Members of the 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenase (2OG oxygenase) superfamily catalyse a wide range of oxidative reactions in biology. 2OG oxygenases require Fe(II) and atmospheric oxygen for their activity, and couple substrate oxidation with the decarboxylation of 2OG into succinate and carbon dioxide. There are more than sixty known 2OG oxygenases in the human genome; they modify small molecules, nucleic acids and proteins implicated in diverse biological processes. Importantly, the seemingly disparate functions of 2OG oxygenases often converge to regulate gene expression. 2OG oxygenases have been shown to affect epigenetic reprogramming, chromatin remodelling, transcription factor activity and mRNA splicing. Emerging evidence indicates that 2OG oxygenases are also involved in the translational control of gene expression. Oxygenases TYW5, ALKBH8, ALKBH5 and FTO were found to catalyse modifications of tRNA and mRNA. The work in this thesis extends these observations by demonstrating that 2OG oxygenase-catalysed protein hydroxylations also play an important role in protein synthesis. The catalytic activities of two oxygenases belonging to the JmjC-only family, NO66 and JMJD4, are described. NO66 catalyses the histidinyl hydroxylation of 60S ribosomal subunit protein L8. NO66 is part of a conserved group of ribosomal protein hydroxylases that can be traced back to prokaryotes. JMJD4 is a lysyl hydroxylase of eRF1, the eukaryotic release factor responsible for translation termination. The hydroxylation of eRF1 takes place on a conserved NIKS motif important for release factor activity, and promotes effcient translational termination. JMJD4 is further implicated in cell growth and cancer, though the link between its activity and tumourigenesis remains to be determined. These results highlight the potential of 2OG oxygenases as regulators of protein synthesis, and further extend the scope of 2OG oxygenase function. The small molecule inhibition of 2OG oxygenases presents a novel therapeutic possibility targeting translational control in cancer and other diseases.

Master of Science / Department of Animal Sciences and Industry / Barry Bradford / Angiopoietin-like protein 4 (ANGPTL4) is a 55-kDa secreted glycoprotein which is an important factor for regulation of energy and lipid metabolism. Plasma ANGPTL4 has the ability to inhibit lipoprotein lipase (LPL) function by preventing it from catalyzing hydrolysis of lipoprotein triglyceride, which contributes to ANGPTL4’s ability to decrease fat storage. Furthermore, research in mice suggests that gut microbes suppress gastrointestinal ANGPTL4 production, and that decreased plasma ANGPTL4 concentrations promote fat storage. In our previous work, we found that bovine ruminal epithelial cells expressed ANGPTL4 to a greater extent than liver hepatocytes, which are usually considered the predominant source of circulating ANGPTL4. Therefore, 3 studies were conducted to evaluate the hypothesis that ruminal expression and plasma concentrations of ANGPTL4 could be influenced by alterations in ruminal fermentation. The first and second studies utilized dietary treatments intended to alter ruminal fermentability. Diets with relatively low or high forage content were fed to 12 non-lactating dairy cows (study 1) and 8 beef cattle (study 2) prior to collection of ruminal fluid and ruminal tissue samples. The results suggested that increasing the dietary concentrate decreased ruminal expression of ANGPTL4 but did not significantly alter plasma ANGPTL4 concentrations. The third study was designed to assess whether effects of diet fermentability on ruminal ANGPTL4 synthesis are mediated by changes in volatile fatty acid concentrations. In this study, 6 lactating cows were infused with acetate, propionate, or butyrate in a Latin square design. Results showed that ANGPTL4 expression was not significantly altered by volatile fatty acid infusions, but that expression was correlated with ruminal pH and total volatile fatty acid concentration. The mechanism by which ANGPTL4 regulates intracellular lipid metabolism also remains unclear. Although ANGPTL4 is known to associate with β1 and β5 integrins, it is unknown if these extracellular matrix proteins mediate the effects of ANGPTL4 in adipose tissue or muscle. The objective of the last experiment was to detect the ANGPTL4 receptor or mediator in muscle satellite cells and adipose tissue. We successfully expressed recombinant bovine ANGPTL4 with a cell free glycoprotein synthesis system. However, we did not detect the ANGPTL4–receptor complex following exposure to bovine adipose tissue explants or cultured bovine muscle satellite cells. Overall, these research projects determined that the ruminal ANGPTL4 production is influenced by fermentation, but it remains unclear whether fermentation products or direct host/microbe interactions are responsible. Finally, it will be important to identify the ANGPTL4 receptor or mediator to better understand the downstream regulatory mechanisms involved in mediating the metabolic effects of ANGPTL4.

Pacientes com diabetes mellitus apresentam perda de massa e força muscular esquelética. O treinamento de força é prescrito aos pacientes diabéticos como parte do tratamento, pois melhora o controle glicêmico além de promover aumento da massa muscular. Foram investigados os mecanismos envolvidos na hipertrofia muscular induzida por sobrecarga mecânica durante o estabelecimento do estado diabético do tipo I induzido por estreptozotocina em ratos. Os experimentos foram realizados nos músculos com predominância de fibras oxidativas (sóleo) ou glicolíticas (extensor digital longo - EDL). Avaliou-se a modulação da via de síntese de proteínas PI3K-AKT-mTOR sete dias após indução de hipertrofia dos músculos sóleo por tenotomia do músculo gastrocnêmio e do EDL pela ablação do músculo tibial. Determinou-se também a expressão de mRNA de outras vias de sinalização que controlam a hipertrofia muscular: mecanotransdução (FAK), Wnt/β-catenina e miostatina e folistatina. Os músculos sóleo e EDL quando submetidos à sobrecarga funcional sofreram hipertrofia semelhante em animais controles e diabéticos. O aumento das forças tetânica e isotônica, absolutas e específicas, ocorreu na mesma magnitude que a hipertrofia muscular. A hipertrofia do músculo EDL nos animais diabéticos envolveu principalmente a via PI3K-AKT-mTOR além da redução no conteúdo de AMPK e diminuição da expressão de miostatina. No músculo sóleo, a hipertrofia foi mais pronunciada nos animais diabéticos por ativação mais intensa da via pelas proteínas rpS6 e aumento na expressão de mRNA de IGF-1, MGF e folistatina além de diminuição nos conteúdos de miostatina, MuRF-1 e atrogina-1. As modificações relacionadas à sinalização permitiram ao músculo sóleo alcançar valores de força e massa muscular similares ao grupo controle. / Patients with diabetes mellitus have reduction in skeletal muscle mass and strength. Strength training is prescribed to diabetic patients as part of the treatment since it improves glycemic control and promotes an increase of skeletal muscle mass. The mechanisms involved in the overload-induced muscle hypertrophy during the establishment of the type I diabetic state, induced by streptozotocin, were investigated in rats. The experiments were performed in muscles with predominance of oxidative (soleus) or glycolytic (EDL) fibers. PI3K/AKT/mTOR protein synthesis pathway was evaluated seven days after the overload-induced hypertrophy of the soleus muscle by tenotomy of the gastrocnemius muscle and of the EDL muscle by tibialis anterior muscle ablation. The mRNA expression of genes associated with different signaling pathways that control muscle hypertrophy was also evaluated: mechanotransduction (FAK) signaling, Wnt/β-catenin, myostatin and follistatin. The soleus and EDL muscles when submitted to overload had similar hypertrophic responses in control and diabetic animals. The increase of twitch and tetanic, absolute and specific, forces had the same magnitude as the muscle hypertrophic response. Hypertrophy of the EDL muscle from diabetic animals mostly involved mechanical loading-stimulated PI3K/AKT/mTOR pathway in addition to the reduced activation of AMPK and decrease of myostatin expression. Hypertrophy was more pronounced in the soleus muscle of diabetic animals due to a more potent activation of rpS6 and increased mRNA expression of IGF-1, MGF and follistatin, and decrease of the myostatin, MuRF-1 and atrogin-1 contents. The activated signaling pathways enabled the soleus muscle mass and force of the diabetic rats to reach the values of the control group.

O presente projeto analisou a expressão gênica de Amblyomin -X, uma proteína com potencial terapêutico expressa em Escherichia coli BL21(DE3), por intermédio de um vetor de expressão plasmideal induzido por IPTG ( Isopropyl α-D-1-thiogalactopyranoside). A quantificação da expressão gênica foi realizada utilizando a técnica de RT-qPCR absoluta. A molécula Amblyomin-X corresponde a um inibidor de serino protease do tipo Kunitz, que apresenta atividade antitumoral e anticoagulante. Devido a sua importância terapêutica, fez-se necessário implementar um protocolo de escalonamento de produção, utilizando biorreatores. A informação sobre expressão gênica pode ajudar a interpretar o funcionamento do microrganismo durante os cultivos em frascos agitados e biorreatores, auxiliando na obtenção de protocolos de cultivo. A partir do momento da indução do gene Amblyomin-X, a bactéria Escherichia coli recombinante direciona grande parte de seu metabolismo para a expressão e produção dessa proteína, que é expressa em forma de corpos de inclusão insolúveis. As proteínas em forma de corpos de inclusão insolúveis permitem uma fácil extração do produto, porém, dificulta na quantificação por técnicas cromatográficas, por esse motivo, buscou-se também uma correlação entre a expressão gênica e a síntese protéica de Amblyomin-X, com o objetivo de validar a quantificação por RT-qPCR como uma forma indireta de quantificação desse tipo de produto protéico. Poucos estudos abordam a correlação existente entre a expressão gênica e a síntese protéica de um gene e sua proteína, e essa comparação poderia, também, revelar aspectos importantes entre a transcrição e a tradução de proteínas heterólogas em microrganismo. Foram realizados experimentos em frascos agitados em duas diferentes temperaturas indução (37°C e 30°C), com a concentração do indutor IPTG de 1 mM e concentração da biomassa (X) de 0,2 g/L. Para os experimentos em biorreatores utilizou-se o ponto central do planejamento experimental com concentração de IPTG 1mM e mais duas condições com concentração de IPTG 0,1 mM e 2mM. Foi encontrada uma grande similaridade entre os gráficos das análises cinéticas referentes a expressão gênica e a velocidade específica de formação do produto (qP). Não foi possível Identificar uma semelhança entre o perfil referente a análise cinética da expressão gênica comparada com a síntese protéica. Esses resultados sugerem que a expressão gênica apresenta uma cinética semelhante a velocidade específica de formação do produto e não necessariamente a concentração de proteína. Não foram encontradas correlações significativas nas análises de cada momento após a indução, exceto pelo ponto t4 do biorreator 14, quando foram correlacionadas a expressão gênica e a velocidade de formação do produto (r=0,998 e valor P=0,03). / The present work analysed the gene expression of Amblyomin-X, a potential therapeutic protein expressed in a recombinant Escherichia coli with a plasmid vector inducible by IPTG ( Isopropyl β-D-1- thiogalactopyranoside). The gene expression was quantified by absolute RT-qPCR method. Amblyomin-X is a Kunitz-type serine protease inhibitor which demonstrates antitumor and anticoagulant activity. Due to Amblyomin therapeutic importance, it was necessary to implement a production protocol using bioreactors. The information on gene expression can helps to understand the microorganism behavior during the cultures in shaken flasks and bioreactors. From the moment of the induction of the gene Amblyomin-X, the recombinant Escherichia coli directs its metabolism for expression and production of this protein. The Amblyomin-X protein is expressed as insoluble inclusion bodies. Insoluble inclusion bodies proteins can be easyly extracted from the cell, however it difficults its quantification by chromatographic techniques. Because of that, besides analysis of the gene expression, by RT-qPCR method, we tried to correlate the gene expression with their protein levels concentration, whose objective was implement the RT-qPCR as an indirect method to quantify these type of protein product. Few studies correlate the gene expression in their protein, and this comparison can contribute to reveal important aspects between transcription and translation of heterologous proteins in Escherichia coli, for the achievement of scale up process in biorreactors. Therefore, the quantification by RT-qPCR, represents a most secure way to understand the expression of heterologous proteins in recombinant systems. Experiments were performed in shaken flasks at two different induction temperatures (37 °C and 30 °C), 0,2 g/L of biomass (X) at the induction moment with IPTG 1mM. Biorreactors are performed with three different IPTG concentrations (0,1 mM, 1 mM and 2 mM). A great similarity was found between the graphs of the kinetic analyzes concerning gene expression and the specific rate of product formation (qP). It was not possible to identify a similarity between the profile concerning kinetic analysis of gene expression compared to protein synthesis. These results suggest that gene expression exhibits similar kinetics to the specific rate of product formation and not necessarily to the protein concentration. were found no significant correlations in the analyzes of each moment after induction, except for point t4 of the bioreactor 14, when the gene expression and the rate of the product formation were positively correlated (r = 0.998 and P value = 0.03).

Foi isolada uma sub-fração de microsomas, constituída por membranas do retículo endoplasmático, através do tratamento do sobrenadante pós-mitocondrial por detergente. Em testes de microscopia eletrônica e ultracentrifugação analítica, esta preparação de membranas apresentou-se livre de contaminação por outras organelas celulares. Quando incubada em condições adequadas, a fração de membranas incorpora vários aminoácidos em um produto insolúvel em TCA a quente. O tratamento do material incorporado com enzimas proteolíticas acarreta a perda de cerca de 60% da radioatividade derivada de aminoácidos marcados. Não há liberação de radioatividade por tratamento com RNase, DNase, lecitinase ou α-amilase. A remoção de aminoácidos terminais não implica em diminuição considerável de radioatividade. As melhores condições de pH e concentração de Mg para o processo são próximas às fisiológicas. O requerimento de ATP e enzima pH 5 no meio de incubação não é absoluto, mas sua adição estimula o processo. Há indicações de que a independência de fornecimento externo de GTP para o processo resulta de um conteúdo endógeno da partícula. o processo de incorporação é inibido em parte por RNase, NaF, puromicina e anisomicina. / Not available

Foi isolada uma sub-fração de microsomas, constituída por membranas do retículo endoplasmático, através do tratamento do sobrenadante pós-mitocondrial por detergente. Em testes de microscopia eletrônica e ultracentrifugação analítica, esta preparação de membranas apresentou-se livre de contaminação por outras organelas celulares. Quando incubada em condições adequadas, a fração de membranas incorpora vários aminoácidos em um produto insolúvel em TCA a quente. O tratamento do material incorporado com enzimas proteolíticas acarreta a perda de cerca de 60% da radioatividade derivada de aminoácidos marcados. Não há liberação de radioatividade por tratamento com RNase, DNase, lecitinase ou α-amilase. A remoção de aminoácidos terminais não implica em diminuição considerável de radioatividade. As melhores condições de pH e concentração de Mg para o processo são próximas às fisiológicas. O requerimento de ATP e enzima pH 5 no meio de incubação não é absoluto, mas sua adição estimula o processo. Há indicações de que a independência de fornecimento externo de GTP para o processo resulta de um conteúdo endógeno da partícula. o processo de incorporação é inibido em parte por RNase, NaF, puromicina e anisomicina. / Not available

Antibacterial agents specific for the 50S ribosomal subunit not only inhibit translation but also prevent assembly of that subunit. I examined the 30S ribosomal subunit in growing Escherichia coli cells to see if antibiotics specific for that subunit also had a second inhibitory effect. I used the aminoglycoside antibiotics paromomycin and neomycin, which bind specifically to the 30S ribosomal subunit. Both antibiotics inhibited the growth rate, viable cell number, and protein synthesis. I used a 3H-uridine pulse and chase assay to examine the kinetics of ribosome subunit assembly in the presence and absence of each antibiotic. Analysis revealed a concentration dependent inhibition of 30S subunit formation in the presence of each antibiotic. Sucrose gradient profiles of cell lysates showed the accumulation of an intermediate 21S translational particle. Taken together this data gives the first demonstration that 30S ribosomal subunit inhibitors can also prevent assembly of the small subunit.

Barley, cornstarch, crested wheatgrass, glucose, molasses and solka floc were fermented with rumen fluid obtained from sheep that were adapted or unadapted to feed grade biuret (Kedlor) in their diet. Each rumen fluid and substrate combination was subjected to three nitrogen treatments: (1) control, (2) nitrogen added as feed grade biuret and (3) reagent grade biuret in a factoral arrangement of treatments. In vitro fermentations were terminated at 0, 12, 24, and 36 hours and the protein nitrogen insoluble in trichloroacetic acid (TCA) was determined. Molasses and cornstarch fermentation residues increased while barley, crested wheatgrass, glucose and solka floc decreased in TCA insoluble protein nitrogen. The average of all treatments decreased in TCA insoluble protein nitrogen during the first 12 hours and increased during the remaining 24 hours. Unadapted rumen fluid with simple carbohydrates from glucose and molasses and adapted rumen fluid with complex carbohydrates from barley, crested wheatgrass, cornstarch and solka floc gave the largest gains in TCA insoluble protein nitrogen. No significant differences occurred due to the addition of non-protein nitrogen.

This thesis examines the signaling pathways involved in the regulation of milk protein synthesis in the lactating mammary gland and their control. The protein synthetic machinery can be regulated during the transcription, translation and degradation stages of mRNA processing. Translation control in eukaryotes involves changes in the activity or other functional properties of the translation factors. These include proteins involved in initiation, peptide-chain elongation and termination of mRNA processing. Changes in the nutritional, physiological and hormonal status of the body are sensed by receptors that signal to a central protein, known as mammalian target of rapamycin (mTOR). The mTOR signaling pathway then activates or inhibits the activity of translation factors and kinases involved in the initiation and elongation stage of translation. A major objective of this thesis was to elucidate which genes and pathways are involved in the regulation of milk protein synthesis in the mammary gland and the mechanism(s) that regulate their action. The results presented here show that changes in milk protein production occurring during lactation in response to external stimuli are potentially regulated at the level of translation or subsequent processing rather than by transcriptional regulation (mRNA abundance). The results also show that in response to growth hormone (GH) treatment, which increased the yield of milk protein, the phosphorylation status of the ribosomal protein S6 (S6) is increased as well as the protein abundance of eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E (eIF4E). These results suggest an important relationship between milk protein yield and changes in the initiation and elongation stages of translation. Another major finding was the elucidation that mTOR is involved in the signaling pathways activated by GH and that this effect involves signaling through the PI-3 kinase pathway. In these experiments, increased protein synthesis was potentially achieved with the use of GH. Thus, this study suggests the mTOR signaling pathway is a key mediator of the GH effects in protein synthesis stimulation. Finally, the requirement for a functional mTOR signaling (TOS) motif in the eukaryotic initiation factor 4E binding protein (4E-BP1) was identified. This finding could help the identification of other proteins that may be controlled by mTOR and consequently are regulators of mRNA translation. In summary, this thesis unveils key signaling pathways involved in the regulation of milk protein synthesis and provides further insight into the control of the mTOR signaling pathway. These findings open new frontiers for the manipulation of milk composition.

Exploring and understanding how the smallest scale features of a cell affect biochemical reactions has always been a challenge. Nanoscale fabrication advancements have allowed scientists to create small volume reaction containers that resemble the physical scale of cell membranes. Engineers seek to use biological design principles to manipulate information and import new functionality to such synthetic devices, which in turn, play a crucial role in allowing them to explore the effects of physical transport and extreme conditions of temperature and pH on reaction systems. Engineered reaction containers can be physically and chemically defined to control the flux of molecules of different sizes and charge. The design and testing of such a container is described here. It has a volume of 19 pL and has defined slits of 10-200 nm. The device successfully contains DNA and protein molecules and has been used to conduct and analyze enzyme reactions under different substrate concentrations and a continuous cell-free protein synthesis. The effect of DNA concentration and slit size on protein yield is also discussed. Glucose oxidase and horseradish peroxidase were loaded in the small volume container and fed with a solution containing glucose and Amplex Red™ to produce Resorufin. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Enzyme kinetics were characterized and compared with conventional scale results. Continuous cell free protein synthesis in arrays of nanoporous, picoliter volume containers has also been achieved. A multiscale fabrication process allows for the monolithic integration of the containers and an addressable microfluidic network. Synthesis of enhanced green fluorescent protein (eGFP) in the nanoporous containers continues beyond 24 hours and yields more than twice the amount of protein, on a per volume basis, than conventional scale batch reactions. These picoliter, nanoporous containers provide new ways for quick determination of enzyme kinetics and continuous protein synthesis in microfluidic systems. They can be used in a wide variety of applications such as drug discovery, clinical diagnostics and high-throughput screening.