During protein synthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes in a GTP-dependent manner. To enable future studies on the functional and structural requirement of EF-Tu’s function, a Cysteine-free variant of EF-Tu was constructed suitable for subsequent labelling of the protein and use in kinetic studies. Here, the kinetic properties of three Cysteine-less EF-Tu variants are reported, demonstrating that only the variant with the Alanine substitution in position 81 retains wild-type activity with respect to the interaction with guanine nucleotides, aa-tRNA and the ribosome. To explore a possible tRNA independent pathway for the GTPase activation signal, three residues in domain II of EF-Tu (Arginine 279, Glycine 280, Arginine 283) were mutated; the activity of EF-Tu variants were analyzed. Results suggest that these residues are indeed required for efficient ribosome-dependent stimulation of the GTPase activity of EF-Tu. / x, 85 leaves : ill. (some col.) ; 29 cm

We have previously shown that non-specific blockade of the cyclooxygenase (COX) enzymes in skeletal muscle eliminates the normal increase in muscle protein synthesis following resistance exercise. The current study tested the hypothesis that this COX-mediated increase in postexercise muscle protein synthesis is specifically regulated by the COX-2 isoform. Sixteen males (23 ± 1 yr, 177 ± 2 cm, 81.5 ± 3.4 kg) were randomly assigned to one of two groups that received three doses of either a specific COX-2 inhibitor (celecoxib; 200 mg per dose, 600 mg total) or a placebo during the 24 hours following a single bout of resistance exercise with the knee extensors. Skeletal muscle fractional synthesis rate (FSR) was measured at rest and 24 hours postexercise using a primed constant infusion of [2H5]phenylalanine coupled with muscle biopsies of the vastus lateralis. Mixed muscle FSR was increased following exercise to a greater extent (206%, P<0.05) in the COX-2 group (0.052 ± 0.014 %Ih) as compared with the placebo group (0.017 ± 0.007 %Ih). These results suggest that the specific inhibition of the COX-2 isoform in human skeletal muscle causes a compensatory response in muscle protein synthesis. These data also highlight the involvement of the cyclooxygenase pathways in the regulation of muscle protein synthesis following resistance exercise. / School of Physical Education, Sport, and Exercise Science

The purpose of this study was to compare the anabolic response to acute running exercise in two different leg muscles in endurance-trained men using two different stable isotope tracers. 6 male subjects (26±2 yr; V02max 63±2 ml•kg-' •min-') performed a 45 min treadmill run at 77±1 % intensity. Infusions of d3-leucine and d5-phenylalanine were used to measure mixed muscle FSR at rest and 24 hr post-exercise. An additional infusion of 10% amino acid solution was added to the post-exercise infusion to maximize the muscle anabolic response. Muscle biopsies were obtained from the vastus lateralis (VL) and soleus (SOL) at 2 and 6 hr of the infusion for the measurement of isotope incorporation. Additional muscle biopsies were obtained prior to and 4 hr post-exercise for determination of muscle glycogen use. At rest FSR was similar between the VL and SOL using either tracer (p>0.05). At 24 hr post-exercise FSR was elevated in both muscles, independent of the tracer used (p<0.05). Muscle glycogen was decreased to the same extent in both muscles by -31% at 4 hr post-exercise (p<0.05). These data suggest that the VL and SOL muscles are both stimulated similarly during 45 min of level grade running. Additionally, both muscles respond similarly 24 hr post-exercise, independent of the tracer used for the determination of protein synthesis. / School of Physical Education, Sport, and Exercise Science

No description available.

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ?? 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.

Bibliography: leaves 172-185. / xii, 185 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the induction of heat shock protein (HSP) by some of the anti-inflammatory agents and antirheumatic agents used in the management of rheumatoid arthritis. Presents HSP induction in peripheral white blood cells cultured in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1995?

Orientadores: Everardo Magalhães Carneiro, Camila Aparecida Machado de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T15:32:11Z (GMT). No. of bitstreams: 1 CostaJunior_JoseMaria_M.pdf: 1708636 bytes, checksum: 8f21b938177c9819646df6957696b6c4 (MD5) Previous issue date: 2012 / Resumo: Neste trabalho investigamos os efeitos do treinamento físico aeróbio de longa duração, associado ou não com a suplementação de leucina, sobre o metabolismo protéico e glicídico em músculo soleus de camundongos Swiss. Para isso, parte dos camundongos (T) realizou protocolo de 12 semanas de exercício de natação, com 1h de duração, 5 dias/semana, sem sobrecarga, e a outra parte permaneceu sedentária (C). Metade dos animais dos grupos C e T foram suplementados com leucina (1,5%) na água para beber (grupos CL e TL, respectivamente) ao longo do experimento. Os dados foram analisados pela ANOVA Two-Way (variáveis exercício e suplementação com leucina) e o teste post hoc de Newman-Keus foi empregado nos casos de interação das variáveis. Foi adotado um valor de p<0,05 como estatisticamente significativo. Resultados: A área abaixo da curva glicêmica durante o teste de tolerância à glicose foi maior nos grupos suplementados com leucina (CL e TL). Já a sensibilidade à insulina, estimada pelo kITT, foi maior por efeito do exercício. A fosforilação da AS160, etapa distal da cascata de sinalização que leva a captação de glicose no músculo, foi maior no grupo T em relação aos demais tanto na condição basal quanto estimulada com insulina, provavelmente via AMPK, cuja fosforilação foi maior por efeito do exercício em ambas as condições, mas diminuída por efeito da suplementação com leucina após estimulação com insulina. A fosforilação da Akt não foi afetada pelo exercício, mas foi menor no grupo CL em relação aos demais. Apesar do maior peso do músculo soleus por efeito do exercício, a síntese protéica não diferiu entre os grupos, mesmo com a maior fosforilação da mTOR na condição basal no grupo CL, e a redução por efeito do exercício após estimulação com insulina. A degradação protéica no referido músculo, contudo, foi reduzida por efeito do exercício. A expressão gênica de isoformas específicas de E2 e E3 ligases, integrantes da via proteolítica ubiquitina-proteossoma, também foi menor por efeito do exercício. Todos os indicadores de resposta ao treinamento aeróbio foram aumentados por efeito do exercício: tempo até a exaustão em teste de esforço, oconsumo máximo de oxigênio e atividade da enzima citrato sintase. Alguns destes indicadores também sofreram interferência da suplementação. Concluímos que a suplementação com leucina pode prejudicar a homeostase glicêmica e reduzir os efeitos positivos do exercício sobre a sinalização insulínica. O exercício aumentou o peso do músculo soleus ao diminuir a degradação protéica por inibição da via proteolítica ubiquitina-proteossomo, enquanto a síntese protéica não foi afetada por nenhum tratamento (exercício ou suplementação com leucina) / Abstract: We investigated the effects of chronic physical training, associated or not with leucine supplementation, on protein and glucose metabolism in soleus muscle of Swiss mice. Half of the mice (T) performed a 12 weeks protocol of swimming exercise, 1h/day, 5 days/week, bearing no overload, and the other half remained sedentary (C). Additionally, half of the C and T mice were supplemented with leucine (1,5%) into the drinking water (groups CL and TL, respectively) throughout the experiment. Data were analysed by Two-Way ANOVA (variables exercise and leucine supplementation) and the Newman-Keus post hoc test was used in the cases of interaction between the variables. A p<0,05 was considered statistically significant. Results: The area under glucose curve during glucose tolerance test was increased in the leucine supplemented groups (CL and TL). Insulin sensitivity, estimated by kITT, was higher as an effect of exercise. AS160 phosphorylation, a distal step in the signaling pathway which leads to muscle glucose uptake, was increased in the T group compared to the others in both basal and insulin-stimulated condition, probably via AMPK, whose phosphorylation was increased by exercise in both conditions, but it was reduced by an effect of leucine supplementation following insulin stimulation. Akt phosphorylation was not affected by exercise, but it was reduced in the CL group compared to the other groups. Despite the increased weight of soleus muscle caused by an effect of exercise, protein synthesis was similar among the groups, even with the increased mTOR phosphorylation at the basal condition in the CL group, and the reduction caused by exercise following insulin stimulation. Protein degradation in the soleus muscle was reduced by an effect of exercise. Gene expression of specific isoforms of E2 and E3 ligases, members of the ubiquitin-proteosome proteolytic pathway, was also reduced by an effect of exercise. All the indicators of response to aerobic training were increased by exercise: time to exhaustion in the effort test, maximal oxygen uptake and citrate synthase enzyme activity. Some of these indicators were also affected by leucine supplementation. We conclude that leucine supplementation may impair glucose homeostasis and reduce the positive effects of exercise on insulin signaling. Exercise increased soleus muscle weight by reducing protein degradation via an inhibition of the ubiquitin-proteasome proteolytic pathway, whereas protein synthesis was not affected by either of the treatments (exercise or leucine supplementation) / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Translation initiation factor 5A (IF5A) is an essential, highly conserved protein found within all eukaryotic (eIF5A) and archaeal (aIF5A) cells. The IF5A protein is unique in that it contains the amino acid hypusine; a two-step post translational modification of a single, conserved lysine residue. Although hypusination of eIF5A is vital for eukaryotic cell viability, the primary role of the protein and its hypusine side chain remain a mystery. eIF5A, initially identified as a translation initiation factor, is not required for global protein synthesis leading to the prevailing proposal that eIF5A is purely involved in the translation of a select subset of mRNAs. Recently a number of mutational studies have focused on the conserved, hypusine-containing loop region of eIF5A where specific residues have been found to be essential for activity without affecting hypusination. It has been postulated that eIF5A exists as a dimer (40 kDa) under native conditions and that these residues may be at the interface of dimerisation. The aim of this research was therefore to conduct a mutational analysis of the loop region in support of this hypothesis. A functional analysis of the Saccharomyces cerevisiae eIF5A mutant proteins K48D, G50A, H52A and K56A revealed that these substitutions impaired growth to varying degrees in vivo with G50A and K48D mutant proteins displaying the most convincing defects. Gel filtration profiles gave unexpected results determining eIF5A mutant and wild type proteins to have a native molecular weight of 30 to 31 kDa, suggesting that the eIF5A oligomeric state may be transitory and subject to certain conditions. The inconclusive results obtained from using gel filtration studies led to an investigation into the feasibility of producing native, hypusinated peptides for future structural studies using nuclear magnetic resonance. Hypusinated and unhypusinated eIF5A were successfully separated into their domains making this a possibility. Finally, this study proposes a role for eIF5A in eukaryotic IRES-driven translation initiation.

The study of protein synthesis, and the study of gene expression in general, has accelerated in recent years. Following the advent of next-generation RNA sequencing, powerful library preparation paradigms were developed to capture regulatory activity on a genome-wide scale. In particular, ribosome profiling has emerged as a widely-used measurement of translation. In this method, the state of ribosome association across the transcriptome is obtained by isolation and sequencing of the regions of RNA bound by ribosomes, revealing a snapshot of ribosome positions from which gene-specific densities can be calculated. In combination with RNA sequencing for a measurement of baseline transcription in the same samples, ribosome profiling offers a metric of “translation efficiency”, or TE, corresponding to the average ribosome load per given transcript. Ribosome profiling has advanced the study of translation considerably. However, low throughput in the generation of ribosome profiling and RNA sequencing libraries limits the scale of the experiments that can be performed, while issues in the interpretation of aligned ribosome-protected footprints complicate their analysis, especially in systems of complex regulation. The analysis of such regulatory systems would be greatly aided by a high-throughput sequencing method that can capture translational regulation, but current methods of measuring genome-wide translation are inherently limited in scale. This thesis addresses the key issues presented above in separate chapters. Chapter 2 discusses the analysis of elongation and initiation from ribosome profiling and RNA sequencing data in a mouse model of Fragile X Syndrome. In this chapter, several methods of measuring and modeling variability in the distribution of ribosomes along a coding sequence are used alongside analyses of differential RPF and RNA abundances and their ratio, RFApm, which we distinguish from TE to emphasize its dependence on factors other than initiation rate. The chapter summarizes current information regarding the observed effects of FMRP, and proposes a model congruent with these observations and more-recently published studies. Chapters 3 and 4 present approaches to modeling or inferring translational regulatory networks, either by a novel library preparation paradigm or computational inference from publicly-available data. Chapter 3 presents riboPLATE-seq, a high-throughput RNA-seq library construction method based on the existing PLATE-seq method. The method recapitulates significant findings from ribosome profiling and RNA sequencing at a fraction of the per-sample cost, with further advantages in scalability, and could be implemented in a large-scale screen of translational regulators to create a network of their specific targets. Chapter 4 presents an approach to inferring translational regulation from integrative analysis of public ribosome profiling and RNA sequencing data, tailoring the powerful inference engine ARACNe to measure translational interactions. This yields a comprehensive network of translational regulation, assigning target genes to the set of RNA-binding proteins.

While development in flies is well understood, pattem formation and the evolution thereof in arachnids have yet to be clarified. Flies and other metazoans primarily use two families of genes called Hox genes and Pax genes to regulate embryogenesis. Because of the high evolutionary conservation of Hox and Pax proteins, I hypothesize that arachnids also use this system to organize their body pattern. To enable studies of the Westem black widow spider, Latrodectus hesperus, an embryonic eDNA library and a fixation protocol were developed for L. hesperus embryos. The generation of these tools will allow comprehensive analysis of black widow spider development and give insight into whether, and how, spiders use Hox and Pax genes to organize their bodies. Finally, it will provide a more thorough understanding of how different developmental mechanisms have evolved and ultimately how changes in gene expression can lead to a change in overall body plan.