Algorithms and computational tools for metabolic flux analysis

PhD thesis in Biomedical Engineering

Nota preliminar sôbre a absorção e a translocação do radiozinco no cafeeiro [Coffea arabica] cultivado em solução nutritiva

Two water-culture experiments were carried out to study the absorption and the translocation of radiozinc in young coffee plants as influenced by two factors, namely, concentration of heavy metals (iron, manganese, copper and molybdenum) and method of application. Inert zinc was furnished at a uniform rate of 0.05 p.p.m.; the levels of iron supply were 0, 1.0 and 10 p.p.m.; manganese was supplied in three doses 0, 0.5, and 5 p.p.m.; copper - 0, 0.02, and 0.2 p.p.m.; molybdenum - 0, 0.01 and 0.1 p.p.m. When applied to the nutrient solution the activity of the radiozinc was 0.15 microcuries per plant. In the study of the leaf absorption, the radiozinc was supplied at the level of 0.10 microcuries per plant; in this case the material was brushed either on the lower or in the upper surface or both of two pairs of mature leaves. In both experiments the absorption period was 8 weeks. The following conclusions can be drawn: 1. Among the heavy metals herein investigated the iron concentration did not affecc the uptake of the radiozinc; by raising the level of Mn, Cu and Mo ten times, the absorption dropped to 50 per cent and even more whe compared with the control plant; however, when these micronutrients were omitted from the nutrient solution an increase in the uptake of zinc was registered only in the minus - Cu treatment. The effects of high leveds of Mn, Cu and Mo probably indicate an interionic competition for a same site on a common binding substance in the cell surface. 2. The absorption of the radiozinc directly applied to the leaf surface reached levels as high as 8 times that registered when the root uptake took place. Among the three methods of application which have been tried, brushing the lower surface of the leaves proved to be the most effective; this result is easily understood since the stomatal openings of the coffee leaves are preferentially located in the lower surface. In this treatment, about 40 per cent of the activity was absorbed and around 12 per cent were translocated either to the old or to the newer organs. 3. Data herein presented suggest that leaf sprays should be preferred - rather than soil applications - to control zinc deficiency in coffee plants when growing in field conditions.

Sôbre a determinação do nitrogênio, do fósforo e do potássio no mesmo extrato

The determination of total nitrogen, phosphorus, and potassium in plant material can be carried out in a common extract prepared with sulphuric acid and 30 per cent hydrogen peroxide. Nitrogen is estimated by direct nesslerization of a suitable aliquot (1-5 ml of the 50 ml extract made out of 250 mg of dried material); in order to avoid excessive acidity, 10 ml of Nessler's reagent should be employed. An aliquot of 1-5 ml suffices for the colorimetric determination of phosphorus by the molybdenum method; to reduce the phosphomolybdate complex 2 ml of a 2% SnC12 soln are necessary. Potassium is determined by the cobaltinitrite method after elimination of ammonium salts with the aid of aqua-regia.

Tracer studies in the coffee plant (Coffea arabica L.)

Due to the great importance of coffee to the Brazilian economy, a good deal of the work carried out in the "Laboratório de Isótopos", E. E. A. "Luiz de Queiroz", Piracicaba, S. Paulo, Brazil, was dedicated to the study of some problems involving that plant. The first one was designed to verify a few aspects of the control of zinc deficiency which is common in many types of soils in Brazil. An experiment conducted in nutrient solution showed that the leaf absorption of the radiozinc was eight times as high as the root uptake; the lower surface of the leaves is particularly suited for this kind of absorption. Among the heavy metal micronutrients, only iron did not affect the absorption of the radiozinc; manganese, copper, and molybdenum brought about a decrease of fifty per cent in total uptake. In another pot experiment in which two soils typical of the coffee growing regions were used, namely, a sandy soil called "arenito de Bauru" and a heavy one, "terra roxa", only O.l and 0.2 per cent of the activity supplied to the roots was recovered", respectively. This indicates that under field conditions the farmer should not attempt to correct zinc deficiency by applying zinc salts to the soil: leaf sprays should be used wherever necessary. In order to find out the most suitable way to supply phosphatic fertilizers to the coffee plant, under normal farm conditions, an experiment with tagged superphosphate was carried out with the following methods of distribution of this material: (1) topdressed in a circular area around the trees; (2) placed in the bottom of a 15 cm deep furrow made around the plant; (3) placed in a semicircular furrow, as in the previous treatment; (4) sprayed directly to the leaves. It was verified that in the first case, circa 10 per cent of the phosphorus in the leaves came from the superphosphate; for the other treatments, the results ware, respectively: 2.4, 1.7, and 38.0 per cent. It is interesting to mention that the first and the last methods of distribution were those less used by the farmers; now they are being introduced in many coffee plantations. In a previous trial it was demonstrated that urea sprays were an adequate way to correct nitrogen deficiency under field conditions. An experiment was then set up in which urea-C14 was used to study the metabolism of this fertilizer in coffee leaves. In was verified that in a 9 hours period circa 95 per cent of the urea supplied to the leaves had been absorbed. The distribution of the nitrogen of the urea was followed by standard chemical procedures. On the other hand the fate of the carbonic moiety was studied with the aid of the radiochromatographic technique. Thus, the incorporation of C14 in aminoacids, sugars and organic acids was ascertained. Data obtained in this work gave a definite support to the idea that in coffee leaves, as in a few other higher plants, a mechanism similar to the urea cycle of animals does exist.

Estudos sobre a alimentação mineral do cafeeiro: VI Efeitos das deficiências de micronutrientes em Coffea arabica L. var. Mundo Novo cultivado em solução nutritiva

Young coffee plants (Coffea arabica L., var. Mundo Novo) were grown in nutrient solution purified from micronutrients contaminants by the method of MUNNS & JOHNSON (1960). All plants, except those in the control treatment, wer given all macronutrients and all micronutrients except one which was omitted in order to induce its shortage. Symptoms of deficiency were obtained for all known micronutrients but chlorine. Measurements, observations and chemical analysis of leaves allowed the following main conclusions to be drawn. 1. The relative influence of micronutrients in growth-measured by the fresh weight of the entire plant - was as follows: -Fe -Zn -Cu -Mo -Mn complete = -B = -CI. that is: the omission of iron from the nutrient solution caused the severest reduction in growth; lack of B and Cl had no effect. 2. Symptoms of deficiency of B, Fe, Mn, and Zn were found to be in good agreement with those in the literature. Effects of Cu and Mo shortage, however, had not been described so far: In the case of the Cu-deficient plants, the younger leaves were distorted, having an "S" shape, due probably to lack of growth of the veins; they lost their green color and developed rather large, necrotic patches near the margins. When molybdenum was omitted from the nutrient solution yellow spots develop near the margen of subterminal (fully mature) leaves; they became necrotic; there was a characteristic downward curling of the leaf blade along the mid rib so that the opposite edges touched each other underneath. 3. The levels of micronutrients found in normal and deficient leaves are given in Table 4. It is hoped that those values will serve as a basis of judgement of micronutrient contents found in leaves of field grown plants.

Estudos sôbre alimentação mineral do cafeeiro: VIII. Estudo da absorção e da translocação do radiozinco no cafeeiro (Coffea arabica L.)

WATER-CULTURE EXPERIMENTS. Two water-culture experiments were carried out to study the absorption and the translocation of radiozinc in young coffee plants as influenced by two factors, namely, concentration of heavy metals (iron, man ganese, copper and molybdenum) and method of application. Inert zinc was supplied at an uniform rate of 0. 05 p. p. m.; the levels of iron supply were 0, 1.0, and 10.0 p. p.m.; manganese was supplied in three doses 0, 0.5, and 5.0 p. p.m.; copper- 0, 0. 02, and 0. 2 p. p. m.; molybdenum- 0, 0. 01, and 0. 1 p. p. m. When applied to the nutrient solution the activity os the radiozinc (as zinc chloride) was 0. 15 microcuries per plant. In the study of the leaf absorption, Zn65 was supplied at the level of 0. 10 microcuries per plant; in this case the radioative material was brushed either on the lower or on the upper surface or both two pairs of mature leaves. The absorption period was 8 weeks. The radioactivity assay showed the following results: 1 - Among the heavy metals herein investigated the iron concentration did not affect the uptake of the radiozinc; by raising the level of Mn, Cu and Mo ten times, the absorption dropped to 50 per cent and even more when compared with the control plants; when, however, these micronutrients were omitted from the nutrient solution, an increase in the uptake of zinc was registered in the minus Cu treatment only. The effects of high levels of Mn, Cu and Mo probably indicate an interionic competition for a same site on a common binding substance in the cell surface. 2 - The absorption of the radiozinc directly applied to the leaf surface reached levels as high as 8 times that registered when the root uptake took place. Among the three methods of application which have been tried, brushing the lower surface of the leaves proved to be the most effective; this result is easily understood since the stomatal openings of the coffee leaves an preferentially located in the lower surface - in this treatment, about 40 per cent of the activity was absorved and around 12 per cent were translocated either to the old or to the newer organs. Chemical analyses for heavy metals, were carried out only in the plants received Zn65Cl2 in the nutrient solution; the results were as follows; 1 - Control plants had, per 1,000 gm, of dry weight the following amounts in mg.: Zn- 48 in the roots and 29 in the tops; Fe- 165 in the roots and 9 in the tops; Mn- 58 in the roots and 15 in the tops, Cu- 15 in the roots and 1. 2 in the tops; Mo- 2. 8 in the roots and 0. 45 in the tops. 2 - The effect of different levels of micronutrients in the composition of the plants can be summarized as follows: Fe and Zn- when omitted from the nutrient solution, the iron and zinc contents in the roots decreased, no variation being noted in the tops; the higher dosis caused an accumulation in the roots but no apparent effect in the tops; Mn- by omitting this micronutrient a decrease in its content in the roots was noted, where as the concentration in the tops was the same; Mo- no variation in roots and tops contents when molybdenum was omitted; higher dosis of manganese and molybdenum increased the amounts formed both in the roots and in the tops. 3 - The influence of the different concentrations of micronutrients heavy metals on the zinc content of the coffee plants can be described by saying that: Fe and Mo- no marked variation; Mn- no effect when omitted, reduced amount when the high dosis was supplied; Mn- when the plants did not receive manganese the zinc content in roots and tops was the same as in the control plants; a decrease in the zinc content of the total plant occurred when the high dosis was employed; Cu -the situation is similar to that described for manganese. Hence, results showed by the chemical analyses roughly correspond to those of the radioactivity assay; the use of the tracer technique, however, gave best informations along this line. SOIL-POTS EXPERIMENTS. The two types of soils which when selected support the most extensive coffee plantations in the State of São Paulo, Brazil: "arenito de Bauru", a light sandy soil and "terra roxa legitima", a red soil derived from basalt. Besides NPK containing salts, the coffee plants were given two doses of inert zinc (65 and 130 mg ZnCl2 per pot) and radiozinc at a total activity of 10(6) counts/minute. The results of the countings can be summarized as follows: 1 - When plants were grown in "arenito de Bauru" the activity absorbed as per cent of the total activity supplied was not affected by the dosis of inert zinc. The highest value found was around 0. 1 per cent. 2 - For the "terra roxa" plants, the situation is almost the same; there was, however, a slight increase in the absorption of the radiozinc when 130 mgm of ZnClg2 was given: a little above 0. 2 per cent of the activity supplied was absorbed. The results clearly show that the young coffee plants practically did not absorb none of the zinc supplied; two reasons at least could be pointed out to explain such a fact: 1 - Zinc fixation by an exchange with magnesium or by filling holes in the octahedral layer of aluminosilicates, probably kaolinite; 2 - No need for fertilizer zinc in the particular stage of life cycle under which the experiment was set up. The data from chemical analysis are roughly parallel to the above mentioned. When one attempts to compare - by taking data herein reported zinc uptake from nutrient solution, leaf brushing or from fertilizers in the soil, a practical conclusion can be drawn: the control of zinc deficiency in coffee plants should not be done by adding the zinc salts to the soil; in other words: the soil applications used so extensively in other countries seem not to be suitable for our conditions; hence zinc sprays should be used wherever necessary.

Sôbre uma modificação na determinação do Molibdênio em Plantas

The author proposed a new modification on the thiocyanate-stannous chloride method for the determination of molibdenum, when is used a heavier-than-water solvent for extrating the colored molybdenum thiocyanate complex. Carbon tetrachloride - butyl alcohol is the mixture proposed, and the results obtained give a good precision and more sensibility than the other method that use carbon tetrachloride-isoamyl alcohol as extractant.

Absorção de molibdênio pela cana de açúcar variedade Co 419, em função da idade

In this paper the authors describe the results obtained from the determination of molybdenum in sugar cane plant, grown in soils and climate prevailing in Piracicaba, State of São Paulo, Brazil. The molybdenum was determined in samples cut monthly from the 8th to 14th month, from an experiment consisting of 6 plots, 3 fertilized and 3 unfertilized. The fertilized treatment received 40 kg N (ammonium sulfate) 100 kg P2O3, (superphosphate) and 40 kg K2O (potassium chloride) per hectare, just before planting. Molybdenum was determined by thiocyanate-stannous chloride method, using carbon tetrachloride-butyl alcohol misture, for extrating the colored complex. The results obtained show a parallelism in the absorption of molybdenum by the plants of both treatments. The concentration of molybdenum in the stalks have a tendency to decrease, where as it kept more or less constant in leaves, with a exception in the 14° month when it rised probable because of a migration of molybdenum of the stalks to the leaves. The total amount molybdenum taken up was higher with the fertilized plot due its greater mass prodution.

Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

Desenvolupament de la tecnica de deteccio d'HPV en frotis de pell i bulbs pilosos de la cella per a l'aplicació com a marcador de risc en carcinogènesi cutània

Estudi realitzat a partir d’una estada al Department of Pathology de la Vrije Universiteit Medical Center, Holanda, entre agost del 2006 i gener del 2007. Es parteix de la hipòtesi principal que el virus del papil•loma humà (VPH) està implicat com a cofactor en la carcinogènesi del càncer cutani no-melanoma associat a l'exposició solar i a la immunosupressió. L'objectiu principal era la validació d'una tècnica de detecció del VPH en frotis cutanis per a determinar el paper d'aquest en el càncer cutani en pacients trasplantats renals. Es pretenia desenvolupar l'ús dels frotis cutanis per a la detecció del VPH en pell no tumoral i posteriorment establir quines són les mostres més adequades (en quan a localització i tipus d'extracció) per tal de definir el concepte de "portador de VPH". Es recolliren frotis cutanis de zones exposades (front i mà) i no exposades (part interna del braç) al sol, de la zona perilesional així com pèls de cella. Les mostres pertanyen tant a pacients trasplantats renals (immunodeprimits) com a pacients no trasplantats (immunocompetents), de pell normal i de pell cancerosa. Es van emprar diferents tècniques d'extracció de DNA. El DNA del VPH va ser amplificat amb una tècnica de reacció en cadena de la polimerasa (PCR ) específica de tipus cutanis (Beta-Gamma Cutaneous HPV PCR) i es va tipificar amb una hibridació reversa amb sondes específiques (Reverse Line Blotting). Tots els assajos es van fer per triplicat, per tal de poder avaluar la reproduibilitat dels resultats en aquestes mostres. Com a control de la qualitat i la quantitat del DNA les mostres van ser testades per la PCR del gen de la beta-globina. S’ha dectectat el VPH present tant en pell com en pell cancerosa. La tècnica aporta resultats reproduïbes. S’aprecia una bona correlació tant entre els resultats obtinguts dels frotis i el bulbs pilosos, així com de diferents zones raspades d'un mateix pacient.

Purified cofactors and histone H1 mediate transcriptional regulation by CTF/NF-I.

The initiation of RNA polymerase II transcription is controlled by DNA sequence-specific activator proteins, in combination with cofactor polypeptides whose function is poorly understood. Transcriptional cofactors of the CTF-1 activator were purified on the basis of their affinity for the regulatory protein. These purified cofactors were found to be required for CTF-1-regulated transcription, and they counteracted squelching by an excess of activator in in vitro reconstitution experiments. Interestingly, the cofactors possessed an inhibitory activity for basal transcription, which was relieved by the further addition of the activator. Histone H1 also contributes to the regulation of transcription by CTF-1, whereby the activator prevents repression of the basal transcription machinery by the histone. However, histone H1 could not replace the cofactors for CTF-1-regulated transcription, indicating that they possess distinct transcriptional properties. Furthermore, the purified cofactors were found to be required, together with the activator, in order to antagonize the histone-mediated repression of transcription. These results suggest that CTF-1 and its cofactors function by regulating the assembly of the basal transcription machinery onto the promoter when the latter is in competition with DNA-binding inhibitory proteins such as histone H1.

Generation and phenotypic analysis of protein S-deficient mice.

Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros(+/-) heterozygous mice. In the null (-) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros(+/-) mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros(+/-) mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor-induced thromboembolism model. No viable Pros(-/-) mice were obtained through mating of Pros(+/-) parents. Most E17.5 Pros(-/-) embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros(-/-) embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.

A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing.

AbstractBACKGROUND: KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.METHOD: To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.RESULTS: We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.CONCLUSION: Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.

Peroxisome proliferator-activated receptors: insight into multiple cellular functions.

Peroxisome proliferator-activated receptors, PPARs, (NR1C) are nuclear hormone receptors implicated in energy homeostasis. Upon activation, these ligand-inducible transcription factors stimulate gene expression by binding to the promoter of target genes. The different structural domains of PPARs are presented in terms of activation mechanisms, namely ligand binding, phosphorylation, and cofactor interaction. The specificity of ligands, such as fatty acids, eicosanoids, fibrates and thiazolidinediones (TZD), is described for each of the three PPAR isotypes, alpha (NR1C1), beta (NR1C2) and gamma (NR1C3), so as the differential tissue distribution of these isotypes. Finally, general and specific functions of the PPAR isotypes are discussed, namely their implication in the control of inflammatory responses, cell proliferation and differentiation, the roles of PPARalpha in fatty acid catabolism and of PPARgamma in adipogenesis.

Cx36 Is a Target of Beta2/NeuroD1, Which Associates with Prenatal Differentiation of Insulin-producing β Cells.

The insulin-producing β cells of pancreatic islets are coupled by connexin36 (Cx36) channels. To investigate what controls the expression of this connexin, we have investigated its pattern during mouse pancreas development, and the influence of three transcription factors that are critical for β-cell development and differentiation. We show that (1) the Cx36 gene (Gjd2) is activated early in pancreas development and is markedly induced at the time of the surge of the transcription factors that determine β-cell differentiation; (2) the cognate protein is detected about a week later and is selectively expressed by β cells throughout the prenatal development of mouse pancreas; (3) a 2-kbp fragment of the Gjd2 promoter, which contains three E boxes for the binding of the bHLH factor Beta2/NeuroD1, ensures the expression of Cx36 by β cells; and (4) Beta2/NeuroD1 binds to these E boxes and, in the presence of the E47 ubiquitous cofactor, transactivates the Gjd2 promoter. The data identify Cx36 as a novel early marker of β cells and as a target of Beta2/NeuroD1, which is essential for β-cell development and differentiation.