The objective of this study was to optimize an efficient selection system and Agrobacterium mediated transformation of chickpea (Cicer arietinum L.). Cotyledonary node explants of Turkish chickpea cultivar G&ouml / k&ccedil / e were used to determine the effects of selective agents, two antibiotics (Kanamycin, Hygromycin) and two herbicides (PPT, Glyphosate) as well as four antibiotics (Augmentin, Carbenicillin, Cefotaxime, Timentin) for eliminating Agrobacterium on multiple shoot and root induction. Selective agents and antibiotics were applied to explants at different concentrations for one month and numbers of regenerated shoots and roots were recorded. Kanamycin at 100 mg/L, Hygromycin at 20 mg/L, PPT at 3 mg/L and Glyphosate at 5 mg/L were found to be appropriate to select chickpea transformants. Lowest concentrations of all selective agents (50 mg/L Kanamycin, 10 mg/L Hygromycin, 3 mg/L PPT, 1 mg/L Glyphosate) totally inhibited rooting of the regenerated shoots. Among the Agrobacterium-eliminating antibiotics, Cefotaxime and Augmentin each up to 600 mg/L had no adverse effect on shoot induction, whereas Timentin (300 mg/L) significantly increased and Carbenicillin (300 mg/L) significantly decreased shoot induction after four weeks of culture. Augmentin was determined to have no effect on rooting capacities of chickpea shoots. However Cefotaxime at all concentrations significantly decreased root induction. On the other hand only high concentrations of Carbenicillin (300 mg/L) and Timentin (200 mg/L) significantly decreased rooting. Sulbactam in combination with Carbenicillin and Cefotaxime displayed effective inhibition of bacterial growth. Furthermore, Agrobacterium mediated transformation procedure for cotyledonary node explants of G&ouml / k&ccedil / e, was also optimized by monitoring transient uidA expression on 4th, 9th, and 16th days after transformation. Transformation procedure was improved via mechanical injury of axillary region of explants and application of vacuum infiltration at 200 mmHg for 40 minutes.

iv The objective of this study was to optimize tissue culture and regeneration parameters of immature inflorescence culture of Triticum aestivum cv. Bezostaja- 01 and Triticum durum cv. Kiziltan-91. The effects of callus age and vernalisation time of explants on regeneration success were evaluated. For determination of optimum vernalisation time of immature inflorescence, plants subjected to 4 &deg / C for 1, 2, 3, 4, and 5 weeks, respectively. Tillers containing immature inflorescences were collected at the same time. Percentage of inflorescence formed tillers over total explants were reached the highest value, 79 %, at 4 weeks cold treated Kiziltan cultivar and, 73 %, at 5 weeks cold treated Bezostaja cultivar. Isolated immature inflorescences were put onto 2mg /L 2,4-dichlorophenoxyacetic acid and picloram containing callus induction medium for Kiziltan and Bezostaja cultures, respectively. Callus induction rate were found to be 100 % for Kiziltan and Bezostaja. These explants were taken to regeneration after 6, 9, 12 and 15 weeks of dark incubation period. The regeneration capacities of calli were determined as shooting percentage and data were collected after 4, 8, 12, and 15 week regeneration period. The highest shooting percentage of 69 %, were obtained from 6 weeks old calli produced from 4 weeks vernalised explants in Kiziltan cultures at the end of 15 weeks regeneration period. However, shooting percentage was 57.2 % for 9 weeks old calli while it decreases to 37.6 % in 12 weeks old calli and 44.2 % in 15 weeks old calli at the end of 15 weeks regeneration period. This showed that prolonged dark incubation period decreased regeneration capacity of the callus. However, there was no significant difference in regeneration capacities of calli produced from Bezostaja immature inflorescence and the highest shooting percentage was obtained from 9 weeks old calli produced from 5 weeks vernalised explants, 27.4 %. Besides regeneration studies, optimization of transformation parameters for winter wheat cultivars Kiziltan and Bezostaja by using Agrobacterium tumefaciens AGLI containing binary vector pALl56 was performed. Transformation efficiencies were determined by monitoring the transient expression of uidA gene via histochemical GUS assay. Three to four weeks old calli were found to be more responsive to Agrobacterium-mediated transformation in Kiziltan cultures. However, four to five weeks old calli were found to be more responsive to Agrobacterium-mediated transformation in Bezostaja cultures. Different transformation protocols were used. It was found that MGL based and MMA based protocols could be used for Bezostaja and Kiziltan transformation, respectively. The highest GUS expression, 84%, was obtained from 28 weeks old calli produced from 5 weeks vernalised explants in Bezostaja cultures.

Modern agriculture often employs broad-spectrum herbicides combined with herbicide-resistant crops. Usually, this is achieved by altering the crop genom (genetic modification) by inserting of a specific gene coding for resistance to specific herbicide or group of herbicides. Apllying such herbicide thus results in minor crop damage. The resistence of crops against broad-spectrum herbicides depends on the genes, which were inserted into the genom. The gene bar is often used as a resistence -providing element. It mediates resistance against a broad spectrum of herbicides, such as glufosinate and Bialahops. For the selection of transformants and preliminary assessment of the target transgen expression level a suitable marker gene is simultaneously inserted. For this purpose, gene for bacterial ??glucuronidase (GUS) is often used. It is considered to be one of the most frequently used reporter systems for the assessment of transgen expression and also allows to analyze the expression on the tissue, cell and whole cell organelles levels. In this diploma thesis the PCR method and histochemical detection of the enzyme glucuronidase presence were used to detect and evaluate transgenic plants of cv. Golden Promise spring barley, modified by genes bar and gus. The presence of gus gene was determined in different parts of plants.

Les films de Gus Van Sant peuvent être regroupés selon plusieurs périodes distinctes, où les différents circuits de production qui ont permis leur financement (ceux du cinéma indépendant, des grands studios, de la télévision câblée) ont coïncidé avec des approches spécifiques de la vitesse (dynamique,« classique», lente). La notion de rythme semble pertinente pour appréhender ces écarts formels dans l'œuvre du réalisateur. Les travaux de nombreux théoriciens, comme ceux d'Emile Benveniste ou d'Henri Meschonnic, ont cependant montré que le rythme ne pouvait se réduire à des effets ou impressions de vitesse; une approche esthétique de celui-ci doit permettre de saisir la manière dont les sens (relatifs à la signification, l'orientation et la sensation) peuvent être construits par le discours filmique. Dans les longs métrages de Gus Van Sant, le rythme est lié à la représentation des personnages, à travers leurs déplacements physiques ou la dynamique de leur imagination. Mais le rythme est également une force structurante, aussi bien dans la diégèse (un cadre spatio-temporel précis, un contexte, qui s'impose aux personnages) que pour le texte filmique lui-même. Indissociable du sujet qui le provoque ou de celui qui le perçoit, le rythme dépend d'une certaine subjectivité: l'analyse des motifs dans l'œuvre de Gus Van Sant peut permettre de cerner une poétique du réalisateur et d'envisager les possibilités, pour le spectateur, de ressentir ou de définir l'image dans une expérience rythmique personnelle. / Gus Van Sant's films can be classified in three main periods : the various production methods used to finance the films (independent cinema, majors, cable TV), coincide with specific approaches of the speed (rapid, classic cinema, slow). The notion of rhythm seems appropriate to study the different times in Gus Van Sant's filmography. The work of many theorists, Emile Benveniste or Henry Meschonnic for instance, showed however that the rhythm cannot be reduced to effects or feeling of speed ; an aesthetic approach of rhythm enables to understand how the senses (related to meaning, orientation and feeling) can be built by the movie telling. ln Gus Van Sant's full-length film, rhythm is tied to the way the characters appear, through their physical moving but also through their imagination at work. Beyond, rhythm is also a structural part of films, in the fiction story (a specific place and temporality, a context the characters have to deal with), as well as in the film in itself. Rhythm depends on subjectivity as it is related to the character who acts or to the spectator who watches: motifs' analysis in Gus Van Sant's films enables to identify a poetic from the movie director and to explore the different ways the spectator can feel or interpret the image in a persona! rhythmic experience.

[EN] Abstract Fruit set is the transition from a quiescent ovary of the flower to a developing fruit after pollination. This process is associated with the content of gibberellin (GAs) and auxins in the ovary. Therefore the development of parthenocarpic fruit (seedless fruit) can be induced by applying both kinds of hormones. In tomato it has been described that auxin action during fruit set is mediated by GAs, by inducing different biosynthesis pathway genes, among them SlGA20ox, which codify important enzymes for GAs biosynthesis regulation. The SlGA20ox gene family is composed of 4 genes, and SlGA20ox1 seems to play a key role in fruit set. In order to deepen the understanding of this process, it has been investigated in the ovary of tomato: a) the site of expression of SlGA20ox1 and its regulation; b) the localization of IAA (auxin). For this, we have obtained transgenic lines of the tomato Micro-Tom (MT) cultivar and various hormonal mutants (procera, 35S::CsGA20ox1 Dwarf, entire and dgt) with the transgene pSlGA20ox1::GUS. Transgenic lines do not differ phenotypically from the original lines and they have been used to analyze the expression of SlGA20ox1, using the GUS gene as reporter. Localization of SlGA20ox1 transcripts was also investigated in situ. SlGA20ox1 expression in ovary after pollination was located mainly in the funiculus, placenta and embryo. Auxin and brassinosteroids (BR) increased the expression of SlGA20ox1. On the other hand, auxin location in the ovary was investigated using the transgenic line DR5::GUS (expressing the auxin-response gene DR5), and analyzing directly the IAA content by immunolocalization. In pollinated ovaries, GAs and auxin co-localize in the egg (embryo and embryo-sac) and placenta, supporting the hypothesis that both hormones interact during fruit set. Phenotypic analysis of the mutants used in this work showed that GAs inhibit the development of axillary buds, through the DELLA protein. This inhibitory effect is reversed, at least partially, by BR. Furthermore, the introduction of wild gen Dwarf in MT (normalizing its content of BR) increases plant height, but does not extend its vegetative cycle, nor increases parthenocarpy in MT. / [ES] Resumen La fructificación, paso del ovario en reposo a fruto en crecimiento tras la polinización, asociada al aumento de contenido de giberelinas (GAs) y auxinas en el ovario, es un proceso clave para la producción. Por ello se puede inducir también el desarrollo de frutos partenocárpicos (sin semillas) aplicando ambos tipos de hormonas. En tomate se ha descrito que parte de la acción de las auxinas en la fructificación está mediada por GAs, induciendo distintos genes de la ruta biosintética, entre ellos los que codifican SlGA20ox, enzimas importantes para la regulación de la síntesis de GAs. De los 4 genes que forman la familia, SlGA20ox1 parece ser clave en la fructificación. Con objeto de profundizar en el conocimiento de este proceso, se ha investigado en el ovario de tomate: a) la localización de la expresión de SlGA20ox1 y su regulación; b) la localización de IAA (auxina). Para ello, se han obtenido líneas trangénicas del cultivar Micro-Tom (MT) de tomate y diversos mutantes hormonales (procera, 35S::CsGA20ox1 Dwarf, dgt y entire) con el transgén pSlGA20ox1::GUS. Las líneas transgénicas no difieren fenotípicamente de las líneas originales y se utilizaron para analizar la expresión de SlGA20ox1 con el gen delator GUS. Se investigó además la localización de los transcritos de SlGA20ox1 in situ. La expresión de SlGA20ox1 en el ovario tras la polinización se localizó, principalmente, en el funículo, la placenta y el embrión. Tanto las auxinas como los brasinosteroides (BR) aumentaron la expresión de SlGA20ox1. Por otro lado se investigó la localización de auxinas en el ovario usando una línea transgénica DR5::GUS (que expresa el gen de respuesta a auxinas DR5), y analizando también directamente el contenido de IAA mediante inmunolocalización. En ovarios polinizados, la localización de las GAs y auxinas coincidió en el óvulo (embrión y saco embrionario) y la placenta, apoyando la hipótesis de que ambas hormonas interaccionan durante la fructificación. El análisis fenotípico de los mutantes utilizados mostró que las GAs inhiben el desarrollo de los brotes axilares por GAs, a través de la proteína DELLA. El efecto inhibidor es revertido, al menos parcialmente, por BR. Por otro lado, la introducción del gen silvestre Dwarf en MT (normalizando así su contenido en BR) aumenta la altura de la planta, pero no prolonga su ciclo vegetativo, ni aumenta la capacidad partenocárpica de MT. / [CAT] Resum La fructificació, pas de l'ovari en repòs a fruit en creixement després de la pol·linització, associada a l'augment de contingut de giberelines (GAs) i auxines en l'ovari, és un procés clau per a la producció. Per això es pot induir també el desenvolupament de fruits partenocàrpics (sense llavors) aplicant tots dos tipus d'hormones. En el tomàquet s'ha descrit que part de l'acció de les auxines en la fructificació està intervinguda per GAs, induint diferents gens de la ruta biosintètica, entre ells els que codifiquen SlGA20ox, enzims importants per a la regulació de la síntesi de GAs. Dels 4 gens que formen la família, SlGA20ox1 sembla ser clau en la fructificació. A fi d'aprofundir en el coneixement d'aquest procés, s'ha investigat en l'ovari del tomàquet: a) la localització de l'expressió de SlGA20ox1 i la seva regulació; b) la localització de l'IAA (auxina). Per a això, s'han obtingut línies transgèniques de conrear Micro-Tom (MT) de tomàquet i diversos mutants hormonals (procera, 35S::CsGA20ox1 Dwarf, dgt i entire) amb el transgèn pSlGA20ox1::GUS. Les línies transgèniques no difereixen fenotípicamente de les línies originals i es van utilitzar per analitzar l'expressió de SlGA20ox1 amb el gen delator GUS. Es va investigar, a més, la localització dels transcrits de SlGA20ox1 in situ. L'expressió de SlGA20ox1 en l'ovari després de la pol·linització es va localitzar, principalment, en el funicle, la placenta i l'embrió. Tant les auxines com els brasinosteroides (BR) van augmentar l'expressió de SlGA20ox1. D'altra banda es va investigar la localització d'auxines en l'ovari usant una línia transgènica DR5::GUS (que expressa el gen de resposta a auxines DR5) i analitzant també directament el contingut de IAA mitjançant immunolocalització. En ovaris polinitzats, la localització de les GAs i auxines va coincidir en l'òvul (embrió i sac embrionari) i la placenta, refermant la hipòtesi que ambdues hormones interaccionen durant la fructificació. L'anàlisi fenotípic dels mutants utilitzats va mostrar que les GAs inhibeixen el desenvolupament dels brots axil·lars per GAs, a través de la proteïna DELLA. L'efecte inhibidor és revertit, almenys parcialment, per BR. D'altra banda, la introducció del gen silvestre Dwarf en MT (normalitzant així el seu contingut de BR) augmenta l'alçada de la planta, però no perllonga el seu cicle vegetatiu, ni augmenta la capacitat partenocàrpica d'MT. / Gallego García, M. (2016). Papel de la GA 20-oxidasa en la fructificación del tomate [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61958 / TESIS

1998 wurde Psycho neu verfilmt; und das Besondere an diesem Remake war nicht, dass (wie bei Remakes eigentlich üblich) ein anderer Regisseur den Stoff neu interpretiert. Vielmehr sollte sich dieses Remake als nahezu exakte Replik des Originals herausstellen und rief damit kontroverse Diskussionen unter Filmkritikern hervor. Die Idee, ein so werkgetreues Remake zu produzieren, ist einzigartig in der Filmgeschichte. In dieser Studie soll der Fragestellung nachgegangen werden, ob und in welchem Maße das Remake als exakte Replik auch den gleichen thrill wie das Original bieten kann.

Vitamin C (ascorbic acid, AsA), an important primary metabolite of plants, functions as an antioxidant, an enzyme cofactor, and a cell-signaling modulator in a wide array of crucial physiological processes including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, senescence, and growth. To identify genes that may regulate vitamin C levels in plants, about 3000 activation-tagged Arabidopsis lines were treated with ozone, which is a power oxidizing agent. Two mutants were selected for identification of potential genes involved in the regulation of vitamin C synthesis. A putative F-box gene, VCF1, and a purple acid phosphatase, AtPAP15, were identified for further characterization. Two homozygous SALK T-DNA knockouts in the open reading frame (ORF) of VCF1 exhibited high tolerance to ozone when treated with 450 ppb for 3 hours and the AsA levels of these mutants were 2 to 3 fold higher than wild-type (wt) plants. Developmental studies, using RT-PCR, indicated that foliar expression of the VCF1 gene increased with plant age from 1 to 5 weeks, whereas AsA decreased during this same period. The expression of VCF1 was higher under a low-light condition in which AsA was reduced considerably. The AsA levels in two VCF1 overexpressing lines were only 50 to 70% of wt plants. These results suggested that the putative F-box gene functions as a negative regulator of leaf ascorbate content. Overexpression of AtPAP15 with the CaMV 35S promoter resulted in up to 3-fold higher AsA levels than wt plants, where two independent SALK T-DNA insertion mutants in AtPAP15 had 50% less AsA than wt plants. Enzyme activity of bacterially expressed GST:AtPAP15 was greatest with phytate as a substrate indicating that AtPAP15 is a phytase. Phytase catalyzes hydrolysis of phytate (myo-inositol hexakisphosphate) to yield myo-inositol and free phosphate. Thus, AtPAP15 may regulate AsA levels by controlling the input of myo-inositol into this branch of AsA biosynthesis in Arabidopsis. AtPAP15 was expressed in all tested organs in wt plants and suggests that the enzyme may have functions other than phytate degradation during seed germination. / Ph. D.

O emprego de novas tecnologias, como a transgenia, associadas ao melhoramento convencional de cana-de-açúcar apresenta grande potencial no combate a pragas e desenvolvimento de novas variedades. Entretanto, a falta de especificidade na expressão temporal e/ou local dos genes introduzidos tem se mostrado um fator limitante para o sucesso de produtos derivados da transgenia. Os promotores das metalotioneínas, por exemplo, podem representar uma alternativa ao uso de promotores constitutivos, particularmente, aqueles de metalotioneínas do tipo 1 (MT1) por apresentarem níveis elevados de expressão, em diferentes tecidos/órgãos e serem responsivos a estresses bióticos e abióticos. O uso de promotores sintéticos, contendo apenas elementos-cis, como GCG-like, W boxes e JERE, tem sido relevante por induzirem a expressão gênica local em resposta ao ataque de agentes bióticos. Este trabalho teve por objetivo a caracterização funcional de promotores de genes de metalotioneínas de cana-de-açúcar e o uso de elementos regulatórios sintéticos e, em paralelo, avaliou-se o uso de promotores sintéticos no controle da expressão gênica em cana-de-açúcar. Após as análises de expressão transiente, verificou-se que o promotor SoMT1b apresentou atividade GUS e GFP em epitélios de cebola e os promotores sintéticos, 4X Wbox, 4X GCC-like, 4X JERE e 4xW 4xS-box, e o promotor SoMT1b foram capazes de dirigir a expressão do gene repórter uidA (GUS) em calos embriogênicos de cana-de-açúcar. Em análise de expressão estável, o promotor SoMT1b foi capaz de dirigir a expressão do gene GUS para os frutos e sementes de tomate \'Micro-Tom\', mas não foi responsivo a estresse por herbivoria, cádmio e cobre. Também foi realizada a transformação de plantas de cana-de-açúcar, as quais ainda estão sendo analisadas. Os resultados obtidos demonstram a funcionalidade do promotor SoMT1b / New technologies, like genetic transformation, associated with conventional breeding of sugarcane have a large potential in developing new varieties tolerant to biotic and abiotic stress. However, the lack of specificity in the spatial and/or temporal expression of the introduced genes has been a limited factor for the success of products derived from transgeny. Metallothionein promoters, for instance, can represent an alternative to the use of constitutive promoters, particularly those from metallothionein type 1 (MT1) because they present high expression levels in different tissues / organs and are responsive to biotic and abiotic stress. Another alternative is the use of synthetic promoters which contain only cis elements, as GCG-like, W-box and JERE, which induces local gene expression in response to pathogen attack. In this work, we aimed to make the functional characterization of sugarcane metallothionein promoters and synthetic regulatory elements, in parallel, we evaluated the use of synthetic promoters in the control of gene expression in sugarcane. After transient expression analyzis, it was found that SoMT1b promoter was able to control the expression of the reporter genes to GUS and GFP in onion epithelium and GUS in sugarcane embryogenic calli. Additionally, synthetic promoters 4X Wbox, 4X GCC-like, 4X JERE and 4xW 4xS-box were able to direct the expression of the gene uidA (GUS) in embryogenic calli of sugarcane. In stable transformation analysis, SoMT1b promoter was capable of directing uidA expression in fruits and seeds of tomato cv. \'Micro-Tom\', but it was not responsive to herbivory, cadmium and copper stress. It was also carried out the transformation of sugarcane plants with the construction containing the SoMT1b promoter, but these are still being analyzed. The results demonstrate the functionality of the SoMT1b promoter in sugarcane and tomato

ResumenEn esta tesis se ha optimizado un protocolo de regeneración a partir de material varietal de 'Helena' y 'Canino'. Mediante el estudio de los diversos factores que afectan la transformación de material adulto, se ha establecido por primera vez un protocolo eficiente de transformación mediada por Agrobacterium tumefaciens de una variedad comercial de albaricoquero.El diseño de una estrategia de selección gradual con paromomicina ha permitido la regeneración de plántulas transformadas con los genes marcadores nptII y sgfp o gus, con las eficiencias más elevadas que se han publicado hasta el momento para transformar material varietal en especies del género Prunus, aunque la baja viabilidad de las yemas transformadas redujo el número final de plantas obtenidas.El protocolo establecido en esta tesis sienta las bases que permitirán la introducción de genes de interés agronómico y comercial, modificando de manera discreta variedades élite aceptadas y establecidas en el mercado. / In this thesis a protocol of regeneration has been optimized from leaf explants of the cultivars 'Helena' and 'Canino'. By means of the study of the diverse factors that affect the transformation of adult material, an efficient protocol of Agrobacterium tumefaciens-mediated transformation has been established for the first time for a commercial cultivar of apricot.The design of a gradual selection strategy with paromomycin has permitted the regeneration of transformed shoots with the marker genes nptII and sgfp or gus, with the highest efficiencies that have been published up to now from adult material in Prunus, although the low viability of the transformed buds reduced the final number of plants obtained. This protocol establishes the bases that will permit the introduction of agronomic and commercial interesting genes, modifying discreetly commercial cultivars accepted and established in the market.

Made available in DSpace on 2016-06-02T20:23:14Z (GMT). No. of bitstreams: 1 5081.pdf: 5248422 bytes, checksum: dcbec39bdc1f1a1766bcfd2c71f4c065 (MD5) Previous issue date: 2012-08-29 / This research is an analysis of Gus Van Sant s Last Days (2005) soundtrack. From the concept of mis-en-bande, that emphasizes the interactions between the different sounds of the film narrative, we propose to consider the details of the Last Days s sound organization and its crucial contribution to the proposed narrative discourse. / Esta pesquisa consiste em uma análise da banda sonora do filme Last Days (2005), de Gus Van Sant. A partir do conceito de mis-en-bande, noção que valoriza as interações entre os diversos sons que compõem a narrativa cinematográfica, propomos pensar as nuances da organização sonora do filme e sua decisiva contribuição para os discursos narrativos propostos.