Response of soybean genotypes to the expression of green seed under temperature and water stresses

Soybeans grown under water stress associated with high temperatures during seed maturation and pre-harvest may produce green seed (GS) with expressive reduction in seed quality. The objectives of this study were to evaluate the response of different soybean cultivars grown under these stressful conditions regarding their susceptibility to GS production and to determine the chlorophyll retention levels and the chlorophyllase activity in the seeds. Seeds of four soybean cultivars [BRS 133, CD 206, MG/BR 46 (Conquista) and BRSMG 251 (Robusta)] were grown under greenhouse conditions until R5.5. At R6, the plants were transferred to phytotrons under temperature stress (from 28ºC to 36ºC) and with water stresses of 10% gravimetric moisture, no water and normal supply. Seeds were harvested at R9 when the percentage of GS and weight of 100 seeds were determined. The contents of a, b and total chlorophylls and the chlorophyllase activity were also determined. The expression of GS production under these conditions varied among cultivars: Conquista and Robusta were considered more susceptible to the production of GS compared to 'BRS 133' and 'CD 206'. These cultivars produced lower GS levels, lower chlorophyll retention and higher chlorophyllase activity compared to Robusta and Conquista. Soybean plants submitted to water and temperature stresses produced high levels of GS, which were small, light and had high chlorophyll contents and low chlorophyllase activity. The contents of a, b and total chlorophylls in GS were inversely proportional to the chlorophyllase activity.

Physiological and enzymatic alterations in papaya seed during storage

This study was done to evaluate the physiological and enzymatic alterations in papaya (Carica papaya L.) seeds during storage period. Seeds were extracted from mature fruits of Formosa group papaya hybrid Tainung 01. The sarcotesta was removed by rubbing the seeds on a wire screen under running water and then dried to the moisture content (MC) of 5, 8 or 11% The seeds were packed in multilayer paper bags, polyethylene bags, aluminum foil pouch and metallic canisters and stored for 15 months under laboratory conditions. Seeds were evaluated, at three month interval, for MC, germination, and the activity of acid phosphotase (AP) and malate dehyrogenase (MDH) was evaluated with the use of amide gel (12%) electrophoresis. The fresh seeds had post-harvest dormancy, which was broken after six month storage. Independent of the package type, the seeds could be stored for 12 months with 8 or 11% MC under ambient conditions. There was no association between seed deterioration and alterations in AP and MDH activity.

Physiological maturity of fruits and seeds of Poincianella pyramidalis (Tul.) L.P. Queiroz

The Leguminosae family is one of the most representative botanical families of the Caatinga, with 80 endemic species, highlighting the catingueira (Poincianella pyramidalis). The objective of this research work was to study the maturation process of P. pyramidalis seeds based on the physiological maturity. Five harvest of fruits and seeds were carried out, with 15 days interval each, in a period from July to September 2010. The harvests began 75 days after anthesis (d.a.a.) and lasted until 135 d.a.a. Fruits and seeds were subjected to the following assessments: size, moisture content, and dry mass of fruits and seeds; and germination and vigor of seeds (first count of germination, germination speed index, length and dry mass of seedling). Under the environmental conditions of municipality of Soledade, State of Paraiba, Northeast Brazil, the point of physiological maturity of P. pyramidalis seeds occurs at 125 d.a.a., when the maximum accumulation of dry mass is 1.993 g and moisture content is 21%. The ideal point of harvest is between 130 d.a.a and 135 d.a.a., before natural dehiscence, when the moisture content of seeds is between 13.0% and 5.0%.

Survey, survival and control of Alternaria alternata in wheat seeds

The fungus Alternaria alternata was quantified in 75 wheat seed samples collected from three different regions of southern Brazil for Cropping and Use Value (CUV) I, II and III. Fungal presence was evaluated in two hundred disinfested seeds per sample before sowing in a potato-dextrose-agar medium + antibiotic (PDA+A). Fungus survival was evaluated every 45 days for 180 days for three seed batches from six wheat cultivars stored in propylene bags in a storehouse, with air temperature varying between 18 to 22 °C and relative air humidity around 60%. The efficacy of carboxin+thiram, difenoconazol, thiram, triadimenol, triticonazol and triticonazol + iprodione fungicides to control A. alternata was determined. A. alternata was detected in all the samples with an incidences of 39.6 %, 38.8% and 35.9% for the CUV I, CUV II and CUV III regions, respectively. The highest mean incidence of the fungus was found in the CUV I region, the coolest and most humid, and was significantly different from the other two regions. The average reduction in A. alternata viability in the wheat cultivar seeds was 49.5% during the 180 days of storage (inter-harvest period), demonstrating that infected seeds are the primary inoculum source for the fungus. The triticonazol + iprodione fungicide mixture efficiently controls A. alternata.

Physiological maturity of eggplant seeds

Determination of seed physiological maturity and ideal moment for harvesting fruits to extract their seeds are important aspects to produce seeds with high quality. To identify the best period for harvesting eggplant fruits, associated with ideal resting period of the fruit for extracting seeds, an eggplant production field was installed in municipality of Ijaci, in the State of Minas Gerais, Southwestern Brazil. The fruits were harvested at periods of 49, 56, 63, 70, and 77 days after pollination (DAP). The seeds of fruits harvested in each period were manually extracted immediately after harvest or after a post-harvest resting period of seven days, under a shed. The physiological quality of seeds was assessed by tests of: germination percentage; germination and emergence speed indexes; and electrical conductivity; which were carried out in the Central Seed Laboratory, Federal University of Lavras. Electrophoretic analyses of isoenzymes: catalase (CAT); esterase (EST); superoxide dismutase (SOD); and peroxidase (PO), were also therein performed. Results of germination and vigor of seeds have showed that the best period for harvesting the fruit is around 70 DAP; and that seeds should be extracted immediately after harvest. Electrophoretic analysis of enzymes has showed immaturity for eggplant seeds, harvested after 49 DAP.

Juridisen kaksinkertaisen verotuksen problematiikka kansainvälisessä yritystoiminnassa

Tässä tutkielmassa tarkastellaan kansainvälisessä yritystoiminnassa esiintyviä juridisen kaksinkertaisen verotuksen ongelmatilanteita suomalaisten yritysten näkökulmasta. Tutkielmassa pohditaan lisäksi yritysten omaa roolia osana kaksinkertaisen verotuksen tilanteita. Juridista kaksinkertaista verotusta muodostuu, kun sama yhtiö maksaa samasta tulosta veroa kahteen valtioon. Teoriaosio etenee verotusoikeuden määrittämisestä kaksinkertaisen verotuksen muodostumiseen. Kaksinkertainen verotus pyritään poistamaan poistomenetelmällä, joka on Suomessa tavallisesti hyvitysmenetelmä. Ongelmatilanne muodostuu, kun kaksinkertaista verotusta ei saada tehokkaasti poistettua tulon lähdevaltion ja yhtiön asuinvaltion välillä. Tämä tutkielma on laadullinen tutkimus ja tutkimusmenetelmänä on käytetty teemahaastattelua. Tutkimustulokset osoittavat, että kvalifikaatiokonflikteissa juridisen kaksinkertaisen verotuksen ongelmatilanne esiintyy pääsääntöisesti rojalti-liiketulo -luokitteluristiriidan seurauksena. Kiinteän toimipaikan kvalifikaatio on harvinaisempi ongelmatilanne. Hyvitysmenetelmän suurimmat ongelmat esiintyvät puolestaan pääliikkeen tappiollisen liiketoiminnan johdosta. Toinen hyvitysmenetelmän ongelmakohta esiintyy, kun lähdeveron alainen tulo huomioidaan bruttotulosta, mutta ulkomaisen tulon hyvitys suoritetaan nettotulosta. Haastatteluiden perusteella on selvää, että yritysten oma rooli kaksinkertaisen verotuksen ehkäisyssä on merkittävä. Yrityksen omia vaikutusmahdollisuuksia ovat muun muassa riittävä ennakointikyky, dokumentointi, selkeät sopimukset ja asiantuntijuus.

Recueil qui paraît avoir été formé par Nicolas Hennequin, seigneur de Perey, notaire et secrétaire du roi, pour lui servir de protocole. Beaucoup de pièces, non relevées ci-dessous, concernent la famille Hennequin et la famille Sallard, qui lui était alliée ; elles sont comprises entre les années 1541 et 1578. Les autres pièces sont les suivantes.

Contient : 1 « Anticipation », pour « Ollivyer Petit, phrestre à Sainct Aulbin Chasteauneuf », contre « Françoys Chevillon ». Paris, 23 octobre 1555. Acte de HENRI II ; 2 « Respit à ung an » pour Pierre Le Febvre. Paris, 18 novembre 1555. Acte de HENRI II ; 3 Lettre pour faire le « terrier » des seigneuries de « Marsangy, Chuelles, La Thommerye », etc., en faveur de « maistre Ollyvier Symonnet », lieutenant du bailli d'Orléans à Château-Renard, tuteur de Florimond de Sailly, son neveu. Paris, 14 avril 1556, après Pâques. Acte de HENRI II ; 4 « Provision d'une bourse theologale au collège de Navarre », pour Nicolas Hue, du diocèse de Soissons. Paris, 13 juillet 1557. En latin. Acte de HENRI II ; 5 Vers latins et français sur « monseigneur d'Anguean » ; 6 « Don de bourses theologales au collège de Navarre » à Jean Nodot, maître ès-arts. Paris, 3 mars 1558 n. st. En latin. Acte de HENRI II ; 7 Confirmation du privilège pour les notaires et secrétaires de la maison et couronne de France d'être exemptés du logement des gens de guerre. Fontainebleau, 9 avril 1558 n. st. Acte de HENRI II ; 8 Lettre de créance du roi HENRI II pour le cardinal de Châtillon et le sieur Du Mortier. Villers-Cotterets, 26 février 1559 n. st ; 9 Mariage d'Élisabeth, fille de Henri II, et mort de ce roi en 1559 ; 10 Lettre de FRANÇOIS II au prévôt de Paris pour ordonner de faire publier le commandement fait à tous ceux qui ont obtenu quelques privilèges des rois précédents, d'en venir prendre des lettres de confirmation. Saint-Germain-en-Laye, 15 août 1559 ; 11 Lettre de FRANÇOIS II au sujet des officiers comptables alternatifs, incomplète. Villers-Cotterets, septembre 1559 ; 12 Lettre du même au sujet du commerce des grains et de celui des vins. Chambord, 20 décembre 1559. Le commencement manque ; 13 Pièces relatives à la police des pauvres à Paris, comprenant ; a Vers ; b Lettre par laquelle « MONTAIGNE » adresse le traité qui suit « au cardinal de Tournon ». Saint-Germain-des-Prés, 22 juin ; c Traité sur la police des pauvres de Paris ; d « Les sept oeuvres de misericorde spirituelles... temporelles » ; e « Evangile selon S. Luc, chap. XVI » ; f « Exhortation au peuple pour ayder et secourir les paouvres. St Mathieu, XXVe chapitre ». Vers ; 14 ; Nomination par RAOUL SPIFAME, « garde general des pièces, bastons et munitions » de l'artillerie du roi, de « Denys Clerey, Sr de Vaubercey », en qualité de « commis à la garde de l'artillerye et munitions du magazin estably en la ville de Troyes, pour le gouvernement de Champaigne ». Paris, 22 février 1555 n. st ; Lettre du roi HENRI II à ce sujet. Fontainebleau, 14 mars 1555 n. st ; 15 Confirmation par le roi FRANÇOIS II du privilège de « ses officiers domesticques et commenceaulx », en vertu duquel ils sont exemptés du logis des gens de guerre. Saint-Germain-en-Laye, 23 septembre 1560 ; 16 Lettre du roi CHARLES IX pour un emprunt. Saint-Germain-en-Laye, 15 juillet 1561 ; 17 Lettre du roi CHARLES IX au prévôt de Paris, lui ordonnant de contraindre les curés des églises de la prévôté de Paris et leurs paroissiens, à fournir à leurs dépens lesdites églises des ornements et livres nécessaires. Paris, 24 décembre 1562 ; 18 Lettre du roi CHARLES IX pour un emprunt. Amboise, 30 mars 1563 n. st ; 19 Lettre de rémission en faveur de « Jehan Mailly, notaire ou conté de Bryenne ». Novembre 1563. Acte de CHARLES IX ; 20 Lettre de JEAN LORMIER, curé de Perey ; 21 « Litera de emolumento signationis cartarum regiarum equaliter inter omnes de collegio » notariorum « presentes, dividendo quod antea soli notario signanti competebat ». Paris, 24 mai 1389. Acte de CHARLES VI ; 22 Lettre du roi CHARLES IX aux élus de l'élection de Paris, pour la mise aux enchères des fermes des aides. Paris, 27 juin 1565 ; 23 Lettre du roi CHARLES IX, au sujet de l'armement des habitants de Paris. Paris, 15 octobre 1567 ; 24 Éloge du roi ; 25 Extrait des registres de la chambre des comptes, au sujet de la nomination de Hugues Charreton comme trésorier des cent gentilshommes de la maison du roi, sous la charge du duc de Rouannois, 1569 ; 26 Lettre de « JEH. LORMYER, phrestre vicaire » de Perey. 6 novembre ; 27 Lettre du roi CHARLES IX pour un emprunt. Saint-Maur-des-Fossés, 4 janvier 1570 n. st ; 28 Lettre du roi CHARLES IX en faveur des conseillers, notaires et secrétaires de la maison et couronne de France. Blois, mai 1572 ; 29 Autre copie de la même lettre ; 30 « Extraict des registres du conseil privé du roy ». Affaire entre Nicolas Aubelin, Sr de Favelle, secretaire des finances de Monsieur, frère du roi, et Jean de Saint-Germain, jouissant de l'office de notaire et secretaire du roi, maison et couronne de France. Le roi donne gain de cause au Sr de St-Germain. 20 mai 1575 ; 31 « Rolle des esleus establiz en l'election de Paris pour faire l'assiete et departement des tailles sur les paroisses de leur eslection... suivant la commission du roy ... commenceant en l'an mil VC. XLI, au moys d'octobre... » ; 32 « C'est le papier et registre du bail des fermes des aydes des villes (sic) et eslection de Paris pour l'année commenceant le premier janvier VC.XL (sic), finissant le dernier decembre ensuivant VC.XL (sic)... » ; 33 Lettres du roi PHILIPPE VI pour obliger les sénéchaux, baillis et receveurs à rendre compte aux termes ordonnés, etc. Paris, 31 juillet 1338 ; 34 Vers latins sur le roi ; 35 Autres vers latins ; 36 Maximes et préceptes divers, etc., sur la justice ; 37 Table des pièces qui suivent ; 38 « Ordonnances royaulx sur le faiet des finances ». Saumur, 25 septembre 1443. Acte de CHARLES VII ; 39 « Mandatum regis pro exonerationibus regis levandis per magistros Stephanum de Bonney et Stephanum Petit super receptoribus particularibus suarum receptarum generalium ». Saumur, 25 novembre 1443. Acte de CHARLES VII ; 40 Ordonnance sur le fait des finances. Nancy, 10 février 1445 n. st. Acte de CHARLES VII ; 41 Ordonnance pour l'éclaircissement des deux ordonnances précédentes. Bourges, 26 novembre 1447. Acte de CHARLES VII ; 42 Ordonnance relative à la chambre des comptes. Méhun-sur-Yèvre, 23 décembre 1454. Acte de CHARLES VII ; 43 « Rolle des messrs les presidens et maistres des comptes à Paris... en janvier mil VC quarente quatre », ancien style ; 44 Mandement pour que les « gaiges d'officiers, fiefz et aulmosnes » soient payés avant toute autre chose. « Au Bouchat, près Sainct Poursan », 30 janvier 1456 n. st. Acte de CHARLES VII ; 45 Décision de la chambre des comptes au sujet des officiers comptables. 16 février 1460 n. st ; 46 Autre mandement, au sujet de celui ci-dessus n° 44. Les Roches-Tranchelion, 21 avril 1460. Acte de CHARLES VII ; 47 « Assignations faictes aulx officiers comptables de la duché de Normandie, tant de dommainne, aydes, greniers que tailles, par vertu des lettres closes et mandemens de messeigneurs des comptes, pour venir compter du faict de leurs entremises » ; 48 Ordonnance portant réorganisation de l'administration des finances. Blois, 28 décembre 1523. Acte de FRANÇOIS Ier ; 49 « Sermens des officiers qui font le serment en la chambre » des comptes ; 50 « La maniere et les parolles que doibvent estre dictes en recepvant aulcung ou aulcune à faire les foy et honmaige au roy, nostre sire, au bureau de sa chambre des comptes... » ; 51 « Les noms et les ans que ont regné les roys de France », de Pharamond à Henri II ; 52 Vers français ; 53 « Ensuivent les nons des notaires et secretaires du roy, de la maison et couronne de France, tant boursiers que gaigiers, extraict au registre des immatriculations faict et à commencer le premier jour de janvier mil VC. cinquante neuf » anc. st

Formulaire de chancellerie, formé d'actes du règne de LOUIS XI, parmi lesquels sont à signaler les suivants, qui tous, sauf indication contraire, émanent de ce prince.

Contient : 1° D'une table des pièces, qui commence au feuillet 134, se continue aux feuillets 135 et 136, et s'achève aux feuillets 131 v° et 132 r° ; 2° D'une ballade, dont le refrain est : « Faulte d'argent la douleur non pareille » (fol. 137 r°) ; 3° D'un petit glossaire latin (fol. 138 r°) ; 4° D'une ballade, dont le refrain est : « Tost est deffait qui autrui veult deffaire » (fol. 138 v°) ; 5° D'un petit glossaire latin-latin et latin-français (fol. 139 r°) ; 6° De deux lettres ; l'une de BONIFACE VIII à Philippe-le-Bel, l'autre de PHILIPPE-LE-BEL à Boniface VIII (fol. 139 v°)

Protocoles et recueils de pièces, à l'usage des secrétaires du roi.

Contient : 1 Formulaire ou protocole rédigé sous le règne de Charles VIII, mais formé d'actes émanés la plupart de Charles VII ; 2 Lettres de sauvegarde données par JACQUES D'ESTOUTEVILLE, garde de la prévôté de Paris, conservateur des privilèges de l'Université, pour maître Henry de Manreville, écolâtre de Tournay et écolier étudiant en ladite Université ; 3 « Noctes et enseignemens touchant l'office des notaires et secretaires du roy. Et premièrement, le notaire du roy, quelque autre... » ; 4 Protocole de lettres closes au pape, à un cardinal, à un archevêque, etc., de par le roi et autres (fol. 188-198) ; 5 Traité relatif à la rédaction des lettres du roi et exemples. Ce traité commence ainsi : « Licterarum regiarum quedam diriguntur subdictis regni, quedam non subdictis... » et finit par « ... ad Verbum, pro salute humani generis incarnatum, etc. Et sic de similibus. Explicit ». En latin ; 6 Formule d'une lettre d'anticipation empruntée à un acte de Charles VIII, daté de 1497

Physiological reactivity and the perception of emotional stimuli as they relate to social adaptive functioning after traumatic brain injury / y Melonie Jane Hopkins.

Traumatic brain injury (TBI) often affects social adaptive functioning and these changes in social adaptability are usually associated with general damage to the frontal cortex. Recent evidence suggests that certain neurons within the orbitofrontal cortex appear to be specialized for the processing of faces and facial expressions. The orbitofrontal cortex also appears to be involved in self-initiated somatic activation to emotionally-charged stimuli. According to Somatic Marker Theory (Damasio, 1994), the reduced physiological activation fails to provide an individual with appropriate somatic cues to personally-relevant stimuli and this, in turn, may result in maladaptive behaviour. Given the susceptibility of the orbitofrontal cortex in TBI, it was hypothesized that impaired perception and reactivity to socially-relevant information might be responsible for some of the social difficulties encountered after TBL Fifteen persons who sustained a moderate to severe brain injury were compared to age and education matched Control participants. In the first study, both groups were presented with photographs of models displaying the major emotions and either asked to identify the emotions or simply view the faces passively. In a second study, participants were asked to select cards from decks that varied in terms of how much money could be won or lost. Those decks with higher losses were considered to be high-risk decks. Electrodermal activity was measured concurrently in both situations. Relative to Controls, TBI participants were found to have difficulty identifying expressions of surprise, sadness, anger, and fear. TBI persons were also found to be under-reactive, as measured by electrodermal activity, while passively viewing slides of negative expressions. No group difference,in reactivity to high-risk card decks was observed. The ability to identify emotions in the face and electrodermal reactivity to faces and to high-risk decks in the card game were examined in relationship to social monitoring and empathy as described by family members or friends on the Brock Adaptive Functioning Questionnaire (BAFQ). Difficulties identifying negative expressions (i.e., sadness, anger, fear, and disgust) predicted problems in monitoring social situations. As well, a modest relationship was observed between hypo-arousal to negative faces and problems with social monitoring. Finally, hypo-arousal in the anticipation of risk during the card game related to problems in empathy. In summary, these data are consistent with the view that alterations in the ability to perceive emotional expressions in the face and the disruption in arousal to personally-relevant information may be accounting for some of the difficulties in social adaptation often observed in persons who have sustained a TBI. Furthermore, these data provide modest support for Damasio's Somatic Marker Theory in that physiological reactivity to socially-relevant information has some value in predicting social function. Therefore, the assessment of TBI persons, particularly those with adaptive behavioural problems, should be expanded to determine whether alterations in perception and reactivity to socially-relevant stimuli have occurred. When this is the case, rehabilitative strategies aimed more specifically at these difficulties should be considered.

The effect of pH and ethanol on the astringent sub-qualities of red wine, and the implications for optimum grape maturity /

The first objective of this study was to identify appropriate sensory descriptors to assess the astringent sub-qualities of red wine. The influence of pH and ethanol on the sensation of astringency in red wine was evaluated, using a de-alcoholized red wine. A portion of the wine was adjusted to the pH values of 3.2, 3.4, 3.6 and 3.8, and another portion was adjusted to ethanol concentrations of 0%, 6%, 12%, and 15%. In addition, the pH 3.4 and 3.6 treatments were adjusted to an ethanol concentration of 12% and 15% all wines were then assessed sensorially and seventeen terms were identified, through panel discussion, to describe the mouth-feel and taste qualities: velvet, aggressive, silk/satin, dry, fleshy, unripe, pucker viscosity, abrasive, heat, chewy, acidity, grippy/adhesive, bitter, balance, overall astringency, and mouth-coat. Descriptive analysis profiling techniques were used to train the panel and measure the intensity of these attributes. It was found that decreasing pH values (averaged across all ethanol concentrations) showed an increase in the overall astringency of the wine. The combined treatments of ethanol and pH, real wine parameters (pH 3.4 and 3.6; 12% and 15% ethanol) did not have an effect on the perception of the astringent sub-qualities of the wine. A time intensity study was also included using the pH and ethanol adjusted wines, which showed that as the ethanol level of the wines increased so did the time to maximum intensity. The second objective was to identify appropriate sensory descriptors to evaluate the influence of grape maturity and maceration technique (grape skin contact) on the astringency sub-qualities of red vinifera wines from Niagara. The grapes were harvested across two dates, representing an early harvest and a late harvest. A portion of the Cabernet Sauvignon grapes wine was divided into three maceration treatments of oneweek maceration, standard two-week maceration, three-week maceration, and MCM. Another portion of both the early and late harvest Cabernet Sauvignon grapes were chaptalized to yield a final ethanol concentration of 14.5%. The wines were assessed sensorially and thirteen terms were identified, through panel discussion, to describe the mouth-feel and taste qualities: carbon dioxide, pucker, acidity, silk/chamois, dusty/chalky/powdery, sandpaper, numbing, grippy/adhesive, dry, mouthcoat, bitter, balance and, overall astringency. Descriptive analysis techniques were used to train the panel and measure the intensity of these attributes. The data revealed few significant differences in the mouth-feel of the wines with respect to maturity; which included differences in overall astringency and balance. There were varietal differences between Cabernet Sauvignon, Cabernet Franc, and Pinot Noir and differences for Cabernet Sauvignon wines due to the length and manner of maceration and as a result of chaptalization. Statistical analysis revealed a more complex mouth-feel for the Pinot Noir wines; and an increase in the intensity of the astringent sub-qualities as a result of the addition of sugar to the wines. These findings have implications for how processing decisions, such as optimum grape maturity and vinification methods may affect red wine quality.

Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

The Catharanthus roseus 16-hydroxytabersonine O-methyltransferase involved in vindoline biosynthesis /

Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex dimeric anticancer alkaloids vinblastine and vincristine that are derived by coupling vindoline and catharanthine monomers. This thesis describes the novel application of carborundum abrasion (CA) technique as a tool for large scale isolation of leaf epidermis enriched proteins. This technique was used to facilitate the purification to apparent homogeneity of 16-hydroxytabersonine-16-0-methyltransferse (l60MT) that catalyses the second step in the 6 step pathway that converts tabersonine into vindoline. This versatile tool was also used to harvest leaf epidermis enriched mRNAs that facilitated the molecular cloning of the 160MT. Functional expression and biochemical characterization of recombinant 160MT enzyme showed that it had a very narrow substrate specificity and high affinity for 16-hydroxytabersonine, since other closely related monoterpene indole alkaloids (MIAs) did not act as substrates. In addition to allowing the cloning of this gene, CA technique clearly showed that 160MT is predominantly expressed in Catharanthus leaf epidermis, in contrast to several other OMTs that appear to be expressed in other Catharanthus tissues. The results provide compelling evidence that most of the pathway for vindoline biosynthesis including the 0- methylation of 16-hydroxytabersonine occurs exclusively in leaf epidermis, with subsequent steps occurring in other leaf cell types. Small molecule O-methyltransferases (OMTs) (E.C. 2.1.1.6.x) catalyze the transfer of the reactive methyl group of S-adenosyl-L-methionine (SAM) to free hydroxyl groups of acceptor molecules. Plant OMTs, unlike their monomeric mammalian homologues, exist as functional homodimers. While the biological advantages for dimer fonnation with plant OMTs remain to be established, studies with OMTs from the benzylisoquinoline producing plant, Thalictrum tuberosum, showed that co-expression of 2 recombinant OMTs produced novel substrate specificities not found when each rOMT was expressed individually (Frick, Kutchan, 1999) . These results suggest that OMTs can fonn heterodimers that confer novel substrate specificities not possible with the homodimer alone. The present study describes a 160MT model based strategy attempting to modify the substrate specificity by site-specific mutagenesis. Our failure to generate altered substrate acceptance profiles in our 160MT mutants has lead us to study the biochemical properties ofhomodimers and heterodimers. Experimental evidence is provided to show that active sites found on OMT dimers function independently and that bifunctional heterodimeric OMTs may be fonned in vivo to produce a broader and more diverse range of natural products in plants.

Localization and functional characterization of iridoid biosynthetic genes in Catharanthus roseus /

Catharanthus roseus is the sole biological source of the medicinal compounds vinblastine and vincristine. These chemotherapeutic compounds are produced in the aerial organs of the plant, however they accumulate in small amounts constituting only about 0.0002% of the fresh weight of the leaf. Their limited biological supply and high economical value makes its biosynthesis important to study. Vinblastine and vincristine are dimeric monoterpene indole alkaloids, which consists of two monomers vindoline and catharanthine. The monoterpene indole alkaloids (MIA's) contain a monoterpene moiety which is derived from the iridoid secologanin and an indole moiety tryptamine derived from the amino acid tryptophan. The biosynthesis of the monoterpene indole alkaloids has been localized to at least three cell types namely, the epidermis, the laticifer and the internal phloem assisted parenchyma. Carborundum abrasion (CA) technique was developed to selectively harvest epidermis enriched plant material. This technique can be used to harvest metabolites, protein or RNA. Sequencing of an expressed sequence tagged (EST) library from epidermis enriched mRNA demonstrated that this cell type is active in synthesizing a variety of secondary metabolites namely, flavonoids, lipids, triterpenes and monoterpene indole alkaloids. Virtually all of the known genes involved in monterpene indole alkaloid biosynthesis were sequenced from this library.This EST library is a source for many candidate genes involved in MIA biosynthesis. A contig derived from 12 EST's had high similarity (E'^') to a salicylic acid methyltransferase. Cloning and functional characterization of this gene revealed that it was the carboxyl methyltransferase imethyltransferase (LAMT). In planta characterization of LAMT revealed that it has a 10- fold enrichment in the leaf epidermis as compared to the whole leaf specific activity. Characterization of the recombinant enzyme revealed that vLAMT has a narrow substate specificity as it only accepts loganic acid (100%) and secologanic acid (10%) as substrates. rLAMT has a high Km value for its substrate loganic acid (14.76 mM) and shows strong product inhibition for loganin (Kj 215 |iM). The strong product inhibition and low affinity for its substrate may suggest why the iridoid moiety is the limiting factor in monoterpene indole alkaloid biosynthesis. Metabolite profiling of C. roseus organs shows that secologanin accumulates within these organs and constitutues 0.07- 0.45% of the fresh weight; however loganin does not accumulate within these organs suggesting that the product inhibition of loganin with LAMT is not physiologically relevant. The limiting factor to iridoid and MIA biosynthesis seems to be related to the spatial separation of secologanin and the MIA pathway, although secologanin is synthesized in the epidermis, only 2-5% of the total secologanin is found in the epidermis while the remaining secologanin is found within the leaf body inaccessable to alkaloid biosynthesis. These studies emphasize the biochemical specialization of the epidermis for the production of secondary metabolites. The epidermal cells synthesize metabolites that are sequestered within the plant and metabolites that are secreted to the leaf surface. The secreted metabolites comprise the epidermome, a layer separating the plant from its environment.

Assessing the influence of irrigation and fertigation on fruit composition, vine performance and wine quality in a cool, humid climate /

A study was devised to evaluate influences of irrigation and fertigation practices on Vitis vinifera and Vitis labruscana grapes in the Niagara Peninsula. A modified FAO Penman- Monteith evapotranspiration formula was used to calculate water budgets and schedule irrigations. Five deficit irrigation treatments (non-irrigated control; deficits imposed postbloom, lag phase, and veraison; fiiU season irrigation) were employed in a Chardonnay vineyard. Transpiration rate (4-7 /xg H20/cmVs) and soil moisture data demonstrated that the control and early deficit treatments were under water stress throughout the season. The fiiU season irrigation treatment showed an 18% (2001) and 19% (2002) increase in yield over control due to increased berry weight. Soluble solids and wine quality were not compromised, and the fiiU season treatment showed similar or higher °Brix than all other treatments. Berry titratable acidity andpH also fell within acceptable levels for all five treatments. Irrigation/fertigation timing trials were conducted on Concord and Niagara vines in 2001- 02. The six Concord treatments consisted of a non-irrigated control, irrigation fi^om Eichhom and Lorenz (EL) stage 12 to harvest, and four fertigation treatments which applied 70 kg/ha urea. The nine Niagara treatments included a non-irrigated control, two irrigated treatments (ceasing at veraison and harvest, respectively) and six fertigation treatments of various durations. Slight yield increases (ca. 10% in Concord; 29% in Niagara) were accompanied by small decreases in soluble solids (1.5°Brix), and methyl anthranilate concentrations. Transpiration rate and soil moisture (1 1.9-16.3%) data suggested that severe water stress was present in these Toledo clay based vineyards.