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.

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.

Orientador: Elenice Aparecida de Moraes Ferrari / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T21:27:10Z (GMT). No. of bitstreams: 1 Sperandeo_MariaLuizaAntunes_D.pdf: 3058745 bytes, checksum: 13c43e27d3d229473548efb9ae77c620 (MD5) Previous issue date: 2013 / Resumo: O estudo do condicionamento clássico aversivo contribuiu para caracterizar as fases da formação da memória (aquisição, consolidação, reconsolidação, persistência e extinção) e os mecanismos de plasticidade neuronal subjacentes. Entre estes, a ativação de receptores glutamatérgicos NMDA, de transcrição gênica e síntese proteica parecem fundamentais. O fator neurotrófico derivado de cérebro (BDNF) e o gene de expressão imediata (IEG) zenk são ativados no hipocampo de mamíferos durante a reconsolidação e a extinção. Este estudo investigou os efeitos da inibição da síntese proteica e do bloqueio dos receptores NMDA no hipocampo de pombos na reconsolidação e extinção da memória de medo contextual. No Experimento 1, pombos foram condicionados, testados ao contexto (reativação), submetidos à infusão intra-hipocampo de salina (SAL-PR), de anisomicina (ANI-PR) ou de MK-801 (MK-PR) após o teste (pós-reativação; PR), e retestados 2 dias depois (Rt 2d). No Experimento 2, três grupos tiveram um segundo reteste, 9 dias após a infusão (Rt 9d), enquanto que outros três grupos não passaram pelo teste (sem reativação- SR). O treino (20 min) teve três pareamentos som-choque. No teste e no Rt 2d houve exposição ao contexto do condicionamento por 5 min e no reteste Rt 9d, por 30 min. Nos dois experimentos, todos os grupos apresentaram alta ocorrência de congelamento (CONG) nas sessões de treino e de teste, mas no Rt 2d os grupos ANI-PR e MK-PR mostraram uma redução significativa de CONG (p < 0,05). Os grupos SR exibiram alta ocorrência de CONG tanto no Rt 2d, quanto no início do Rt 9d (p > 0,05). No Rt 9d o grupo MK-PR não teve recuperação espontânea de CONG, sugerindo prejuízo irreversível provocado pelo MK-801 na memória de medo contextual. Os grupos, SAL-PR, SAL-SR, ANI-PR, ANI-SR e MK-SR mostraram diminuição gradual de CONG durante a sessão de Rt 9d, evidenciando a extinção. Análises com Western blotting após o Rt 9d indicaram que o conteúdo de BDNF-maduro no hipocampo dos pombos SAL-PR foi significativamente maior em comparação aos demais grupos (p < 0,05), mas não foram observadas diferenças entre os grupos para o Zenk (p > 0,05). Isso sugere a participação do BDNF hipocampal na reconsolidação e na extinção da memória de medo contextual em pombos. Assim, a inibição da síntese proteica e o bloqueio dos receptores NMDA no hipocampo de pombos, após o teste ao contexto, prejudicaram a reconsolidação da memória de medo condicionado ao contexto. Além disso, os prejuízos observados foram dependentes da reativação da memória durante o teste / Abstract: The study of classical aversive conditioning has contributed to the characterization of different phases in memory formation (acquisition, consolidation, reconsolidation, extinction and persistence) and the underlying mechanisms of neuronal plasticity. Among these, the activation of NMDA glutamate receptors, gene transcription and protein synthesis are pointed as essentials. The brain-derived neurotrophic factor (BDNF) and immediate expression gene (IEG) zenk are activated in the hippocampus of mammals during reconsolidation and extinction. This study investigated the effects of protein synthesis inhibition and blockade of NMDA receptors in the hippocampus of pigeons on reconsolidation and extinction of contextual fear memory. In Experiment 1, pigeons were conditioned, tested in the context (reactivation), received intra-hippocampus infusion of saline (SAL-PR), anisomycin (ANI-PR) or MK-801 (MK-PR) after the test (post-reactivation, PR), and had a retest two days later (Rt 2d). Experiment 2, had a retest 9 days after intra-hippocampus infusion (Rt 9d) for three PR groups, and other three groups that were not tested 24h after training served as control for reactivation (no reactivation-NR). Three tone-shock pairings were presented during training (20 min). Pigeons were exposed to the context during 5 min in the test and Rt 2d sessions and during 30 min in Rt 9d. In both experiments, the occurrence of freezing (FRZ) was high in training and testing sessions for all the groups, but ANI-PR and MK-PR pigeons showed a significant decrease in FRZ during the Rt 2d (p < 0.05). The NR groups exhibited high occurrence of FRZ both in the Rt 2d and in the beginning of Rt 9d (p > 0.05). MK-PR pigeons had no spontaneous recovery of FRZ, suggesting irreversible impairment caused by MK-801 in contextual fear memory. The groups, SAL-PR, SAL-NR, ANI-PR, ANI-NR and MK-NR showed a gradual decrease in FRZ during the Rt 9d session, evidencing extinction. Western Blotting analysis indicated that the content of mature BDNF in the hippocampus of SAL-PR group after Rt 9d session was higher than that seen for the other groups (p < 0.05), but no between-group differences for Zenk were observed (p > 0.05). This suggests the involvement of hippocampal BDNF in reconsolidation and extinction of contextual fear memory in pigeons. The present data show that inhibition of protein synthesis and blockade of NMDA receptors in the hippocampus of pigeons, after the testing session to context, impaired reconsolidation of contextual fear conditioning memory and affected the process of extinction. Furthermore, the impairments were dependent on the reactivation of fear memory during the test / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

La perte de masse et de force musculaires liée à l’âge, ou sarcopénie, pourrait être partiellementexpliquée par un défaut de récupération de masse musculaire après des épisodes générateursd’atrophie musculaire. Ainsi, les périodes d’immobilisation qui augmentent avec l’âge (alitement,convalescence, fracture) pourraient être suivies d’une absence de récupération musculaire etcontribuer à la fonte musculaire au cours du vieillissement. Les causes de ce défaut de récupérationimpliquent notamment un déséquilibre du taux de renouvellement protéique et du taux derenouvellement cellulaire. L’objectif de cette thèse a donc été de mettre en évidence les mécanismesresponsables de l’atrophie musculaire chez le rat âgé au cours de l’immobilisation et ceux quiseraient défaillants afin de déceler les mécanismes à cibler pour favoriser la récupérationmusculaire.Des rats âgés ont été immobilisés pendant 8 jours par plâtrage unilatéral de la patte arrière, puislaissés en récupération pendant 40 jours après le déplâtrage. Nous avons montré que chez cesanimaux nourris avec un régime contenant 13% de caséine, l’immobilisation entraîne une atrophiedes muscles immobilisés mais, contrairement au rat adulte, le rat âgé ne récupère jamais la massemusculaire perdue. L’atrophie des muscles immobilisés peut être expliquée par 1/ une augmentationde l’apoptose et de la protéolyse ubiquitine-protéasome-dépendante musculaires, 2/ une diminutionde la régénération des cellules musculaires et 3/ une diminution de la protéosynthèse musculaire àl’état nourri. Tous ces phénomènes pourraient résulter de la présence d’un fort stress oxydant etd’une importante inflammation intramusculaire. Tous ces paramètres sont normalisés dès 10 jours derécupération, ce qui permet de stopper l’atrophie mais ne permet pas d’initier la phase derécupération musculaire. Nous avons donc testé l’effet de différentes supplémentationsnutritionnelles au cours de la période de récupération afin de favoriser un gain de masse musculairepost immobilisation. Des supplémentations en leucine (acide aminé bien connu pour stimuler laprotéosynthèse et inhiber la protéolyse) ont ainsi été réalisées. Chez les rats supplémentés, uneamélioration de la synthèse protéique et une normalisation plus précoce des activités protéolytiquesdu protéasome ont été observées. Cependant cette amélioration du métabolisme protéique ne s’estpas traduite par un gain de masse musculaire. Par contre, la modulation qualitative et quantitative desapports en protéines a pu permettre d’obtenir une récupération significative de masse musculaire :ainsi des régimes contenant 13% de lactosérum et des régimes hyper-protéinés ont permis de gagner50% de la masse perdue et ce, dès 20 jours de récupération.Nos résultats montrent que l’immobilisation chez le rat âgé aggrave la sarcopénie. Une fortealtération du métabolisme protéique permet d’expliquer la perte de muscle et la seule normalisationde la protéolyse et de la protéosynthèse permet d’expliquer l’absence de récupération musculaire.Nous avons montré que la modulation des apports en protéines au cours de la phase de récupérationpouvait permettre un gain de protéines. / Sarcopenia, the age-related muscle mass loss, might be partially explained by an impairedmuscle mass recovery of skeletal muscle mass after a catabolic state. Thus, immobilization periodswhich increase with aging could induce a muscle atrophy followed by a lack of muscle massrecovery. An imbalance of protein and cellular metabolisms are certainly involved in this absenceof recovery. The aim on this Ph.D thesis was to explore the mechanisms involved in muscle massatrophy during immobilization and their possible alteration during the recovery period in old rats.Old rats were immobilized for 8 days by unilateral hind limb casting and then allowed torecover for 40 days. Our results showed that animals fed a 13% casein diet wasted muscle mass inimmobilized muscles but, contrarily to adult animals, they never recovered the muscle mass loss.Muscle atrophy was due to 1/ an increase of apoptotic and ubiquitine-proteasome-dependentproteolytic pathways, 2/ a decrease of muscle regeneration processes and 3/ a decrease of muscleprotein synthesis at the fed state. These changes paralleled an increase of intracellular inflammationand oxidative stress. As these parameters were only normalized during the recovery period, theresultant nitrogen balance was then not enough positive as required for the muscle protein gain,hence contributing to the age-related incomplete muscle mass recovery. We tested free leucinesupplementation (an amino acid known for its stimulatory effect on protein metabolism) during therecovery period to improve muscle mass gain. This supplementation induced a greater muscleprotein synthesis in supplemented animals, but without any muscle mass gain. However, wedemonstrated here for the first time that muscle protein accretion after immobilization-inducedatrophy could be achieved with whey protein or high protein diets.In conclusion, we demonstrated that immobilization in old rats induced a muscle mass atrophyfollowed by an incomplete recovery, hence contributing to the development of sarcopenia. We alsodemonstrated that this lack of recovery cannot be overcome by a dietary free leucinesupplementation, despite a positive effect on protein metabolism, contrarily to high protein andwhey protein diets.

The work discussed in this thesis deals with the significance of initiator tRNA gene copy number in Escherichia coli. A summary of the relevant literature discussing the process of protein synthesis, initiator tRNA selection and gene redundancy is presented in Chapter 1. Chapter 2 describes the ‘Materials and Methods’ used in the experimental work carried out in this thesis. The next three chapters address the significance of initiator tRNA gene copy number in E. coli at three levels; at the level of the molecule (Chapter 3), at the level of the cell (Chapter 4) and at the level of the population (Chapter 5). At the end of the thesis are appended three publications, which include two papers where I have contributed to work not discussed in this thesis and one review article. A brief summary of chapters 3 to 5 is provided below: (i) Chapter 3: Can E. coli remain viable without the 3 G-C base pairs in initiator tRNA? Initiator tRNAs are distinguished from elongator tRNAs by several features key among which are the three consecutive and near universally conserved G-C base pairs found in the anticodon stem of initiator tRNAs. These bases have long been believed to be essential for the functioning of a living cell, both from in vitro and in vivo analysis. In this study, using targeted mutagenesis and an in vivo genetics based approach, we have shown that the 3 G-C base pairs can be dispensed with in E. coli, and the cell can be sustained on unconventional initiator tRNAs lacking the intact 3 G-C base pairs. Our study uncovered the importance of considering the relative amounts of molecules in a living cell, and their role in maintaining the fidelity of protein synthesis. (ii) Chapter 4: Can elongator tRNAs initiate protein synthesis? There are two types of tRNAs; initiator tRNA, of which there is one representative in the cell, and elongator tRNAs of which there are several representatives. In this study, we have uncovered initiation of protein synthesis by elongator tRNAs by depleting the initiator tRNA content in the cell. This raises the possibility that competition between initiator and elongator tRNAs at the P site of the ribosome occurs routinely in the living cell, and provides a basis for initiation at several 'start' sites in the genome that may not be currently annotated as such. We speculate that such a phenomenon could be exploited by the cell to generate phenotypic diversity without compromising genomic integrity. (iii) Chapter 5: How many initiator tRNA genes does E. coli need? E. coli has four genes that encode initiator tRNA, these are the metZWV genes that occur at 63.5 min in the genome, and the metY gene that occurs at 71.5 min in the genome. Earlier studies indicated that the absence of metY had no apparent impact on cell growth. In view of the importance of initiator tRNA gene copy number in maintaining the rate and fidelity of protein synthesis, we examined the fitness of strains carrying different numbers of initiator tRNA genes by competing them against each other in both rich and limited nutrient environments. Our results indicate a link between caloric restriction and protein synthesis mediated by the initiator tRNA gene copy number.

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.

While protein therapeutics are indispensable in the treatment of a variety of diseases, including cancer, rheumatoid arthritis, and diabetes, key limitations including short half-lives, high immunogenicity, protein instability, and centralized production complicate long-term use and on-demand production. Site-specific polymer conjugation provides a method for mitigating these challenges while minimizing negative impacts on protein activity. However, the location-dependent effects of polymer conjugation are not well understood. Cell-free protein synthesis provides direct access to the synthesis environment and rapid synthesis times, enabling rapid evaluation of multiple conjugation sites on a target protein. Here, work is presented towards developing cell-free protein synthesis as a platform for both design and on-demand production of next generation polymer-protein therapeutics, including (1) eliminating endotoxin contamination in cell-free reagents for simplified therapeutic preparation, (2) improving shelf-stability of cell-free reagents via lyophilization for on-demand production, (3) coupling coarse-grain simulation with high-throughput cell-free protein synthesis to enable rapid identification of optimal polymer conjugation sites, and (4) optimizing cell-free protein synthesis for production of therapeutic proteins

Therapeutic proteins have become a cornerstone of modern medicine since the FDA approval of recombinant human insulin in 1982. Likewise, biosensors transform chemical detection and disease diagnostics by identifying biomarkers, chemical contaminants, and infective agents. Long-standing methods for creating therapeutics and biosensors employ whole cells such as Escherichia coli (E. coli). Alternatively, cell-free protein synthesis (CFPS) employs the enzymatic reactions necessary for protein production and biosensing within a cell, but in an engineered reactor environment facilitating unprecedented access to and control over biochemical machinery, preservation by cryodesiccation for portable deployment, and functionality in cytotoxic applications. This dissertation reports advances in an E. coli CFPS production platform toward creating therapeutic proteins by this means. First, an endotoxin-free CFPS platform is created by optimizing fermentation and cell-extract harvest of an endotoxin-free E. coli strain. Next, liquid cell growth culture media is specially formulated to change chemical composition during cell culture and provide a streamlined method for producing high-yielding, endotoxin-free E. coli CFPS. Then, novel CFPS bioreactor formats are mathematically validated and developed which employ "hydrofoam" and oxygen to increase therapeutic protein production yield. Additionally, advances are reported in CFPS biosensing technology. First, a chimeric fusion protein incorporating the ligand binding domain of the human estrogen receptor is expressed in CFPS to detect estrogenic chemicals in the presence of human blood and urine. Next, the molecular mechanism of this protein construct is elucidated and the assay readout is optimized with mathematical simulations and CFPS. Then, CFPS is metabolically engineered to create a biosensor of L-glutamine, the most abundant amino acid in the body. Finally, this dissertation reports the development of a synergistic platform for potentially treating Acute Lymphoblastic Leukemia wherein CFPS is engineered to both produce the therapeutic protein crisantaspase and assess its activity in the presence of human serum for improved, potentially even personalized treatment of the disease. It is anticipated that the advances reported herein will contribute to the utility of in vitro or cell-free protein synthesis for therapeutic and diagnostic applications.

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder, which causes dysfunction/loss of lower motor neurons and muscle weakness as well as atrophy. While SMA is primarily considered as a motor neuron disease, recent data suggests that survival motor neuron (SMN) deficiency in muscle causes intrinsic defects. We systematically profiled secreted proteins from control and SMN deficient muscle cells with two combined metabolic labeling methods and mass spectrometry. From the screening, we found lower levels of C1q/TNF-related protein 3 (CTRP3) in the SMA muscle secretome and confirmed that CTRP3 levels are indeed reduced in muscle tissues and serum of an SMA mouse model. We identified that CTRP3 regulates neuronal protein synthesis including SMN via mTOR pathway. Furthermore, CTRP3 enhances axonal outgrowth and protein synthesis rate, which are well-known impaired processes in SMA motor neurons. Our data revealed a new molecular mechanism by which muscles regulate the physiology of motor neurons via secreted molecules. Dysregulation of this mechanism contributes to the pathophysiology of SMA.