Intrinsic noise in gene regulatory networks

Cells are intrinsically noisy biochemical reactors: low reactant numbers can lead to significant statistical fluctuations in molecule numbers and reaction rates. Here we use an analytic model to investigate the emergent noise properties of genetic systems. We find for a single gene that noise is essentially determined at the translational level, and that the mean and variance of protein concentration can be independently controlled. The noise strength immediately following single gene induction is almost twice the final steady-state value. We find that fluctuations in the concentrations of a regulatory protein can propagate through a genetic cascade; translational noise control could explain the inefficient translation rates observed for genes encoding such regulatory proteins. For an autoregulatory protein, we demonstrate that negative feedback efficiently decreases system noise. The model can be used to predict the noise characteristics of networks of arbitrary connectivity. The general procedure is further illustrated for an autocatalytic protein and a bistable genetic switch. The analysis of intrinsic noise reveals biological roles of gene network structures and can lead to a deeper understanding of their evolutionary origin.

Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica.

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.

Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.

ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: implications for ATP driven helicase translocation.

To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S). Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized. The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-crosslinked Rep complexes formed with dT16. Therefore, ATP hydrolysis by both PS and P2S complexes are not coupled to DNA dissociation. This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P2S dimer. However, ATP hydrolysis by the doubly ligated P2S2 Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency). These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA. Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding.

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.

Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein.

Type II restriction endonucleases are dimers of two identical subunits that together form one binding site for the double-stranded DNA substrate. Cleavage within the palindromic recognition site occurs in the two strands of the duplex in a concerted manner, due to the action of two catalytic centers, one per subunit. To investigate how the two identical subunits of the restriction endonuclease EcoRV cooperate in binding and cleaving their substrate, heterodimeric versions of EcoRV with different amino acid substitutions in the two subunits were constructed. For this purpose, the ecorV gene was fused to the coding region for the glutathione-binding domain of the glutathione S-transferase and a His6-tag, respectively. Upon cotransformation of Escherichia coli cells with both gene fusions stable homo- and heterodimers of the EcoRV variants are produced, which can be separated and purified to homogeneity by affinity chromatography over Ni-nitrilotriacetic acid and glutathione columns. A steady-state kinetic analysis shows that the activity of a heterodimeric variant with one inactive catalytic center is decreased by 2-fold, demonstrating that the two catalytic centers operate independently from each other. In contrast, heterodimeric variants with a defect in one DNA-binding site have a 30- to 50-fold lower activity, indicating that the two subunits of EcoRV cooperate in the recognition of the palindromic DNA sequence. By combining a subunit with an inactive catalytic center with a subunit with a defect in the DNA-binding site, EcoRV heterodimers were produced that only nick DNA specifically within the EcoRV recognition sequence.

Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes.

Recent evidence indicates that polyunsaturated long-chain fatty acids (PUFAs) prevent lethal ischemia-induced cardiac arrhythmias in animals and probably in humans. To increase understanding of the mechanism(s) of this phenomenon, the effects of PUFAs on Na+ currents were assessed by the whole-cell patch-clamp technique in cultured neonatal rat ventricular myocytes. Extracellular application of the free 5,8,11,14,17-eicosapentaenoic acid (EPA) produced a concentration-dependent suppression of ventricular, voltage-activated Na+ currents (INa). After cardiac myocytes were treated with 5 or 10 microM EPA, the peak INa (elicited by a single-step voltage change with pulses from -80 to -30 mV) was decreased by 51% +/- 8% (P < 0.01; n = 10) and 64% +/- 5% (P < 0.001; n = 21), respectively, within 2 min. Likewise, the same concentrations of 4,7,10,16,19-docosahexaenoic acid produced the same inhibition of INa. By contrast, 5 and 10 microM arachidonic acid (AA) caused less inhibition of INa, but both n - 6 and n - 3 PUFAs inhibited INa significantly. A monounsaturated fatty acid and a saturated fatty acid did not. After washing out EPA, INa returned to the control level. Raising the concentration of EPA to 40 microM completely blocked INa. The IC50 of EPA was 4.8 microM. The inhibition of this Na+ channel was found to be dose and time, but not use dependent. Also, the EPA-induced inhibition of INa was voltage dependent, since 10 microM EPA produced 83% +/- 7% and 29% +/- 5% inhibition of INa elicited by pulses from -80 to -30 mV and from -150 to -30 mV, respectively, in single-step voltage changes. A concentration of 10 microM EPA shifted the steady-state inactivation curve of INa by -19 +/- 3 mV (n = 7; P < 0.01). These effects of PUFAs on INa may be important for their antiarrhythmic effect in vivo.

Artificial regulation of gene expression in Escherichia coli by RNase P.

Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.

Estudo de modelos para o comportamento a altas queimas de varetas combustíveis de reatores a água leve pressurizada

Este trabalho tem como objetivo estudar as modificações introduzidas, ao longo de sucessivas versões, nos modelos empíricos do programa computacional FRAPCON utilizado para a simulação do comportamento sob irradiação de varetas combustíveis de Reatores a Água Leve Pressurizada (Pressurized Water Reactor - PWR) em regime de estado estacionário e sob condições de alta queima. No estudo, foram analisados os modelos empíricos utilizados pelo FRAPCON e que são apresentados em sua documentação oficial. Um estudo bibliográfico foi conduzido sobre os efeitos da alta queima em combustíveis nucleares visando melhorar o entendimento dos modelos utilizados pelo FRAPCON nestas condições. Foram feitas simulações do comportamento sob irradiação de uma vareta combustível típica de um reator PWR utilizando as versões 3.3, 3.4 e 3.5 do FRAPCON. Os resultados apresentados pelas diferentes versões do programa foram comparados entre si de forma a verificar as consequências das mudanças de modelo nos parâmetros de saída do programa. Foi possível observar que as modificações introduzidas trouxeram diferenças significativas nos resultados de parâmetros térmicos e mecânicos da vareta combustível, principalmente quando se evoluiu da versão FRAPCON-3.3 para a versão FRAPCON-3.5. Nessa ultima versão, obteve-se menores temperaturas na vareta combustível, menores tensões e deformações no revestimento, menor espessura da camada de oxido formada no revestimento a altas queimas na vareta combustível.

Carbon assimilation, herbage accumulation, nutritive value, and grazing efficiency of Mulato II brachiariagrass under continuous stocking

Grazed pastures are the backbone of the Brazilian livestock industry and grasses of the genus Brachiaria (syn. Urochloa) are some of most used tropical forages in the country. Although the dependence on the forage resource is high, grazing management is often empirical and based on broad and non-specific guidelines. Mulato II brachiariagrass (Convert HD 364, Dow AgroSciences, São Paulo, Brazil) (B. brizantha × B. ruziziensis × B. decumbens), a new Brachiaria hybrid, was released as an option for a broad range of environmental conditions. There is no scientific information on specific management practices for Mulato II under continuous stocking in Brazil. The objectives of this research were to describe and explain variations in carbon assimilation, herbage accumulation (HA), plant-part accumulation, nutritive value, and grazing efficiency (GE) of Mulato II brachiariagrass as affected by canopy height and growth rate, the latter imposed by N fertilization rate, under continuous stocking. An experiment was carried out in Piracicaba, SP, Brazil, during two summer grazing seasons. The experimental design was a randomized complete block, with a 3 x 2 factorial arrangement, corresponding to three steady-state canopy heights (10, 25 and 40 cm) maintained by mimicked continuous stocking and two growth rates (imposed as 50 and 250 kg N ha-1 yr-1), with three replications. There were no height × N rate interactions for most of the responses studied. The HA of Mulato II increased linearly (8640 to 13400 kg DM ha-1 yr-1), the in vitro digestible organic matter (IVDOM) decreased linearly (652 to 586 g kg-1), and the GE decreased (65 to 44%) as canopy height increased. Thus, although GE and IVDOM were greatest at 10 cm height, HA was 36% less for the 10- than for the 40-cm height. The leaf carbon assimilation was greater for the shortest canopy (10 cm), but canopy assimilation was less than in taller canopies, likely a result of less leaf area index (LAI). The reductions in HA, plant-part accumulation, and LAI, were not associated with other signs of stand deterioration. Leaf was the main plant-part accumulated, at a rate that increased from 70 to 100 kg DM ha-1 d-1 as canopy height increased from 10 to 40 cm. Mulato II was less productive (7940 vs. 13380 kg ha-1 yr-1) and had lesser IVDOM (581 vs. 652 g kg-1) at the lower N rate. The increase in N rate affected plant growth, increasing carbon assimilation, LAI, rates of plant-part accumulation (leaf, stem, and dead), and HA. The results indicate that the increase in the rate of dead material accumulation due to more N applied is a result of overall increase in the accumulation rates of all plant-parts. Taller canopies (25 or 40 cm) are advantageous for herbage accumulation of Mulato II, but nutritive value and GE was greater for 25 cm, suggesting that maintaining ∼25-cm canopy height is optimal for continuously stocked Mulato II.

Real time optimization in chemical process: evaluation of strategies, improvements and industrial application.

The increasing economic competition drives the industry to implement tools that improve their processes efficiencies. The process automation is one of these tools, and the Real Time Optimization (RTO) is an automation methodology that considers economic aspects to update the process control in accordance with market prices and disturbances. Basically, RTO uses a steady-state phenomenological model to predict the process behavior, and then, optimizes an economic objective function subject to this model. Although largely implemented in industry, there is not a general agreement about the benefits of implementing RTO due to some limitations discussed in the present work: structural plant/model mismatch, identifiability issues and low frequency of set points update. Some alternative RTO approaches have been proposed in literature to handle the problem of structural plant/model mismatch. However, there is not a sensible comparison evaluating the scope and limitations of these RTO approaches under different aspects. For this reason, the classical two-step method is compared to more recently derivative-based methods (Modifier Adaptation, Integrated System Optimization and Parameter estimation, and Sufficient Conditions of Feasibility and Optimality) using a Monte Carlo methodology. The results of this comparison show that the classical RTO method is consistent, providing a model flexible enough to represent the process topology, a parameter estimation method appropriate to handle measurement noise characteristics and a method to improve the sample information quality. At each iteration, the RTO methodology updates some key parameter of the model, where it is possible to observe identifiability issues caused by lack of measurements and measurement noise, resulting in bad prediction ability. Therefore, four different parameter estimation approaches (Rotational Discrimination, Automatic Selection and Parameter estimation, Reparametrization via Differential Geometry and classical nonlinear Least Square) are evaluated with respect to their prediction accuracy, robustness and speed. The results show that the Rotational Discrimination method is the most suitable to be implemented in a RTO framework, since it requires less a priori information, it is simple to be implemented and avoid the overfitting caused by the Least Square method. The third RTO drawback discussed in the present thesis is the low frequency of set points update, this problem increases the period in which the process operates at suboptimum conditions. An alternative to handle this problem is proposed in this thesis, by integrating the classic RTO and Self-Optimizing control (SOC) using a new Model Predictive Control strategy. The new approach demonstrates that it is possible to reduce the problem of low frequency of set points updates, improving the economic performance. Finally, the practical aspects of the RTO implementation are carried out in an industrial case study, a Vapor Recompression Distillation (VRD) process located in Paulínea refinery from Petrobras. The conclusions of this study suggest that the model parameters are successfully estimated by the Rotational Discrimination method; the RTO is able to improve the process profit in about 3%, equivalent to 2 million dollars per year; and the integration of SOC and RTO may be an interesting control alternative for the VRD process.

Photogenerated o-Azaxylylenes: Mechanistic Studies and Synthetic Applications

This research sets out to build upon excited state o-azaxylylene cycloaddition. The mechanism behind the excitation and cycloaddition process of photogenerated o-azaxylylenes was determined experimentally. Time-correlated single-photon counting, steady-state spectroscopy, triplet quenching experiments, and quantum yield studies provided evidence suggesting that excited state intramolecular proton transfer is followed by intersystem crossing and stepwise addition to the tethered unsaturated pendant. In keeping with the principles of diversity oriented synthesis, a modular approach was taken to gain access to a diverse array of N,O,S-Polyheterocycles which were modified postphotochemically via Suzuki coupling to yield fused biaryls. Cycloaddition products, outfitted with halogens in the aromatic ring of the o-azaxylylene, proved to be reactive with a variety of boronic acids resulting in a rapid growth in structural complexity. A novel procedure was developed that utilized multiple o-azaxylylene cores in a photochemical cascade transformation yielding complex scaffolds of unprecedented topology. The photoprecursors were produced in a one-pot two-step sequence from commercially available starting materials, and upon irradiation yield structures containing up to five fused hetrocyclic rings, and showed complete diastereoselectivity.

El impacto de la crisis económica sobre la movilidad internacional de los residentes extranjeros en la provincia de Alicante

Introducción: En este estudio se investigan los efectos de la crisis económica en los flujos migratorios internacionales. Específicamente, se realiza un análisis comparado sobre los comportamientos en la movilidad residencial de los principales grupos de residentes a partir de las Estadísticas de Variaciones Residenciales (EVR) de 2005 a 2010 en Alicante. Método: Se analizan los microdatos de las EVR para comparar las entradas y salidas de inmigrantes procedentes de los países que aglutinan a las personas que se trasladan orientadas por motivos más cercanos a la esfera del ocio que a la del trabajo, con el grupo de nacionalidades que concentra a la mayoría de los inmigrantes laborales que llegaron atraídos por las ofertas de empleo generadas con la expansión del sector inmobiliario. Resultados: Entre los años 2007 y 2009 se redujeron mucho las variaciones residenciales de entrada en la provincia Alicante desde el extranjero, mientras que se incrementaron las variaciones relacionadas con las salidas. Aunque existen diferencias en el balance de entradas y salidas según la zona de la provincia, la nacionalidad y la edad de los migrantes, la crisis parece asociarse con una tendencia a la reducción del número de residentes extranjeros en general. Discusión o Conclusión: Se concluye que la crisis no sólo ha generado un éxodo de los ciudadanos con menos recursos económicos, también ha provocado la salida masiva de aquellos otros inmigrantes cuya presencia en España se asocia con el consumo.

Evidences of the Cerium Oxide-Catalysed DPF Regeneration in a Real Diesel Engine Exhaust

The active phase Ce0.5Pr0.5O2 has been loaded on commercial substrates (SiC DPF and cordierite honeycomb monolith) to perform DPF regeneration experiments in the exhaust of a diesel engine. Also, a powder sample has been prepared to carry out soot combustion experiments at laboratory. Experiments performed in the real diesel exhaust demonstrated the catalytic activity of the Ce–Pr mixed oxide for the combustion of soot, lowering the DPF regeneration temperature with regard to a counterpart catalyst-free DPF. The temperature for active regeneration of the Ce0.5Pr0.5O2-containing DPF when the soot content is low is in the range of 500–550 °C. When the Ce0.5Pr0.5O2-containing DPF is saturated with a high amount of soot, pressure drop and soot load at the filter reach equilibrium at around 360 °C under steady state engine operation due to passive regeneration. The uncoated DPF reached this equilibrium at around 440 °C. Comparing results at real exhaust with those at laboratory allow concluding that the Ce0.5Pr0.5O2-catalysed soot combustion in the real exhaust is not based on the NO2-assisted mechanism but is most likely occurring by the active oxygen-based mechanism.

Análise comparativa dos métodos da ISO 13790 e sua adequabilidade na estimativa das necessidades de energia para aquecimento e arrefecimento e da temperatura do ar interior

Tese de mestrado integrado em Engenharia da Energia e do Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016