína transportadora de IGF-1 (IGFBP-3), glicose plasmática, uréia e creatinina séricas, IGF-1 muscular e hepático por kits comerciais de tecnologia multiplex Luminex e aminograma sérico por cromatografia. As análises moleculares foram realizadas para as proteínas chaves envolvidas na via de síntese protéica muscular em sua forma total e fosforilada, sendo estas: IRS-1, Akt, mTOR, 4E-BP1 e p70S6K determinadas por método de western blotting. Resultados: Não foram encontradas diferenças estatisticamente significativas nos parâmetros avaliados com exceção da creatinina que se mostrou mais elevada nos grupos suplementados com arginina. A suplementação de arginina, nas concentrações administradas, bem como o exercício de alta intensidade pelo período determinado não foram capazes de alterar a expressão das proteínas envolvidas na regulação de síntese protéica muscular de ratos nem a sensibilidade celular à insulina. Conclusão: não houve aumento da síntese protéica muscular com a suplementação de arginina, nestas condições experimentais. / The arginine is an amino acid conditionally essential that participates in innumerous metabolic reactions in the body like, for instance, the urea cycle, the synthesis of creatine and production of nitric oxide (NO). Besides those functions the arginine is associated, with the insulin sensitivity, GH secretion and most recently with muscle protein synthesis. The aim of this work was to investigate the effect of L-arginine chronic oral supplementation on the muscle protein synthesis, through mTOR pathway, in order to contribute with new scientific discussions about this broad action amino acid. Methods: Wistar male adult rats were used with about 200g body weight distribute into four groups of fourteen animals named this way: Trained Arginine (TA), sedentary Arginine (SA), Trained Diet-Control (TC) and Sedentary Diet-control (SC). Both diets were elaborated based on the AIN-93 recommendations, considering that the enriched diet with arginine was added 2% of this amino acid and the control diet received a mix of non-essential amino acid in order to ensure that both were isonitrogenous and isocaloric and the proportions of present amino acids in the rations have been checked through aminogram. The animals training consisted of anaerobic exercise with sections composed by four jump series, with one minute rest among these in a PVC cube water. The jumps were performed with a load of 50% of their body weight attached in the animal\'s trunk, five days a week over six weeks. The animals\' body weight evolution as well as the food intake were evaluated three times a week in order to figure a weekly average. Oral glucose test tolerance (OGTT) and insulin test tolerance (ITT) have been done in the beginning and in the end of the experiment in all animals to evaluate insulin sensitive changes. The animals were anesthetized to insulin infusion, gastrocnemic and plantaris muscles, liver and blood collects 72 hrs after the last training section and afterwards sacrificed according to CEA-USP approved protocol. The biochemical analysis were blood determination of insulin, GH, IGF-1 and its binding protein 3 (IGFBP-3), glucose, urea and creatinine, and muscle and liver IGF-1 through commercial kits of multiplex Luminex technology and seric aminogram through chromatography. The molecular analysis were performed for the key proteins of the muscle protein synthesis pathway in its total and phosphorylated form: IRS-1, Akt-1, mTOR, 4E-BP1 and p70S6K determined by western blotting method. Results: Significant statistical differences were not found to all evaluated biomarkers in this experiment except for creatinine which was more elevated in groups supplemented with arginine. The arginine supplementation, in these given doses, as well as the high-intense exercise, failed in stimulate both the expression of the proteins involved in the muscle protein synthesis regulation and the insulin sensitivity in the rats in this condition. Conclusion: There hasn\'t been any increase in the muscle protein synthesis with arginine supplementation, in these experimental conditions.

Esse trabalho compreendeu a avaliação do impacto do jogo Sintetizando Proteínas sobre o processo de ensino-aprendizagem de alunos do ensino médio e sua compreensão conceitual a respeito do tema proteínas, fundamentados na teoria sócio-interacionista de Vygotsky. Os conceitos formulados pelos alunos foram coletados em três etapas diferentes: a primeira, anterior à aplicação do jogo, por meio de um questionário diagnóstico e uma pré-entrevista; a segunda, durante o jogo, por meio de gravações áudio-visuais; a terceira, posterior à aplicação do jogo, por meio de uma pós-entrevista. Os dados levantados pelo questionário diagnóstico permitiram detectar algumas tendências do pensar dos alunos com relação ao conceito de proteína: a) tendência em definir genericamente o papel das proteínas; b) tendência dos alunos em associar proteínas aos alimentos de origem animal; c) tendência em entenderem as proteínas apenas como construtoras de tecidos, portanto uma função prioritariamente estrutural. A análise e interpretação dos dados referentes às pré e pós-entrevistas e das transcrições das gravações áudio-visuais nos mostraram que as interações ocorridas durante o jogo tiveram um importante papel na evolução de alguns conceitos antes incompreendidos pelos alunos. Dessa forma o jogo, enquanto um modelo que simula o processo de síntese protéica dentro da célula, proporcionou aos alunos experiências que os permitiram vivenciar esse processo, estabelecendo uma ponte entre o abstrato e o concreto, possibilitando aos mesmos o alcance de níveis de desenvolvimento mais elevados e a evolução de conceitos antes incompreendidos. / The main goal of this study was evaluating the impact of the game Synthesizing Proteins on the teaching-learning process of high school students and their conceptual understanding on the subject proteins, based on Vygotskys sociointeractionist theory. The concepts of the students were collected in three different stages: before the application of the game, using a diagnostic questionnaire and a pre-interview; during the game, by means of audio-visual recordings; after the application of the game, by a post-interview. The data raised from the diagnostic questionnaire had allowed to detect some trends of students thinking with regard to the protein concept: a) the trend in generically defining the role of proteins to the organism (human) b) students trend in only associating proteins to foods of animal origin c) trend in only understanding proteins as tissue constructors, so a function primarily structural. The analysis and interpretation of data collected by pre- and post-interviews and the transcripts of the audio-visual recordings showed that the interactions occurred during the game had an important role in the evolution of some concepts that were previously misunderstood by students. Thus, the game as a model that simulates the process of protein synthesis inside the cell, allowed the students experience this process, establishing a bridge between the abstract and concrete and allowing them to achieve high levels of development.

O aumento mundial na incidência da obesidade está associado com um aumento significante com os custos com a saúde. Somente nos Estados Unidos, os gastos com os tratamentos associados a obesidade superam 9% dos custos anuais com a saúde, em torno de $147 bilhões de dólares por ano. Os efeitos da obesidade no músculo esquelético estão relacionados com o desenvolvimento da resistência à insulin (IR), diabetes e piora da qualidade de vida. Trabalhos rescentes tem demonstrado que a dieta hiperlipídica (DHL) diminui a capacidade do músculo esquelético responder a sinais de crescimento. Este efeito negativo relacionado a diminuição na ativação da via Akt/mTOR e aumento nd estresse de retículo endoplasmático (ERE) é denominado de resistência anabólica. Por outro lado, estudos têm demonstrado um possível efeito benéfico da suplementaçãoo com o ácido graxo polinsaturado ômega 3 (Ag-w3) e do treinamento físico aeróbico em diversos parâmetros como, melhora da capacidade oxidativa, sistema imunológico, síntese proteica e degradação em saudáveis ou com alguma patologia associada. Dessa maneira, o presente trabalho demonstra que 8 semanas de DHL contribuíram para o desenvolvimento da obseidade. Por outro lado, o protocolo de TA promoveu um efeito protetor ao ganho de peso nos animais obesos. A suplementação com o AG-w3 foi capaz de prevenir alguns parametros analisados e a associação do óleo de peixe não foi capaz de potencializar os efeitos benéficos encontrados com o TA. / There is a worldwide increase in the incidence of obesity that is associated with significant increases in medical costs. In the United States alone, treatment of obesity related health problems accounts for up to 9% of the total annual cost of healthcare, an estimated $147 billion per year. The effects of obesity on skeletal muscle are correlated with insulin resistance (IR), diabetes and decreased of quality of life. Recent work has demonstrated that a high fat diet (HFD) decreases the ability of skeletal muscle to hypertrophy in a model of increased mechanical load. This negative effect on muscle growth is correlated with a decrease in activation of the Akt/mTOR signaling pathway and an increase in endoplasmic reticulum stress.

Com o objetivo de estudar a espécie bubalina quanto ao desempenho de machos bubalinos do nascimento ao abate em regime de pastejo e as características de carcaça em dois pesos de abate, assim como o metabolismo ruminal de dietas com diferentes níveis de proteína e energia e a produção de gases in vitro, o presente trabalho apresenta-se avaliações feitas em quatro partes. A parte 1 foi realizada com búfalos em crescimento criados à pasto, do nascimento até atingirem dois pesos distintos de abate (517 e 568 kg). Avaliações do desempenho foram realizadas com medição do peso vivo, perímetro torácico, altura de cernelha e comprimento corporal e as avaliações das características da carcaça e carne com determinação do rendimento de carcaça quente e fria, perda no resfriamento, peso da gordura, peso do fígado, temperatura e pH da carcaça, área de olho de lombo, espessura de gordura subcutânea, marmorização, maciez e coloração. A segunda parte avaliou dietas com três níveis de proteína (9%, 12% e 15%) no metabolismo ruminal. Os Itens analisados foram: consumo de nutrientes, pH, amônia e ácidos graxos voláteis no rúmen e degradabilidade in situ. Na parte 3, foram avaliadas dietas com dois níveis de proteína (9 e 15%) e dois de energia (65 e 69% NDT) no metabolismo ruminal. Além dos Itens avaliados na parte 2 foram ainda analisados a digestibilidade com uso de marcador, taxa de passagem de liquido ruminal e volume do rúmen e síntese de proteína microbiana. Na última parte foi realizada avaliação de produção de gases in vitro com estudo da cinética da degradabilidade in vitro no tempo de 72 horas. Animais abatidos com diferentes pesos apresentaram desenvolvimento ponderal diferenciado desde o início do crescimento. Não houve diferença entre os dois grupos de animais nas características de carne e carcaça, mas os búfalos abatidos mais pesados (568 kg) apresentaram maior deposição de gordura interna. Níveis de proteína de 9%, 12% e 15% não influenciaram na degradabilidade in situ dos nutrientes e no pH ruminal. A concentração de amônia e AGV foram maiores com níveis de 15% de proteína na dieta. Os níveis de energia (alta ou baixa) combinados com teores de proteína (alto ou baixo) e as correlações entre os níveis de energia e proteína não promoveram efeitos significativos sobre o pH ruminal concentração de amônia, taxa de passagem de líquido e volume ruminal em búfalos, entretanto, dieta com teor de 15% de proteína bruta, independente dos níveis de energia na dieta apresentaram melhores degradabilidades efetivas dos nutrientes. Os níveis de energia não influenciaram significativamente na concentração amônia ruminal ao contrário dos níveis de proteína em que a maior quantidade de proteína na dieta produziu maior concentração de amônia. Não houve diferença significativa na taxa de passagem e volume ruminal entre as quatro dietas fornecidas aos animais. Dietas com diferentes níveis de energia e proteína não influenciaram na qualidade do inóculo para a produção de gases in vitro. / With the aim of studying the buffalo on the performance of males from birth to slaughter in buffalo grazing and carcass characteristics in two slaughter weights, as well as the metabolism of diets with different levels of protein and energy and the production of gases in vitro, this work presents evaluations conducted in four parts. Part 1 was performed with buffalo raised in pasture from birth until they reach two different slaughter weights (517 and 568 kg). Performance assessments were performed with measurement of body weight, chest girth, height and body length and evaluations of carcass characteristics and meat with determining the hot and cold carcass, the cooling loss, fat weight, liver weight, temperature and pH of the carcass, ribeye area, fat thickness, marbling, tenderness and color. The second part evaluated diets with three protein levels (9%, 12% and 15%) on rumen metabolism. Items discussed were the amount of nutrients, pH, ammonia and volatile fatty acids in the rumen and degradability in situ. In Part 3, were evaluated diets with two protein levels (9 and 15%) and two energy (65 and 69% of TDN) on rumen metabolism. Besides the items evaluated in Part 2, were also analyzed the digestibility, passage rate and ruminal volume and rumen microbial protein synthesis. In the last part was done evaluation of gas production in vitro with study of the kinetics of degradation in 72 hours. Animals slaughtered at different weights showed differential weight performance since the beginning of growth. There were no differences between the two groups of animals on meat and carcass characteristics, but the buffaloes slaughtered heavier (568 kg) had higher deposition of internal fat. Protein levels of 9%, 12% and 15% did not influence the in situ degradability of nutrients and rumen pH. The concentration of ammonia and VFA levels were higher with 15% protein diet. Energy levels (high or low) combined with protein levels (high or low) and the correlations between the levels of energy and protein did not cause significant effects on rumen pH, ammonia concentration, liquid passage rate and ruminal volume in buffalo, however, dietary content of 15% crude protein, independent of the energy levels in the diet showed better effective degradability of nutrients. Energy levels did not significantly modify the rumen ammonia concentration unlike the protein levels where in the higher protein diet resulted in higher ammonia concentration. There was no significant difference in passage rate and ruminal volume between the four diets fed to the animals. Diets with different levels of energy and protein did not influence the quality of inoculum for the gas production in vitro.

<p>Initiation of protein synthesis in <i>E.coli </i>involves several steps, which lead to the formation of the first peptide bond. This process requires three initiation factors: IF1, IF2 and IF3. Using a novel technique of combined light scattering and stopped-flow, we elucidated the importance of IF2•GTP conformation for the recruitment of 50S to 30S pre-initiation complex. Moreover, GTP hydrolysis is essential for IF2 release and later binding of ternary complex. Interestingly, a switch in IF2 affinity to G-nucleotides is induced during 30S pre-initiation complexes formation. </p><p>We found that IF1, previously with unknown functions in vitro, increases the rate of naked 70S dissociation by a factor 80 and acts as a fidelity factor in preventing 70S formation containing elongator tRNA instead of fMet-tRNA^fMet. We showed that RRF/EFG/IF3 split both naked and post-termination complexes while IF1/IF3 split only naked ribosomes. The mechanisms of action of RRF/ EFG, the order of their binding to 70S, as well as, the three different conformation of EF-G on the ribosomes are emphasized. Interestingly, 70S formation rate is dependent on the concentration of IF3 and not linear with 50S subunits concentration. We demonstrated that the rate-limiting step in 70S formation is IF3 dissociation from 30S complexes.</p><p>The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNA^fMet (initiator tRNA), Met-tRNA^fMet(non-formylated initiator tRNA) and Phe-tRNA^Phe (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNA^fMet, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes.</p><p>Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNA^fMet binding to 30S subunits, subsequently; the stabilization of fMet-tRNA^fMet by IF2 increases the rate of IF3 dissociation. Later, IF2 in GTP conformation allows 50S docking to 30S pre-initiation complex free of IF3 followed by GTP hydrolysis allowing IF2 release for ternary complex to bind and start elongation of protein synthesis. </p>

<p>Macrolides are a large group of clinically relevant antibiotics that inhibit protein synthesis by binding to the large ribosomal subunit in the peptide exit tunnel, close to the peptidyl transferase center (PTC). We have shown that the peptide length of the resulting peptidyl-tRNA drop-off products is proportional to the distance between the PTC and the respective macrolide in the tunnel. This indicates that macrolides act by sterically blocking the nascent peptide exit path.</p><p>A substantial amount of read-through into full-length product was observed for some macrolides and depends on the relation between the dissociation rate constants for peptidyl-tRNA and the macrolide, respectively. The dissociation rate constant for josamycin is 60 times lower than the dissociation rate constant for erythromycin, which explains why no read-through is seen for josamycin in contrast to erythromycin.</p><p>Macrolides do not compete with binding of ternary complexes, hence they are non-competitive inhibitors. However, the text-book description is not valid for macrolide antibiotics, and we show that this is due to the equilibrium assumption generally used to describe non-competitive inhibitors. Our results suggest that a more thorough mechanistic investigation is required to classify inhibitors than what has been proposed previously.</p><p>Further, we have examined the phenomenon of peptide mediated resistance to macrolides. Our results show that expression of a resistance peptide increases the dissociation rate constant for erythromycin.</p><p>In addition, we have examined the accuracy of protein synthesis on three different levels: (<i>i</i>) How do the three initiation factors accomplish fast and accurate initiation of protein synthesis, (<i>ii</i>) how does proof-reading work on the isoleucyl-tRNA synthetase, and (<i>iii</i>) what is the accuracy in the tRNA selection and how is it accomplished? Our data propose a change of the view on all these mechanisms.</p><p>In conclusion this thesis presents new results on protein synthesis, macrolide antibiotics and macrolide resistance.</p>