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.

The Tobacco mosaic virus (TMV) is a positive sense single stranded RNA virus and is found across the world. TMV can impact the overall yield and quality of the crop resulting in an economic loss. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, mottling, necrotic lesions and stunted growth. Historically, TMV has caused controversy on whether this economically significant virus is seedborne or seed transmitted. The objective of this study is to track TMV infection from infected seeds to seedlings to determine the percentage of seed transmission. This experiment used three pods from three different TMV infected cultivar K 326 flue-cured tobacco plants. Seeds from each pod were germinated in a growth chamber for approximately ten days. Samples were separated into seed coat, root and leaves after germination. Total RNA was extracted from each part and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample. Endpoint RT-PCR was used to determine a conservative threshold value from the RT-qPCR results. These results demonstrated that TMV influenced percent germination with a range from 94% to 50%. Seed coats had a significantly higher virus titer concentration (P < 0.05) when compared to the roots and leaves. Statistical analysis revealed highly significant (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Results demonstrate that TMV is seed transmitted in flue-cured tobacco. / Master of Science / The Tobacco mosaic virus (TMV) is an RNA virus that occurs globally in areas where tobacco is grown. TMV is a tobamovirus and infects over 350 plant species. TMV can reduce the yield and quality of the crop which will result in an economic loss for the grower. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, necrotic lesions, and stunted growth, and there are no effective ways to eradicate the virus. There has been controversy on whether to categorize TMV as a seedborne virus or a seed-transmitted virus because the location of the virus within a seed is unknown. This study examined seeds from three pods grown on three different TMV-infected flue-cured tobacco plants of cultivar K 326 to track TMV infection from infected seeds to seedlings. Seeds from each pod were germinated in a growth chamber for ten days and samples were separated into leaves, root and seed coat. Each sample had total RNA extracted and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample since this technology can detect small amounts of virus. Endpoint RT-PCR was used to conservatively determine an infection threshold value from the RT-qPCR results. Percent germination of TMV infected seeds ranged from 94% to 50%. Seed coats had a significantly higher virus titer (P < 0.05) when compared to the roots and leaves in each pod. Statistical analysis showed (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in iv leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Therefore, results show that TMV is seed-transmitted in flue-cured tobacco.

El Pepino mosaic virus (PepMV) es un Potexvirus que fue descrito por primera vez en pepino dulce (Solanum muricatum Ait.) en Perú (Jones et. Al., 1980). En 1999 se detectó en Holanda, infectando a tomate (Van der Vlugt et al., 2000) mostrando una variada sintomatología. Desde entonces, el PepMV se ha expandido rápidamente por las principales áreas productoras de tomate del Mundo. Este virus se ha convertido en uno de los principales problemas en la producción de tomate en Europa donde produce importantes pérdidas económicas. La rápida expansión del PepMV en las zonas afectadas ha estado facilitada por su eficaz transmisión mecánica con las operaciones de cultivo y mediante los insectos polinizadores (Lacasa et al., 2003), sin embargo no se ha detectado la existencia de ninguna especie de insecto capaz de actuar como vector del mismo. El PepMV podría permaneces en el campo de una cosecha a la siguiente infectando a la flora arvense que podría actuar como reservorio de la enfermedad. Asimismo se ha comprobado la transmisión por semilla que podría constituir la forma de dispersión del virus a la larga distancia (Córdoba et. A., 2007). Actualmente se conocen diferentes aislados del virus que afectan al tomate. En primer momento, debido a las diferencias biológicas y moleculares observadas entre el aislado del PepMV que infectaba a tomate y el original de pepino dulce, distintos autores consideraron el aislado de tomate como un aislado diferente, denominándolo aislado tipo tomate (Van der Vlugt y Beredsen, 2002). Estudios posteriores demostraron que los aislados de PepMV de Europa, América del Norte y Canadá comparados con el aislado original de pepino dulce presentaban diferencias evidentes en sintomología, así como estructura poblacional del PepMV en cultivo de tomate en España, analizando y secuenciando tres zonas distintas del genoma del virus. / Alfaro Fernández, AO. (2009). Epidemiología y variabilidad patogénica del virus del mosaico del pepino dulce (Pepino mosaic virus). Nuevas enfermedades asociadas a su presencia (torrao o cribado) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/7027

Los virus producen graves pérdidas económicas en la agricultura. Esta problemática es muy dinámica ya que cada año aparecen nuevas virosis y es frecuente los fenómenos de emergencia con una rápida expansión de los virus. El control de las enfermedades víricas resulta poco eficaz en muchos casos porque la población viral es capaz de evolucionar y superar dichas estrategias. Por ello es clave entender la dinámica de las poblaciones y los factores implicados en la evolución de los virus con respecto a distintos aspectos de su biología del ciclo viral: replicación, movimiento dentro de la planta, respuesta a los mecanismos de defensa de la planta, transmisión a otras plantas, etc. El objetivo de esta tesis ha sido el estudio de los factores implicados en la evolución de dos virus que difieren en su variabilidad genética y gama de huéspedes: i) el Virus 1 del marchitamiento del haba (Broad bean wilt virus 1, BBWV-1), del género Fabavirus; y ii) el Virus del mosaico del tabaco (Tobacco mosaic virus, TMV) del género Tobamovirus. Primero se han desarrollado una serie de herramientas metodológicas que han permitido la detección rápida de BBWV-1 mediante hibridación molecular de improntas, la detección y cuantificación de BBWV-1 y TMV y su diferenciación de otras virosis del mismo género mediante RT-PCR cuantitativa a tiempo real. Se ha llevado a cabo la construcción de clones de cDNA del genoma completo de BBWV-1 para obtener transcritos infecciosos que puedan ser usados para estudiar la biología molecular, evolución y epidemiología. Una vez desarrollado esta metodología se ha usado para evaluar la eficacia biológica de BBWV-1 en el huésped y el efecto de algunos factores: concentración del inóculo, estado de desarrollo de la planta, tipo de huésped, aplicación de un activador de la defensa de la planta, y la infección con otro virus. Así mismo se han estudiado los factores relacionados con la eficacia biológica del virus durante su transmisión por pulgones: título viral / Ferriol Safont, I. (2012). FACTORS INVOLVED IN THE EVOLUTION OF BROAD BEAN WILT VIRUS 1 AND TOBACCO MOSAIC VIRUS [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16000

Abstract Ficus carica L. is considered one of the oldest fruit trees in the Mediterranean basin and is widely grown and harvested for the consumption of its fruits dry and fresh. This species is affected by different virus diseases, especially by Fig mosaic disease (FMD), for which Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) and Fig fleck-associated virus (FFkaV) are associated. FMD is the most widespread disorder of this species, which represents a threat and a constraint for healthy fig production and germplasm exchange. Thus, the objective of the present doctoral research was the establishment of an efficient and rapid in vitro F. carica propagation, sanitation and conservation of free-FMD plant material for future large-scale commercialization. Initially, FMD-related viruses distribution was screened within the different fig plant organs (buds, leaves, syconia and seeds) of 14 Mediterranean genotypes (Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto nero, Catalanisca, Houmairi, Triboiti and Turca 'Serilop') which were utilized afterward as in vitro plant source material. RT-PCR assays revealed that all the aforementioned viruses were present without any exception in seeds, whereas only 4 viruses (FBV, FFkaV, FLMaV-1 and FMV) were detected in buds, leaves and syconia with highly variable infection rates. Moreover, encapsulation technology proved to be a powerful multiplication technique to sustain standard fig tissue culture protocol for three cultivars (Catalanisca, Palazzo and Bifera) and it gave high, almost similar, viability, regrowth and conversion rates. Microcutting rooting in one-step was achieved and conversion rate was comparable for the three cultivars. Furthermore, in order to eliminate FMD associated viruses, with the exception of FBV-1 which resisted to all the sanitation attempts, Caulogenesis and Meristem Tip Culture Protected by the Synthetic Seeds technique (MTC-SS) gave the best sanitation rates. Finally, F. carica (cv. Houmairi) artificial seeds conservation, for final delivery, was achieved. A high viability and moderate regrowth rates were registered with a lesser conversion rate strictly related to the plant growth regulators (PGRs) used. Keywords: Fig, mosaic, RT-PCR, virus distribution, cytokinins, encapsulation, micropropagation, synthetic seed. / Resumen La higuera (Ficus carica L.) es considerada como uno de de los árboles frutales más antiguos de la cuenca mediterránea y es ampliamente cultivado y cosechado para el consumo de sus frutos tanto secos como en fresco. Esta especie se ve afectada por diversas enfermedades virales, especialmente por la denominada "Fig mosaic disease" (FMD) asociada actualemnte a los virus: Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) y Fig fleck-associated virus (FFkaV). Esta enfermedad representa una amenaza y un obstáculo para la producción de higos y el intercambio de germoplasma. El principal objetivo del presente trabajo fue establecer un método de propagación de higuera in vitro para el saneamiento y la conservación de material vegetal libre de FMD para su posterior comercialización. Inicialmente, se estudió la distribución de los virus implicados en la enfermedad en diversos órganos de 14 genotipos de F. carica (Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto nero, Catalanisca, Houmairi, Triboiti y Turca 'Serilop'), los cuales fueron utilizados posteriormente como fuente material vegetal in vitro. Los resultados obtenidos mediante RT-PCR revelaron que todos los virus mencionados estaban presentes sin excepción en las semillas, mientras que sólo cuatro de ellos (FBV, FFkaV, FLMaV-1 y FMV) fueron en brotes, hojas y siconios con tasas de infección variables. Además, la tecnología de encapsulación demostró ser una técnica de multiplicación eficaz para poder aplicar el protocolo estándar de cultivo de tejidos de higo para tres cultivares (Catalanisca, Palazzo y Bifera) dando altas tasas de viabilidad, rebrote y conversión. Se logró el enraizamiento de microcortes en un solo paso y el índice de conversión fue comparable para los tres cultivares. La callogénesis y el culñtivo de meristemos con la técnica de la semilla sintética (MTC-SS) fueron las técnicas que proporcionaron mayores tasas de desinfección para los virus estudiados a excepción de con FBV-1, entidad viral que no fue eliminada con ninguna de las técnicas ensayadas. Por último, se logró la conservación de las semillas artificiales de higuera (cv Houmairi), registrándose una alta viabilidad y tasas de rebrote moderadas con un menor grado de conversión estrictamente relacionado con hormonas utilizadas. Palabras clave: Higuera, mosaico, RT-PCR, la distribución de los virus, hormonas, encapsulación, micropropagación, y la semilla sintética. / Resum La figuera (Ficus carica L.) és considerada un dels arbres fruiters més antics de la conca mediterrània i és àmpliament conreat i collit per al seu consum fresc i sec. Les malalties virals, especialment "Fig mosaic disease" (FMD), associada amb els viruses: Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) i Fig fleck-associated virus (FFkaV). Esta malaltia representa una amenaça per a la producció de figues i l'intercanvi de germoplasma. El principal objectiu d'aquest treball va ser estableixerun mètode de propagació de figuera in vitro per al sanejament i la conservació de material lliure de FMD per a su posterior commercialització. Inicialment, es va estudiar la distribució dels virus associats a FMD en diversos òrgans en 14 genotips de F. carica (Palazzo, Severoni Precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto diners, Catalanisca, Houmairi, Triboiti i Turca 'Serilop'), els quals van ser utilitzats posteriorment com a font de material vegetal in vitro. Els resultats obtinguts del anàlisis realitzats per RT-PCR van revelar que tots els virus eren presents sense excepció en les llavors, mentre que només quatre virus (FBV, FFkaV, FLMaV-1 i FMV) van ser detectats en brots, fulles i siconis amb taxes d'infecció variables. A més, la tecnologia d'encapsulació va demostrar ser una tècnica de multiplicació eficaç per poder aplicar el protocol estàndard de cultiu de teixits de figa per a tres cultivars (Catalanisca, Palazzo i Bifera) donant taxesadequades de viabilitat, rebrot i conversió. Es va aconseguir l'arrelament de microtalls en un sol pas i l'índex de conversió va ser comparable per als tres cultivars. La calogènesi i el cultiu de meristems protegits per llavors sintètiques (MTC-SS)van ser les tècniques que proporcionarem millores tases de desinfecció per als virus estudiats amb l'excepció de FBV-1 que es va resistir a tots els mètodes de sanejament. Finalment, es va aconseguir la conservació de la llavors artificials de figuera (cv. Houmairi), registrant-ne una alta viabilitat i taxes de rebrot moderades amb un menor grau de conversió estrictament relacionat amb hormones utilitzades. Paraules clau: Figuera, mosaic, RT-PCR, la distribució dels virus, hormones, encapsulació, micropropagació, i la llavor sintètica. / Yahyaoui, E. (2017). Use of standard and setup of non conventional techniques for the elimination of viruses associated with Fig Mosaic Disease (FMD) in fig germplasm (Ficus carica L.) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79876

No / Yellow leaf syndrome (YLS) of sugarcane has been associated with Sugarcane yellow leaf virus (ScYLV) and has been reported from most sugarcane growing countries around the world. As sugarcane is vegetatively propagated, it is important to use effective and sensitive detection methods to screen new propagating material. Virus detection in symptomatic tissue is currently achieved using enzyme linked immunosorbent assay (ELISA), tissue blot immunoassay (TBIA) or a conventional RT-PCR based assay. This paper reports the development of an improved assay based on multiplex real-time fluorescent RT-PCR. The new assay is 100-fold more sensitive than conventional RT-PCR, and incorporates a novel `RNA specific¿ internal positive control (based around the intron of the caffeic acid 3-o-methyltransferase gene) to guard against false negative results. The paper also describes the comparison of eight RNA extraction methods for sugarcane tissue giving a number of alternatives for different laboratory situations. The sensitivity of this assay has allowed the detection of ScYLV in many samples that were thought to be healthy following conventional testing (RT-PCR, ELISA or TBIA). The detection of ScYLV using this TaqMan assay can be applied to the production of ScYLV-free plants and prevents its spread through the propagation material.

Diplonema papillatum est un organisme unicellulaire qui vit dans l’océan. Son génome mitochondrial possède une caractéristique spéciale: tous les gènes sont brisés en de multiples fragments qui s’appellent modules. Chaque module est codé par un chromosome différent. L’expression d’un gène exige des épissages-en-trans qui assemblent un ARN messager complet à partir de tous les modules du gène. Nous avons précédemment montré que le gène cox1 est encodé dans neuf modules avec six Us non encodés entre le module 4 et le module 5 de l’ARN messager mature [1]. Nous n’avons identifié aucune séquence consensus connue de site d’épissage près des modules. Nous spéculons qu’un ARN guide (gRNA) a dirigé l’épissage-en-trans du gène cox1 par un mécanisme qui est semblable à l’édition d’ARN par l’insertion/la suppression des Us chez les kinétoplastides, le groupe sœur des diplonémides. Nous avons trouvé que les six Us sont ajoutés au bout 3’ de l’ARN d’une façon semblable à ceux ajoutés par le TUTase lors de l’édition de l’insertion des Us chez les kinétoplastides. Nous avons construit des profils de gRNA de l’épissage-en-trans avec les expressions régulières basé sur notre connaissance des gRNAs dans l’édition d’ARN chez les kinétoplastides. Selon la complémentarité partielle entre le gRNA et les deux modules adjacents, nous avons généré des amorces pour RT-PCR visant à détecter des séquences qui sont assorties à un des profils de gRNA. Une expérience pilote in vitro n’a pas permis de reconstituer l’épissage-en-trans des modules 3, 4, et 5, suggérant que nous devons améliorer nos techniques. / Diplonema papillatum is a single cellular organism that lives in the ocean. Its mitochondrial genome possesses a special feature: all genes are fragmented in multiple pieces that are called modules and each module is encoded by a different chromosome. Expression of a gene requires trans-splicing that successfully assemble a full-length mRNA from all modules of the gene. It was previously shown that the cox1 gene is encoded in nine modules that are all located on different chromosomes; moreover, a stretch of six non-encoded Us exist between Module 4 and 5 in the mature mRNA [1]. No consensus sequence of known splicing sites was identified near the modules. We speculate that trans-splicing of the cox1 gene is directed by guide RNAs (gRNAs) via a mechanism that is similar to U-insertion/deletion editing in kinetoplastids, the sister group of diplonemids. We have detected populations of small RNA molecules that could come from mitochondrial. We found that the six Us were added to the 3’ end of Module 4 in a similar way to the Us added by the TUTase in kinetoplastid U-insertional editing. Sequence profiles of possible trans-splicing gRNAs were constructed in regular expressions based on our knowledge of known gRNAs in kinetoplastid RNA editing. According to the complementarity between the gRNA and the two adjacent modules, primers were designed for RT-PCR that aims to detect gRNA sequences. Among the results, we identified sequences that match or partially match the gRNA profiles. A pilot in vitro assay did not reconstitute trans-splicing of module 3, 4 and 5, suggesting that further technical improvements are needed.

Heat shock proteins (Hsp) are highly conserved proteins that are part of the universal stress response of the cell. Their main function is to protect cells against structural and functional damage. Organisms exposed to different forms of stress, such as e.g. a lack of nutrients or water, hypoxia, infection or inflammation, demonstrated an increased gene expression of these proteins. Pregnancy complications cause stress conditions for maternal and fetal organism, which may result in an increased gene expression of Hsp. In my thesis, I examined the concentration of extracellular mRNA for five different heat shock proteins (Hsp27, Hsp60, Hsp70, Hsp90, HspBP1) in the plasma of pregnant women and wheather this concentration is affected by possible pregnancy complications (preeclampsia, fetal growth restriction and gestational hypertension). I also investigated a possible correlation between mRNA plasma concentration for Hsp and pulsatility index values (PI) obtained by Doppler ultrasound. This research should help to invent a new predictive method for pregnancy complications, based on a detection of specific biomarkers in the first trimester of pregnancy. The research was conducted on plasma samples obtained from peripheral blood of pregnant women, whose collection was performed during clinical manifestations of...

Prostate carcinoma is considered to be one of the main medical problem in male population. Prostate carcinoma is the most frequently diagnosed malignancy in men and the death rate has the second position within all diagnosed malignancies in Czech Republic (ÚZIS). There is only one reliable diagnostic tool: PSA (prostate specific antigen). Level of PSA is often elevated in men with prostate carcinoma. This diploma thesis is focused on study of changes in gene expression in prostate carcinoma. Three candidate genes were analyzed: VCL (vinculin), SHB (Src homology 2 binding protein) and OCT3 (organic cation transporter 3). According to recent publications, these genes are related to tumor progression and they could have prognostic significance. In this thesis the following methodological approaches were used: 82 prostatic specimens were collected from patients and mRNA was isolated from these specimens; then RT-PCR was used to obtain cDNA, fragments were detected by electrophoresis. At the end statistical methods were used for evaluation. Relative expression of the genes in prostate carcinoma tissue was compared to relative expression of the genes in BPH (benign prostatic neoplasia) tissue. Results showed higher expression of genes SHB and OCT3 in prostate carcinoma tissue in compariscon to BPH...

4 Abstract Prostate cancer is one of the major medical problems within the male population in the Czech Republic and in the world. It is on second place among cancer illnesses with respect to mortality in czech male population. Its incidence strongly increases with age. Prostate cells have a unique ability to accumulate zinc in high concentrations compared to other tissues of human body. It is necessary for the proper physiological function of the prostate. There was detected loss of this accumulation ability in prostate cancer cells, which seems to be a condition to carcinogenesis in prostate cells. In this thesis was investigated the expression of four genes involved in the maintenance of homeostasis of zinc in prostate cells. Genes ZIP1 and ZIP7 encode zinc transporters, genes MT1-F and MT2 encode metallothioneins. There was collected 90 biopsy specimens from patients with prostate cancer or with benign prostatic hyperplasia. mRNA was isolated from these samples, cDNA was obtained by RT-PCR. This cDNA was detected by gel electrophoresis and the results were statistically evaluated. Several correlations was found between gene expression and the clinical data of patients. The most important result, there was found lower levels of expression of genes MT1- F and ZIP1 in samples of patients with cancer...