A proteína colibistina (CB), uma Rho GEF neuro-especifica, apresenta papel importante na formação e funcionamento das sinapses inibitórias do sistema nervoso central por interagir com a proteína scaffold gefirina e com receptores GABAA e promover o agrupamento e transporte dessas proteínas para a membrana pós-sináptica. Recentemente, nosso grupo de pesquisa identificou interação de CB com um complexo proteico que controla o início da tradução em eucariotos (complexo eIF3), o que sugeriu pela primeira vez que essa proteína pode estar envolvida também na regulação da tradução em células neurais. Ainda, já havia sido descrito que gefirina, parceira funcional de CB, interage com mTOR, uma quinase que desempenha papel fundamental no controle do início da tradução. Contudo, até o momento não havia estudos adicionais investigando o papel de CB neste cenário. Assim sendo, o presente trabalho teve como objetivo investigar o envolvimento da proteína CB no controle do início da síntese proteica mediada pela via de sinalização mTORC1. Foram utilizados dois modelos experimentais: i) um sistema de expressão heteróloga - superexpressão de CB em células HEK293T, e ii) um modelo endógeno de expressão - células neuroprogenitoras derivadas de células-tronco pluripotentes induzidas (iNPCs) provenientes de indivíduos controles e de um paciente com deleção no gene da CB. Por meio de experimentos de coimunoprecipação nós verificamos que CB interage fisicamente com mTOR nos dois modelos experimentais utilizados. Ainda, nossos resultados mostraram que CB parece modular a atividade da via mTORC1, e nas iNPCs derivadas do paciente a ausência de CB leva a um aumento na ativação desta via de sinalização. Em concordância com esses resultados, nós observamos aumento em neo-síntese proteica nas iNPCs provenientes do paciente, o que pode ser um mecanismo patofisiológico contribuindo para as alterações cognitivas e comportamentais observadas no paciente. Embora estudos adicionais sejam necessários para melhor entender os mecanismos moleculares deste controle de início de tradução mediado por CB, nós sugerimos um modelo no qual CB, por interagir fisicamente com mTOR e eIF3, sequestra estas proteínas e impede que mTOR ative seus alvos e desencadeie a formação do complexo de inicio de tradução. Em conclusão, nossos resultados oferecem novas evidências do envolvimento de CB no controle da síntese proteica / Collybistin (CB), a neural specific RhoGEF, plays key roles in inhibitory synapse formation and function that cluster and localize the scaffold protein gephyrin and GABAA receptors to the neural postsynaptic membrane. We have recently reported that CB interacts with a protein complex that controls translation initiation in eukaryotic cells (eIF3 complex), which suggested for the first time that this protein may also act as regulator of protein synthesis in neural cells. Moreover, it has been previously described that gephyrin, the CB functional partner, interacts with mTOR, a kinase that plays a pivotal role in the control of translation initiation. However, until now there were no further studies investigating the role of CB in this scenario. The purpose of this study was to investigate if CB is involved in the control of translational initiation mediated by the mTORC1 signaling pathway. Two experimental models were used: i) a heterologous expression system - overexpression of CB in HEK293T cells, and ii) an endogenous expression model - neural progenitor cells derived from induced pluripotent stem cells (iNPCs) from control individuals and a patient with a deletion of the entire CB gene. We performed coimmunoprecipitation experiments and verified that CB physically interacts with mTOR both in 293T cells and in control iNPCs. In addition, our results show that CB appears to modulate the activity of mTORC1 pathway, and the absence of CB leads to increased mTORC1 signaling activation in patient\' iNPCs. In agreement with these results, we observed increased de novo cap-dependent translation in patient cells, which could be a pathophysiological mechanism contributing to cognitive and behavioral abnormalities observed in the patient. Although further studies are needed to better understand the molecular details of CB-mediated translational control, we suggest a model whereby CB, by physically interacting with mTOR and eIF3, sequesters these proteins, thereby preventing both the ability of mTOR to activate its targets and the formation of the translational initiation complex. In conclusion, our results offer new insights into the role of CB in protein synthesis control

No description available.

Objectives: The discovery of new antibiotic targets is important to stem the increase in antibiotic resistance to most currently used antimicrobials. The bacterial ribosome is a major target for a large number of antibiotics that inhibit different aspects of translation. Most of these antimicrobial agents also inhibit ribosomal subunit formation as a second cellular target. Precise subunit assembly requires the activity of several distinct RNases for proper rRNA processing. The present work shows that the vanadyl ribonucleoside complex (VRC) inhibited RNases in Staphylococcus aureus involved in ribosomal subunit formation without an effect on translation. Methods: Methicillin-susceptible and -resistant strains of S. aureus were examined for the inhibitory effects of VRC on cell viability by colony counting. Protein synthesis rates were measured by isotopic methionine incorporation. Ribosome synthesis was measured by radiolabelled uridine incorporation into ribosomal subunits as displayed on sucrose gradients. Pulse and chase radiolabelling was used to measure subunit synthesis rates. RNA turnover was determined by a gel on a chip assay. Results: The rates of subunit synthesis and the amounts of both subunits were significantly reduced in the presence of the compound. Ribosomal RNA was degraded and cell viability was reduced as a consequence. VRC also stimulated the inhibitory effects of a macrolide and an aminoglycoside antibiotic on ribosome formation. Conclusions: Bacterial ribosomal subunit synthesis was specifically impaired in VRC-treated cells, with the rates and amounts of both subunits reduced. Cell viability was significantly reduced and rRNA turnover was stimulated.

The hippocampus supports the initial formation and recall of episodic memories, as well as the consolidation of short-term into long-term memories. The ability of hippocampal neurons to rapidly change their connection strengths during learning and maintain these changes over long time-scales may provide a mechanism supporting memory. However, little evidence currently exists concerning the long-term stability of information contained in hippocampal neuronal activity, likely due to limitations in recording extracellular activity in vivo from the same neurons across days. In this thesis I employ calcium imaging in freely moving mice to longitudinally track the activity of large ensembles of hippocampal neurons. Using this technology, I explore the proposal that long-term stability of hippocampal information provides a substrate for episodic memory in three different ways. First, I tested the hypothesis that hippocampal activity should remain stable across days in the absence of learning. I found that place cells – hippocampal neurons containing information about a mouse’s position – maintain a coherent map relative to each other across long time-scales but exhibit instability in how they anchor to the external world. Furthermore, I found that coherent maps were frequently used to represent a different environment and incorporated learning via changes in a subset of neurons. Next, I examined how learning a spatial alternation task impacts neuron stability. I found that splitter neurons whose activity patterns reflected an animal’s future or past trajectory emerged relatively slowly when compared to place cells. However, splitter neurons remained more consistently active and relayed more consistent spatial information across days than did place cells, suggesting that the utility of information provided by a neuron influences its long term stability. Last, I investigated how protein synthesis, known to be necessary for long-term maintenance of changes in hippocampal neuron connection strengths and for proper memory consolidation, influences their activity patterns across days. I found that along with blocking memory consolidation, inhibiting protein synthesis induced a profound, long-lasting decrease in neuronal activity up to two days later. These results combined demonstrate the importance of rapid, lasting changes in the hippocampal neuronal code to supporting long-term memory.

To study the effect of NaCl stress on gene expression in alfalfa (Medicago sativa L.), greenhouse and laboratory experiments were conducted with 22 cultivars during germination and post-germination growth. The ability of alfalfa cultivars to germinate at the different NaCl concentrations was not related to their postgermination performance (salt tolerance) under those conditions. Genetic effects were evident for Na and Cl ion uptake and accumulation in alfalfa shoots and roots. The strategies of alfalfa cultivars to cope with NaCl stress includes exclusion of Na from shoots and Cl from roots or from the whole plant depending on cultivar and NaCl level. The reduction in shoot dry weight was not related to water stress or reduction in Ca, Mg, or chlorophyll concentrations. Results of this study indicated that the reduction in K ion uptake and more directly the toxic effects of high Na and Cl in plant tissues contributed to the reduction in shoot dry weight . The possible subpartitioning of Na and Cl ions between different tissues, cell types, cell components, and/or the different changes in protein structure and enzyme activity at the high Na and Cl concentrations, independent of total concentration of ions, might also contribute to differences in salt tolerance (shoot dry weight) among cultivars. The factors determining shoot dry weight in alfalfa grown under the different NaCl treatments of this study were not the same for all cultivars. Sodium chloride stress induced qualitative and quantitative changes in shoot and root proteins of alfalfa. These changes were dependent on cultivar and salt concentration, as well as length of exposure to salt stress.

Both resistance and endurance exercise elicit an increase in muscle protein synthesis during recovery from exercise. Ingestion of amino acids augments the exercise-induced stimulation of muscle protein synthesis following resistance exercise. Our work showed that 8 wk of unilateral resistance training induced muscle hypertrophy only in the exercised limb. Importantly, using this unilateral model we showed that muscle hypertrophy was confined to the exercised leg and occurred without measurable changes in circulating anabolic hormones. We then went on to use the unilateral leg resistance exercise model to study how animal-derived (milk) and plant-derived (soy) proteins impacted acute post-exercise protein turnover. We observed that ingestion of soy or milk protein resulted in a positive net protein balance following resistance exercise. Moreover, milk promoted a greater net protein balance and muscle protein synthesis than soy protein. In the final study, a key finding was that acute endurance and resistance exercise differentially stimulated myofibrillar and mitochondrial protein synthesis and also differentially affected cellular signaling proteins involved in the regulation of the protein synthetic response. Specifically, the acute, untrained state response showed that resistance exercise stimulated myofibrillar and mitochondrial protein synthesis while endurance exercise stimulated mitochondrial protein synthesis. Following resistance training only myofibrillar protein synthesis increased after exercise, while mitochondrial protein synthesis was unchanged. Endurance exercise training did not affect the acute protein synthetic response and so following training mitochondrial protein synthesis was stimulated as it was acutely, prior to training. In conclusion, the studies within this thesis provided novel insights on the impact of intact dietary proteins and differing modes of exercise on the control skeletal muscle protein metabolism. / Thesis / Doctor of Philosophy (PhD)

The formation of synapses in cell cultures of rat cerebellum was examined in the presence of the protein synthesis inhibitor cycloheximide. First, cell survival in the presence of 25 μg/ml cycloheximide was determined by phase contrast microscopy, trypan blue exclusion, total protein and uptake of [3H]gamma-aminobutyric acid (GABA). Neurons with 24 h incubation in cycloheximide appeared normal with little cell death, but by 48 h incubation the first signs of cell death were found. Some viable neurons were still found in cultures incubated continuously in cycloheximide for 72 h. Normally, the number of synapses seen in cerebellar cultures with the electron microscope shows an increase during the first several weeks in culture. However, the number of synapses in cultures treated with cycloheximide decreased, indicating that inhibition of protein synthesis at least partially inhibited synaptogenesis. Cycloheximide also inhibited the maintenance of synapses already formed as seen by the decrease in the number of synapses from the time the cycloheximide was added. To determine the sensitivity of the forming presynaptic element to cycloheximide, the development of apparent presynaptic elements was investigated. In cultures treated with polylysine-coated sepharose beads, neurites grew and formed apparent presynaptic elements with the bead taking the position of the postsynaptic element. Cultures pretreated with cycloheximide for 1 h followed by 24 h incubation with both cycloheximide and coated beads showed a normal number of apparent presynaptic elements. The first decrease in numbers was seen after 12 h preincubation and 12 h incubation with both cycloheximide and coated beads. Even after 72 h continuous incubation some apparent presynaptic elements could be formed although at reduced levels. Results presented here suggest that continuous protein synthesis is not necessary for the formation of the presynaptic element, but that active protein synthesis is required for neurons to form and maintain postsynaptic elements.

Missing page 192. Page 194 was repeated, therefore one was omitted. / <p> The synthesis and degradation of proteins were studied during aging of cultured human fibroblasts. Equations were derived to yield expressions for the rates of protein degradation, export, and synthesis during exponential growth and steady state from the approach to equilibrium method of radioactively labeling protein. Old cells (cells from normal donors at late passage, cells from old donors, or cells from subjects which the accelerated aging phenotypes of Hutchinson-Gilford (progeria) and Werner syndromes) have a reduced growth rate (0.3-1.3%/hour) when cultured at low density compared to young cells (early-passage cells from normal donors) (2.0-2.5%/hour). Prior to the terminal passage in old cultures, this reduction in growth rate is related primarily to an increased rate of protein degradation (0.96-1.3%/hour in old cells compared to less than 0.55%/hour in young cells). Early-passage cells achieve rapid growth in low density cultures by increasing the protein synthetic rate and decreasing the degradation rate. In high density cultures where the net growth rate was close to zero, the rates of degradation and synthesis were similar in young and old cells prior to their terminal passage (1.9-2.5%/hour). In all cases the rate of protein export was small (less than 0.5%/hour) compared to the rate of protein synthesis. </p> Proteins synthesized by young and old cells were analyzed by two-dimensional gel electrophoresis and were found to be essentially identical in molecular weight and isoelectric points. Induction of synthesis of aberrant proteins by histidine starvation in the presence of histidinol did not reveal differences between early- and late-passage cells from young or old normal donors or from subjects with progeria or Wener Syndrome. Furthermore, there was no correlation between in vitro lifespan and the synthesis of aberrant protein. </p> <p> It is concluded that the increased degradation of proteins and the slow net growth of old cells and the reduced lifespan of cells from old normal donors and subjects with progeria or Werner Syndrome are not due to abnormal protein synthesis. This is contrary to the predictions of the error catastrophe theory of aging. </p> <p> The aberrant proteins synthesized during amino acid starvation are believed to result from amino acid substitution. Several observations reported here are consistent with this hypothesis: (i) No turnover of either native or substituted actins synthesized during histidine starvation of cultured human cells was; (ii) Changes in the isoelectric points of native and substituted actins are predicted by analyses based on the presumed changes in their amino acid composition; (iii) Estimates of the protein synthetic error rates during normal protein synthesis can be derived from a computer model of mRNA translation based on the proposed mechanism of mistranslation; these estimates are consistent under a variety of starvation conditions and are close to other estimates obtained independently for the error frequency in mammalian cells. </p> <p> In both young and old cultured human fibroblasts the error frquency at the histidine codon was calculated to be 1.1 ± 0.1 x 10⁻⁴ (mean+S.E.). Three lines of Sv40-transformed human fibroblasts had error frequencies 2-5 fold greater than their untransformed counterparts. Studies with a variety of other human and non-human cell types did not support the conclusion that transformation in general increased in rate of mistranslation. The observation of increased error frequencies in SV40-transformed human cells may be restricted to this viral transformation. </p> <p> The computer simulations of mRNA translation have provided a means of extrapolating error frequencies determined during amino acid starvation to the error frequency during normal protein synthesis. This model is of great interest for its potential use as a method of rapidly quantifying protein synthetic error frequencies in cultured cells. </p> / Thesis / Doctor of Philosophy (PhD)

<p> The experiments described in this thesis were performed to study the: 1. Role of nitrate in the induction of nitrate reductase. 2. Effect of nitrate on the in vivo regulation of nitrate reductase. 3. Role of atrazine on the regulation of nitrate reductase.</p> <p> The nitrate reductase assay system was improved up to 10 fold over that previously used (28). This improvement included the addition of oxalacetic acid and malic dehydrogenase to the system, thereby oxidizing all the NADH at the end of the reaction. NADH, which was a required co-factor in the assay system, interfered with the nitrite colour formation.</p> <p> When the induction kinetics of the enzyme are followed in root tip sections (0 -10 mm) of corn, a 25 minute lag period followed by a period of rapid nitrate reductase synthesis is seen. Neither the lag period nor the rate of increase in enzyme activity is affected by the concentration of the inducer used (1.0, 10.0 or 25.0 mM nitrate), However, with concentrations of nitrate from 0.1 mM to 10.0 mM, there is an increase in the final level of nitrate reductase. Nitrate levels between 10.0 mM to 100.0 mM did not alter this maximally induced level of enzyme.</p> <p> In mature root sections (25-35 mm from the tip), a longer lag time and a slower rate of increase in enzyme activity is seen. The system is not saturated with nitrate concentrations up to 100.0 mM.</p> <p> Cycloheximide, a protein synthesis inhibitor, was added to the roots after the enzyme had been maximally induced. This allowed one to study the in vivo turnover of the enzyme. Nitrate did not alter the in vivo turnover of nitrate reductase in corn roots.</p> <p> Atrazine is a herbicide that has been reported to increase nitrate reductase levels in corn (34). When used in the experiments described here, it was found to be ineffective in altering the nitrate reductase activity of the roots and leaves of corn.</p> / Thesis / Master of Science (MSc)

No description available.