Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery.

The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

THE LYCHEE TREE PROPAGATION BY LAYERING

The aim of the study was to assess the influence of season and different substrates on rooting of air layers of lychee (Litchi chinensisSonn.) for the production of seedlings to ensure the formation of uniform and productive orchards. Air layers were done in plants of the Bengal cultivar using leafy and healthy woody branches, with about 0.010 to 0.015 m in diameter, in which were performed complete girdling with 0.020 m wide at a distance of 0.30 to 0.40 m below the apex. Then the branches were wrapped in moistened substrate. The layering was made at six times of theyear (January, March, May, July, September and November) and two substrates were used (coconut fiber and sphagnum) in a 6 x 2 factorial design in a randomized block with ten replicates. After 90 days, layers were separated from the matrix plant and evaluated for rooting and callus formation, root number, considering only the primary roots, length, area and volume of the roots, beyond the dry weight of roots and calluses. The months of January, March, September and November showed the best results for all analyzed variables related to rooting. With respect to the substrates, the only difference was in January and March to the root number and dry weight of roots, where the sphagnum showed the best results. The month of July was more conducive to the formation of calluses. The period between September and March was more suitable to the propagation of lychee, when there were rooting percentages above 90%, in addition to the formation of large amount of roots.

Propagation through cutting technique of species ocurring in the Lower São Francisco River in Sergipe State with different concentrations of indolbutiric acid

The objective of this work was to evaluate the feasibility of vegetative propagation through cutting technique of seven tree species with strong occurrence in the riparian forest of the Lower São Francisco River in Sergipe State, under different concentrations of indolbutiric acid at 0, 2500, and 5000 mg.L-1, for potentialization of its use in soil bioengineering technique. It was used a complete random block design with three replicates, and a total of twenty-one treatments. The evaluation period was 120 days for each species, and the data collection was made in intervals of fifteen days, in a total of eight evaluations for each species. The evaluated parameters were: Survival Rate, callus formation, and Root Dry matter Weight. Among the studied species, Schinus terebinthifolius Raddi presented the best results related to cutting technique mainly under the indolbutiric acid concentration of 2500 mg.L-1.

CLONAL PROPAGATION OF NEEM (Azadirachta indica A. Juss.) VIA DIRECT AND INDIRECT IN VITRO REGENERATION

ABSTRACTThe study was conducted with shoot tip explants of neem (Azadirachta indica A. Juss) to identify a viable regenerative process. Shoot tips were obtained from neem embryos cultured alternatingly in DKW medium supplemented with BAP and medium without hormones. Initial shoot development was influenced by cotyledon presence. Basal callus, excised from in vitro stem base, also presented organogenic potential. In some cases, plant lines, obtained from each seed, presented different characteristics. The most common characteristic observed in vitro was callus formation at the stem base. However, the rarest characteristics were stem callus formation and leaf senescence. The regenerated shoot tips were further subculture and rooted on a medium supplemented with IBA so that complete plants could be obtained. The rooted plants were transplanted to a greenhouse and successfully acclimatized. No significant differences in in vivo development were observed between neem plants from callus and from shoot tip propagation.

ROOTING MINI-CUTTINGS OF Paulownia fortunei var. mikado DERIVED FROM CLONAL MINI-GARDEN1

ABSTRACTWe aimed to evaluate the technical efficiency of mini-cuttings technique on vegetative propagation of Paulownia fortunei (Seem.) Hemsl. var. Mikado, as well as the possible existence of anatomical barriers to its rooting. Therefore, plants originated from cuttings formed the mini-stumps and, consequently the clonal mini-garden, which was conducted in semi-hydroponic system. We evaluated the survival of mini-stumps and sprouts production for five successive collects, the percentage of mini-cuttings rooting and their anatomical description. The mini-cuttings were prepared with about 6 to 8 cm in length and two leaves reduced by about 50% in the upper third, being remained an area of, approximately 78 cm2 (10 cm diameter). The mini-cuttings were placed in tubes of 53 cm3, with substrate formed with fine vermiculite and carbonized rice hulls (1:1 v/v) and rooted in acclimatized greenhouse. After 30 days we evaluated the percentage of rooted mini-cuttings, radicial vigor (number and length of roots / mini-cutting), callus formation, emission of new shoots and maintenance of the original leaves. The mini-stumps showed 100% survival after five collects and an average production of 76-114 mini-cuttings/m2/month and rooting ranged from 70 to 90%. Mini-cuttings technique is efficient in to propagate adult propagules of the species and there are not anatomical barriers preventing roots emission.

Alloxan-induced diabetes delays repair in a rat model of closed tibial fracture

A closed fracture was performed on the left tibia of 3-month-old Wistar rats weighing 250 to 350 g that were either healthy (N = 24) or made diabetic with alloxan (N = 24) to investigate the effect of alloxan-induced diabetes on the course of bone fracture healing. Histomorphometric analysis of the fracture site was performed at 7, 14, 25, and 35 days. After 7 days, diabetic rats had significantly less cartilage (P = 0.045) and greater fibrous connective (P = 0.006) tissue formation at the fracture site compared to controls. In contrast, marked callus formation was seen in diabetic rats with significant osteogenesis (P = 0.011, P = 0.010, P = 0.010, respectively, for 14, 25, and 35 days) and chondrogenesis (P = 0.028, P = 0.033, P = 0.019) compared to controls. Radiographic analysis revealed a displaced fracture with poor bone fragment alignment and delayed consolidation at these times in the diabetic group. The levels of alkaline phosphatase were significantly higher in diabetic rats at 25 days (P = 0.009). These results suggest that the initial excessive formation of fibrous connective tissue associated with delay in chondrogenesis and osteogenesis may not provide suitable stability of the fractured site, contributing to the inappropriate alignment of fragments and an increase in the volume of callus in later stages of repair. The resulting displaced fracture in diabetic rats requires long periods for remodeling and complete bone consolidation.

The effects of cultural conditions and parasitism on the lipid composition of choanephora cucubitarum (Berk. Rav.) thaxter /|nJohn M. Deven. -- 260 St. Catharines [Ont. : s. n.],

The fatty acid composition of the total, neutral, sterol, free fatty acid and polar-lipid fractions in the mycelium of Choanephora cucurbitarum was determined. The major fatty acids in all lipid fractions were palmitic, oleic, linoleic and y-linolenic acid. Different lipid fractions did not show any particular preference for any individual fatty acid; however, the degree of unsaturation was different in various lipid fractions. Addition of glutamic acid to the malt-yeast extract medium resulted in the biosynthesis of a number of long-chain fatty acids beyond y-linolenic acid. These fatty acids, e.g. C22~1' C24:0 and C26=Q were never observed to be present in the fungus when grown on a malt-yeast extract medium without glutamic acid. Furthermore, thin-layer chromatographic analysis showed a larger and denser spot of diphosphatidyl glycerol from the mycelium grown on the glutamic acid medium than from the control mycelium. Various cultural conditions such as temperature, age, pH, light and carbon:nitrogen ratio in the growth medium used in this study did not alter the qualitative profile of fatty acids normally present in the organism. Neither did these conditions stimulate the production of further long-chain fatty acids (C20 - C26) beyond y-linolenic acid as observed in growth media containing glutamic acid. These cultural conditions influenced the degree of unsaturation, this being due mainly to changes in the concentration of y-linolenic acid. The fatty acid pattern of the lipid fractions though the same qualitatively, differed quantitatively due to the variation in the y-linolenic acid content under different cultural conditions. The degree of unsaturation of various lipid fractions decreased with increases in temperature, light intensity and pH, but within each treatment the same pattern of decreasing degree of unsaturation with increasing age was observed. The cultural conditions, used in this study, are also known to influence the degree and rate of development of the parasite, Piptocephalis virginiana. A direct correlation was observed between the levels of y-linolenic acid in C. cucurbitarum during the early stages of growth (24 h) and the degree of parasitism of P. virginiana. The amount of y-linolenic acid present in the host mycelium was found to be unrelated to either the dry weight of the mycelium or to the total lipid contents. K. virginiana is confined to host species which produce y-linolenic acid in their mycelium. The lipid profile of the host, C. cucurbitarum, did not show a significant qualitative or quantitative change in the lipid profile as a result of infection by the parasite, P. virginiana,e However, an increase in the total lipid was observed in the infected host mycelium. The significance of these results is discussed.

Investigations on the production of long chain fatty acids in Choanephora cucurbitarum (Berk.

The fatty acid composition of the total cellular lipids of Choanephora cucurbitarum incubated for 96 hrs on either glucose-ammonium sulfate or malt-weast extract media was determined. The major fatty acids were palmitic, palmitoleic, stearic and linoleic acids. The saturated fatty acid possessing the longest acyl chain was stearate (C 18:0). The presence of glutamic acid (2.0 x 10-1% or 1.36 x la-2M) in either of the above growth media resulted in increase in percent of 1f-linolenic acid, decrease in percent of linoleic ~iCid and appearance of a new series of fatty acid> C ~8 e.g. C ",,,,'V' C2k:O, C26,O. The addition of glutamic acid had no effect on the lipid yield but slightly decreased the degree of unsaturation. Compounds which duplicated the effect of glutamic acid were acetate, malate, citrate, succinate, 0( -ketoglutarate, prOline, -y -aminobutyric acid and glucose (3%) but not aspartic acid or alanine. ~o correlation was found between glutamic acid pool concentration and the presence in the growth medium of those compounds which stimulate long chain fatty acid production. Four hours of incubation with 27 JJ 1-1 glutamate supported the production of long chain fatty acids. This stimulation is inhibited if 272 .u M isophthalic acid is added with 27 AJ M glutamate. But, long chain fatty acids were detected when 27 JJ M eX -ketoglutarate is also present in the incubation mixture. Five hours of incubation with 100 ,Mg/ml of cycloheximide resulted in over 9CY/o inhibition of cytoplasmic :protein synthesise Glutamate (27 .uM) enhanced the synthesis of long chain fatty acids under these conditions. These findings are discussed in an attempt to provide a plausible explanation COmmon to compounds that support the production of long chain fatty acids.

Rôles de la protéine tyrosine phosphatase DEP-1 dans l'angiogenèse, la perméabilité vasculaire et la progression tumorale

L’angiogenèse et l’augmentation de la perméabilité vasculaire sont des éléments clés pour la croissance et la progression tumorale. Par conséquent, de nombreux efforts sont déployés à comprendre les mécanismes moléculaires impliqués dans la formation et le remodelage des vaisseaux sanguins de manière à identifier de nouvelles cibles thérapeutiques potentielles. De cette optique, les travaux de cette thèse se sont concentrés sur la protéine tyrosine phosphatase DEP-1, initialement identifiée comme un régulateur négatif de la prolifération et de la phosphorylation du VEGFR2 lorsque fortement exprimée dans les cellules endothéliales. Toutefois, en utilisant une approche d’ARNi, il a été démontré que via sa capacité à déphosphoryler la tyrosine inhibitrice de Src (Y529), DEP-1 était également un régulateur positif de l’activation de Src dans les cellules endothéliales stimulées au VEGF. Puisque Src joue un rôle central dans la promotion de l’angiogenèse et la perméabilité vasculaire, nous avons en plus démontré que DEP-1 était un promoteur de ces fonctions in vitro et que la tyrosine phosphorylation de sa queue C-terminale, permettant l’interaction et l’activation de Src, était requise. Les travaux de recherche présentés dans cette thèse démontrent dans un premier temps à partir d’une souris Dep1 KO, dont le développement ne présente aucun phénotype apparent, que la perte de l’expression de DEP-1 se traduit en une inhibition de l’activation de Src et de l’un de ses substrats, la VE-Cadherine, en réponse au VEGF chez la souris adulte. Nos résultats démontrent donc, pour la première fois, le rôle primordial de DEP-1 dans l’induction de la perméabilité vasculaire et de la formation de capillaires in vivo. Conséquemment, la croissance tumorale et la formation de métastases aux poumons sont réduites due à une inhibition de leur vascularisation ce qui se traduit par une diminution de la prolifération et une augmentation de l’apoptose des cellules cancéreuses. De façon intéressante, l’expression élevée de DEP-1 dans les vaisseaux sanguins tumoraux de patientes atteintes du cancer du sein corrèle avec une vascularisation accrue de la tumeur. En plus du rôle de DEP-1 dans la réponse angiogénqiue à l’âge adulte, nos travaux ont également démontré le rôle important de DEP-1 lors de la vascularisation de la rétine, un modèle in vivo d’angiogenèse développementale. Dans ce contexte, DEP-1 inhibe la prolifération des cellules endothéliales et limite leur bourgeonnement et la complexification du réseau vasculaire rétinien en permettant l’expression adéquate du Dll4, un régulateur crucial de l’organisation de la vascularisation développementale. Cette expression du Dll4 découlerait de la stabilisation de la β-caténine par l’inactivation de la GSK3β, un régulateur important de la dégradation de la β-caténine, en réponse au VEGF selon la voie de signalisation VEGFR2-Src-PI3K-Akt-GSK3β. Ainsi, ces travaux identifient DEP-1 comme un régulateur important de l’organisation vasculaire rétinienne. Les rôles positifs de DEP-1 dans les cellules endothéliales découlent principalement de sa capacité à lier et activer la kinase Src. En plus de contribuer à la réponse angiogénique, Src est également un oncogène bien caractérisé notamment pour sa contribution au programme invasif des cellules cancéreuses mammaires. Les travaux de cette thèse illustrent que DEP-1 est préférentiellement exprimée dans les cellules cancéreuses mammaires invasives et qu’il régule l’activation de Src, de voies de signalisation invasives et, par le fait même, de l’invasivité de ces cellules in vitro et in vivo. De façon intéressante, ces observations corrèlent avec des données cliniques où l’expression modérée de DEP-1 est associée à un mauvais pronostic de survie et de rechute. Ces résultats démontrent donc, pour la première fois, le rôle positif de DEP-1 dans l’activation de Src au niveau des cellules endothéliales et des cellules cancéreuses mammaires ce qui permet la régulation du bourgeonnement endothélial, de la perméabilité vasculaire, de l’angiogenèse normale et pathologique en plus de l’invasion tumorale.

Analyse du métabolisme du soufre de la bactérie autotrophique acidophile Acidithiobacillus thiooxidans ATCC 19377

Les impacts environnementaux dues à l'extraction minière sont considérables. C'est l'action des microorganismes, en utilisant leur métabolisme du soufre sur les déchets miniers, qui engendre les plus grands défis. Jusqu'à présent, peu de recherches ont été effectués sur les microorganismes environnementaux pour la compréhension globale de l'action du métabolisme du soufre dans une optique de prévention et de rémédiation des impacts environnementaux de l'extraction minière. Dans cette étude, nous avons étudié une bactérie environnementale, Acidithiobacillus thiooxidans, dans le but de comprendre le métabolisme du soufre selon le milieu de culture et le niveau d'acidité du milieu. Nous avons utilisé la transcriptomique à haut débit, RNA-seq, en association avec des techniques de biogéochimie et de microscopie à électrons pour déterminer l'expression des gènes codants les enzymes du métabolisme du soufre. Nous avons trouvé que l'expression des gènes des enzymes du métabolisme du soufre chez ce microorganisme sont dépendantes du milieu, de la phase de croissance et du niveau d'acidité présent dans le milieu. De plus, les analyses biogéochimiques montrent la présence de composés de soufre réduits et d'acide sulfurique dans le milieu. Finalement, une analyse par microscopie électronique révèle que la bactérie emmagasine des réserves de soufre dans son cytoplasme. Ces résultats permettent une meilleure compréhension de son métabolisme et nous rapprochent de la possibilité de développer une technique de prédiction des réactions ayant le potentiel de causer des impacts environnementaux dus à l'extraction minière.

Studies On The Reproduction Of Indian Whiting Sill/160 5'/Ham/1 (Forskal) (Percoidei, Sillaginidae)

From the point of view of rational exploitation and proper management of the fishery resources as well as for the development of intensive aquaculture of fishes through selective breeding, brood stock development, domestication and genetic improvement, a sound knowledge of reproductive biology and physiology of the candidate species is of great importance. In recent times, a wealth of information on maturity, spawning habits, spawning periodicity, spawning season, size at maturity and fecundity of commercially important fishes has been generated. Gametogenesis involves the transformation of Primordial germ cells in the gonads into specialised cells or gametes, namely ova in the female and sperms in male, through a series of complex morphological and cytological events. The formation of male gamete is known as spermatogenesis. In the female, the primary growth phase involving the formation of primary oocyte from oogonia is known as oogenesis, which would be followed by the secondary growth phase, in which considerable increase in the size of the oocyte occurs, due mainly to accumulation of yolk. This process is known as vitellogenesis, which would be followed by final maturation and ovulation of the ova. In the present work, basic aspects of maturation and spawning, salient features of gametogenesis and associated biochemical changes occurring during these processes in an important cultivable fish, Sillago sihama belonging to the family Sillaginidae have been investigated.

Establishment of Shrimp Cell Lines: Perception and Orientation

Development of continuous shrimp cell lines for effective investigation on shrimp viruses remains elusive with an arduous history of over 25 years. Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. Though improved growth and longevity of shrimp cells in vitro could be achieved by using modified growth media this did not make any leap to spontaneous transformation; probably due to the fact that shrimp cells inhibited neoplastic transformations. Oncogenic induction and immortalization are considered as the possible ways, and an exclusive medium for shrimp cell culture and an appropriate mode of transformation are crucial. In this review status of shrimp cell line development and its future orientation are discussed

The synthesis of high coercivity cobalt-in-carbon nanotube hybrid structures and their optical limiting properties

Magnetic heterostructures with carbon nanotubes having multiple functionalities are fascinating materials which can be manipulated by means of an external magnetic field. In this paper we report our investigations on the synthesis and optical limiting properties of pristine cobalt nanotubes and high coercivity cobalt-in-carbon nanotubes (a new nanosystem where carbon nanotubes are filled with cobalt nanotubes). A general mobility assisted growth mechanism for the formation of one-dimensional nanostructures inside nanopores is verified in the case of carbon nanotubes. The open-aperture z-scan technique is employed for the optical limiting measurements in which nanosecond laser pulses at 532 nm have been used for optical excitation. Compared to the benchmark pristine carbon nanotubes these materials show an enhanced nonlinear optical absorption, and the nonlinear optical parameters calculated from the data show that these materials are efficient optical limiters. To the best of our knowledge this is the first report where the optical limiting properties of metal nanotubes are compared to those of carbon nanotubes

Caracterització de metabòlits produïts per soques de "Pseudomonas fluorescens" efectives en el control biològic de fongs fitopatògens

El principal objectiu d'aquest treball ha estat estudiar la producció de metabòlits amb activitat antibiòtica per soques de l'espècie Pseudomonas fluorescens de la col·lecció EPS, i també avaluar la seva potencialitat com a agents de biocontrol. Es va disposar també de diverses soques de P. fluorescens, cedides per altres investigadors, que van utilitzar-se com a referència perquè algunes són actives en control biològic i produeixen metabòlits secundaris d'interès en el biocontrol de malalties de plantes. La present memòria s'estructura en cinc capítols, que són, introducció al control biològic, descripció de l'etapa de selecció de soques i cerca dels metabòlits produïts, estudi de la producció d'HCN per la soca EPS288, estudi de la producció de l'antibiòtic 2,4-diacetilfloroglucinol (DAPG), i finalment, el darrer capítol, on s'ha estudiat la producció de DAPG sobre material vegetal i la capacitat de colonitzar arrels per diverses soques d'interès. En l'etapa de prospecció, va demostrar-se que un 37% del total de les soques de la col·lecció EPS produïen HCN, totes de l'espècie P. fluorescens, i un 90% d'aquestes provenien de les arrels de plantes. Es va confirmar la producció dels metabòlits secundaris 2,4-diacetilfloroglucinol, àcid fenazina-1-carboxílic, i pirrolnitrina, per diverses soques de la col·lecció EPS seleccionades mitjançant tècniques moleculars. Així, de la col·lecció EPS, les soques EPS317 i EPS808 produeixen DAPG, la EPS263 àcid fenazina-1-carboxílic i pirrolnitrina i, EPS894, EPS895, EPS945 produeixen àcid fenazina-1-carboxílic. La producció d'HCN es va estudiar més exhaustivament en la soca EPS288, seleccionada per la seva activitat antifúngica i candidata a agent de biocontrol contra Stemphylium vesicarium, causant de la estemfiliosi de la perera, i contra Penicillium expansum, causant de la podridura blava en conservació de fruita a postcollita. Per aquest estudi, es va dissenyar i validar un sistema per recollir l'HCN a partir de cultius en medi líquid. S'ha demostrat que la temperatura d'incubació, la concentració cel·lular de sembra i la composició del medi de cultiu afectaven a la producció d'HCN. Els medis complexos i la glicina n'afavorien la síntesi i la font de carboni no afectava. La soca EPS288 va produir més HCN que P. fluorescens CHA0, soca de referència productora d'HCN i descrita com a activa en processos de biocontrol de fongs fitopatògens. En l'estudi de la producció de DAPG, les soques de la col·lecció EPS i de referència, es van comparar en diversos medis de cultiu estudiant l'efecte de la complexitat i consistència del medi, i l'addició de ferro o de glucosa. Va demostrar-se que la producció de DAPG depèn principalment de la soca i de les característiques del medi de cultiu. La glucosa estimula la producció, mentre que el ferro pràcticament no afecta, i en general, el medi sòlid i complex estimula la producció de DAPG. Tanmateix, aquests efectes varien en alguna de les soques assajades donant lloc a comportaments singulars. En el seguiment del creixement amb un sistema automàtic es va comprovar que la velocitat específica de creixement i la concentració cel·lular assolida al final del cultiu, estaven condicionades per la composició del medi de cultiu. En les proves d'antagonisme vers fitopatògens que foren seleccionats com a indicadors, va observar-se que tant l'antagonisme in vitro com la inhibició d'infeccions sobre material vegetal estaven parcialment relacionades amb la producció dels metabòlits secundaris estudiats. La promoció del creixement de portaempelts per aquestes soques depenia de la soca i de l'hoste, però no es pogué establir una relació causa-efecte amb el metabòlits produïts. També es va comprovar que algunes de les soques podien sobreviure en ferides de pomes i de peres, on produïren DAPG. Mutants resistents a rifampicina de diverses soques de la col·lecció EPS i de referència es van inocular en llavors de pomera i de tomatera que es van sembrar i incubar en condicions controlades. Es va fer el seguiment de la població bacteriana total i resistent a rifampicina present a les arrels durant 72 dies. Totes les soques van colonitzar les arrels de les plantes, mantenint una elevada població durant 37 dies, cap d'elles va estimular el creixement ni mostrar efectes fitotòxics, no afectant tampoc signicativament a la població bacteriana espontània de les arrels. La soca EPS808, una de les seleccionades pel treball, va aconseguir uns nivells de producció de DAPG, una velocitat de creixement i una supervivència relativa a les arrels similar a altres soques de referència descrites com a bons agents de biocontrol. En conseqüència, se la considera una candidata a agent de biocontrol que hauria de ser objecte de futurs estudis d'eficàcia.

Method of plant tissue culture and regeneration

Plants may be regenerated from stomatal cells or protoplasts of such cells. Prior to regeneration the cells or protoplasts may be genetically transformed by the introduction of hereditary material most preferably by a DNA construct which is free of genes which specify resistance to antibiotics. The regeneration step may include callus formation on a hormone-free medium. The method is particularly suitable for sugar beet.