The Birnbaum-Saunders (BS) distribution is a positively skewed distribution and is a popular model for analyzing lifetime data. In this thesis, we first develop an improved method of estimation for the BS distribution and the corresponding inference. Compared to the maximum likelihood estimators (MLEs) and the modified moment estimators (MMEs), the proposed method results in estimators with smaller bias, but having the same mean squared errors (MSEs) as these two estimators. Next, the existence and uniqueness of the MLEs of the parameters of BS distribution are discussed based on Type-I, Type-II and hybrid censored samples. In the case of five-parameter bivariate Birnbaum-Saunders (BVBS) distribution, we use the distributional relationship between the bivariate normal and BVBS distributions to propose a simple and efficient method of estimation based on Type-II censored samples. Regression analysis is commonly used in the analysis of life-test data when some covariates are involved. For this reason, we consider the regression problem based on BS and BVBS distributions and develop the associated inferential methods. One may generalize the BS distribution by using Laplace kernel in place of the normal kernel, referred to as the Laplace BS (LBS) distribution, and it is one of the generalized Birnbaum-Saunders (GBS) distributions. Since the LBS distribution has a close relationship with the Laplace distribution, it becomes necessary to first carry out a detailed study of inference for the Laplace distribution before studying the LBS distribution. Several inferential results have been developed in the literature for the Laplace distribution based on complete samples. However, research on Type-II censored samples is somewhat scarce and in fact there is no work on Type-I censoring. For this reason, we first start with MLEs of the location and scale parameters of Laplace distribution based on Type-II and Type-I censored samples. In the case of Type-II censoring, we derive the exact joint and marginal moment generating functions (MGF) of the MLEs. Then, using these expressions, we derive the exact conditional marginal and joint density functions of the MLEs and utilize them to develop exact confidence intervals (CIs) for some life parameters of interest. In the case of Type-I censoring, we first derive explicit expressions for the MLEs of the parameters, and then derive the exact conditional joint and marginal MGFs and use them to derive the exact conditional marginal and joint density functions of the MLEs. These densities are used in turn to develop marginal and joint CIs for some quantities of interest. Finally, we consider the LBS distribution and formally show the different kinds of shapes of the probability density function (PDF) and the hazard function. We then derive the MLEs of the parameters and prove that they always exist and are unique. Next, we propose the MMEs, which can be used as initial values in the numerical computation of the MLEs. We also discuss the interval estimation of parameters. / Thesis / Doctor of Science (PhD)

Cotton has been a world-wide economic staple in textiles and oil production. There has been a concerted effort for cotton improvement to increase yield and quality to compete with non-natural man-made fibers. Unfortunately, cultivated cotton has limited genetic diversity; therefore finding new marketable traits within cultivated cotton has reached a plateau. To alleviate this problem, traditional breeding programs have been attempting to incorporate practical traits from wild relatives into cultivated lines. This incorporation has presented a new problem: uncultivated cotton hampered by photoperiodism. Traditionally, due to differing floral times, wild and cultivated cotton species were unable to be bred together in many commercial production areas world-wide. This worldwide breeding problem has inhibited new trait incorporation. Before favorable traits from undomesticated cotton could be integrated into cultivated elite lines using marker-assisted selection breeding, the markers associated with photoperiod independence needed to be discovered. In order to increase information about this debilitating trait, we set out to identify informative markers associated with photoperiodism. This study was segmented into four areas. First, we reviewed the history of cotton to highlight current problems in production. Next, we explored cotton’s floral development through a study of floral transition candidate genes. The third area was an in-depth analysis of Phytochrome C (previously linked to photoperiod independence in other crops). In the final area of study, we used Genotype-By-Sequencing (GBS), in a segregating population, was used to determine photoperiod independence associated with single nucleotide polymorphisms (SNPs). In short, this research reported SNP differences in thirty-eight candidate gene homologs within the flowering time network, including photoreceptors, light dependent transcripts, circadian clock regulators, and floral integrators. Also, our research linked other discrete SNP differences, in addition to those contained within candidate genes, to photoperiodicity within cotton. In conclusion, the SNP markers that our study found may be used in future marker assisted selection (MAS) breeding schemas to incorporate desirable traits into elite lines without the introgression of photoperiod sensitivity.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M.Sc. (Life Sciences (Microbiology))

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M. Sc. (Life Sciences (Microbiology))

Understanding of chronic sequelae development after infection with foodborne pathogens is limited and an increased understanding could assist with the development of more accurate burden of disease estimates. The purpose of this thesis was to determine via systematic review and meta-analysis of the published international literature, the proportion of cases of Salmonella, Campylobacter and E. coli O157 that will develop the chronic sequelae of reactive arthritis, haemolytic uraemic syndrome, irritable bowel syndrome, inflammatory bowel disease or Guillain Barré syndrome. This information can be used to increase our understanding of the relationship between infection and the development of long term health complications while providing a key piece of information for the development of accurate burden of disease estimates. / Canadian Institutes of Health Research Institute of Population and Public Health/Public Health Agency of Canada, Applied Public Health Research Chair (awarded to Jan M. Sargeant)

The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal. / Group B streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis and has recently been recognized as an increasingly common cause of invasive disease in immunocompromised adults (elderly or chronic diseases). GBS produces a number of extracellular enzymes, several of which interact with the host immune system and are important for the GBS- host interaction and for the development of disease. Previous studies showed that metalloproteases are involved in several metabolic pathways in different cellular types. For this reason, we decided to investigate the possible involvement of GBS metalloproteases during cell interaction and apoptosis/necrosis induced by microorganism in human umbilical vein endothelial cells (HUVEC) and epithelial respiratory cells line (A549). Treatment of GBS with metalloproteases inhibitors (EDTA, EGTA and PHEN) did not induce alteration on bacterial growth, but promoted changes in the expression of surface proteins, adhesive capacity and profile of intracellular survival of the pathogen. The GBS and supernatant of bacterial growth medium (conditioned medium; MC) promoted the death of HUVEC and A549 cells. However, the metalloproteases inhibitors treatment restored the cellular viability induced by GBS and MC, suggesting that GBS metalloproteases are involved in the disruption of cell barrier, promoting bacterial dissemination. This study describes for the first time apoptosis and necrosis induced by GBS and MC in HUVEC and A549 cells after 24h incubation, respectively. We also observe reduction of pro-caspase-3 after infection of HUVEC with GBS and MC, suggesting activation of caspase-3. Moreover, the over-expression of pro -apoptotic protein Bax and decrease of anti-apoptotic protein Bcl-2 levels in HUVEC show the involvement of mitochondrial apoptotic mechanism (intrinsic via). Enhanced understanding of the molecular basis of GBS pathogenesis may pinpoint novel bacterial and host molecules that can represent novel therapeutic or immunoprophylactic targets against disease caused by this foremost of neonatal pathogens.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal. / Group B streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis and has recently been recognized as an increasingly common cause of invasive disease in immunocompromised adults (elderly or chronic diseases). GBS produces a number of extracellular enzymes, several of which interact with the host immune system and are important for the GBS- host interaction and for the development of disease. Previous studies showed that metalloproteases are involved in several metabolic pathways in different cellular types. For this reason, we decided to investigate the possible involvement of GBS metalloproteases during cell interaction and apoptosis/necrosis induced by microorganism in human umbilical vein endothelial cells (HUVEC) and epithelial respiratory cells line (A549). Treatment of GBS with metalloproteases inhibitors (EDTA, EGTA and PHEN) did not induce alteration on bacterial growth, but promoted changes in the expression of surface proteins, adhesive capacity and profile of intracellular survival of the pathogen. The GBS and supernatant of bacterial growth medium (conditioned medium; MC) promoted the death of HUVEC and A549 cells. However, the metalloproteases inhibitors treatment restored the cellular viability induced by GBS and MC, suggesting that GBS metalloproteases are involved in the disruption of cell barrier, promoting bacterial dissemination. This study describes for the first time apoptosis and necrosis induced by GBS and MC in HUVEC and A549 cells after 24h incubation, respectively. We also observe reduction of pro-caspase-3 after infection of HUVEC with GBS and MC, suggesting activation of caspase-3. Moreover, the over-expression of pro -apoptotic protein Bax and decrease of anti-apoptotic protein Bcl-2 levels in HUVEC show the involvement of mitochondrial apoptotic mechanism (intrinsic via). Enhanced understanding of the molecular basis of GBS pathogenesis may pinpoint novel bacterial and host molecules that can represent novel therapeutic or immunoprophylactic targets against disease caused by this foremost of neonatal pathogens.

The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.

[ES] El melón (Cucumis melo L.) es una de las especies hortícolas más demandadas en el mundo, y como tal el mejoramiento vegetal tiene el desafío continuo de generar alternativas para el agricultor por medio del desarrollo de nuevas variedades que presenten, tolerancia/ resistencia a factores de estrés biótico (plagas y enfermedades) y abiótico (temperatura, humedad, suelo) y a su vez atributos de calidad apreciados por el consumidor. El mejoramiento convencional en melón por medio de cruzamientos entre líneas seleccionadas es una estrategia ampliamente utilizada, pero que presenta algunas limitaciones, como las barreras de cruzabilidad entre las variedades comerciales y el germoplasma silvestre, además de los períodos extensos de tiempo requeridos para evaluar las progenies. En este contexto, el injerto y el desarrollo de nuevos patrones están siendo adoptados a nivel mundial como estrategias alternativas de mejora en cultivos hortícolas, que permite aprovechar caracteres de interés presentes en especies no cultivadas o no comerciales sin necesidad de realizar largos y complejos planes de mejora (cruzamiento y retrocruzamiento). El uso de la diversidad intra e interespecífica como portainjertos para sandía ha dado muy buenos resultados, documentados en una amplia bibliografía científica. Sin embargo, las ventajas y beneficios del injerto presentan inconsistencias en los estudios realizados en melón. Esto se explica por los diferentes métodos de cultivos evaluados, así como por la amplia gama de variedades y tipos comerciales de melón existentes; esto exige a su vez, mayor inversión y fortalecimiento de la investigación en esta área del conocimiento. La presente Tesis Doctoral tiene por objetivo la generación de información relacionada al injerto en melón mediante la evaluación de portainjertos desarrollados en los grupos de mejora del Instituto de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), donde se ha llevado a cabo este trabajo, utilizando para ello diferentes condiciones de cultivo, y por lo tanto estrés, y utilizando dos tipos de melón muy consumidos en España y Europa como el ‘Piel de Sapo’ y el ‘Cantalupo’, además de una variedad tradicional muy apreciada por su calidad, el ‘Meló d’Or d’Ontinyent’. La recuperación de variedades tradicionales es un reto para la agricultura moderna y gracias al injerto validamos el potencial de esta técnica para producir ‘Meló d’Or d’Ontinyent’ bajo condiciones de estrés por la presencia de un hongo patógeno como Monosporascus cannonballus Pollack et Uecker en el suelo. El patrón experimental UPV-PRMc, desarrollado por el grupo de mejora de cucurbitáceas del COMAV de la Universidad Politécnica de Valencia, mediante la hibridación de un melón tipo ‘Piel de Sapo’ y una accesión de un melón silvestre del tipo agrestis, mostró su capacidad para proporcionar resistencia y vigor a la variedad tradicional, que sin el uso de un portainjerto resistente sería incapaz de culminar su ciclo de cultivo bajo estas condiciones. Ensayos de campo complementarios permitieron evaluar el comportamiento de diferentes portainjertos experimentales y comerciales de Cucurbita y Cucumis, en condiciones de estrés bajo sistemas de producción convencional en campo abierto. Con relación al vigor y al desarrollo de las plantas injertadas, los patrones de Cucurbita mostraron un retraso en el vigor temprano del cultivo y tanto Ma x Mo EF como Pe x Pe ZS tuvieron los más bajos rendimientos. Destaca el uso del híbrido Ma x Ec como una buena alternativa a los híbridos comerciales normalmente utilizados entre C. maxima x C. moschata. Por su parte los portainjertos de C. melo mostraron una mejor afinidad y mayor vigor consistentemente a lo largo de los 3 años de estudio, al igual que los híbridos interespecíficos Fi x My y Fi x An. Adicionalmente al estudio de la compatibilidad entre el melón Piel de Sapo y los diferentes patrones experimentales y comerciales, también se abordó el impacto sobre la calidad del fruto. Este aspecto es quizá el más controvertido en relación al injerto por el uso de patrones filogenéticamente distantes que pueden pertenecer a otra especie o género distinto al melón. Nuestros datos reflejaron que los patrones de Cucurbita tuvieron efecto sobre la forma del fruto y la cavidad seminal, mientras que los patrones de Cucumis melo presentaron un menor impacto sobre los parámetros de calidad del fruto. También es importante destacar que los patrones de especies silvestres o no cultivadas de Cucumis no afectaron significativamente la calidad, a pesar de que C. metuliferus E. Meyer ex Naudin mostró signos de colapso en el segundo año de evaluación y consistentemente produjo los frutos más pequeños en comparación a las plantas sin injertar. El estudio complementario sobre el perfil metabólico y aromático de los frutos ha permitido ampliar los criterios de selección de portainjertos de melón. Se observó la variación en el contenido de ácidos y azúcares principalmente debido al grado de maduración y en menor medida a una combinación de patrón-variedad específica. La modificación del perfil de compuestos volátiles mostró un efecto claro de los portainjertos de Cucurbita sobre la variedad de melón, con el incremento de compuestos aromáticos relacionados a la calabaza y la reducción de otros compuestos claves en el aroma del melón Piel de Sapo. Con la perspectiva de aprovechar el germoplasma silvestre como portainjerto, en el Capítulo 2 de la presente tesis doctoral, presentamos los estudios enfocados en la caracterización de especies silvestres o no cultivadas del género Cucumis, que son una fuente de resistencias a patógenos y enfermedades de importancia económica como nematodos, oído o Fusarium, y que están ausentes en las variedades comerciales de melón. La estandarización de metodologías para optimizar la germinación de accesiones de Cucumis silvestres ha sido un primer paso para maximizar el rendimiento de la semilla como material de partida, ya que la dormancia presente en el germoplasma no cultivado suele ser un factor limitante. Basados en los estudios preliminares de campo en los que se observó la diferencia de vigor entre una accesión de C. metuliferus y el melón ‘Piel de Sapo’, se estudió si los híbridos interespecíficos desarrollados por el grupo de mejora suponían una opción para el incremento del vigor y la combinación de características deseables en una misma planta capaz de soportar el injerto de una variedad comercial de melón. Las barreas de incompatibilidad, impidien los cruzamientos viables con C. metuliferus (filogenéticamente separado del resto de Cucumis africanos) pero los híbridos interespecíficos de los cruzamientos entre Cucumis ficifolius A. Rich. x Cucumis anguria L. y Cucumis ficifolius x Cucumis myriocarpus Naudin son viables. Estos materiales F1, mostraron un mayor vigor, un incremento en el diámetro de su hipocótilo y una buena compatibilidad con melones de tipo ‘Cantalupo’ y ‘Piel de Sapo’, bajo condiciones de invernadero y a campo abierto. Un alto grado de resistencia a Fusarium oxysporum f. sp. melonis como resultado de la combinación de sus genomas parentales, así como una buena tolerancia al estrés osmótico bajo condiciones in vitro, destacan como características deseables en estos dos híbridos interespecíficos. Utilizando especies silvestres e híbridos de Cucumis, se evaluaron distintas metodologías para la obtención de poliploides, con el objetivo de potenciar el vigor de las plántulas (tamaño y diámetro del hipocótilo) y estudiar los efectos de la poliploidización. La colchicina fue el compuesto antimitótico más efectivo aplicado sobre semillas pregerminadas de dos híbridos interespecíficos de Cucumis. En líneas homocigóticas de C. anguria, C. ficifolius, C. myriocarpus, o C. metuliferus, la duplicación del genoma no fue estable en ningún caso. La evaluación del nivel de ploidía confirmó que los alopoliploides obtenidos fueron hexaploides (6n=72). Los nuevos materiales obtenidos, son alopoliploides sintéticos que resultaron de la duplicación de un genoma híbrido. A diferencia de sus antecesores triploides, los alopoliploides mostraron la recuperación de su fertilidad, asociado posiblemente a la presencia de SNPs concretos relacionados a genes que codifican procesos reproductivos. La mejora de características de interés en especies silvestres (mayor diámetro de los hipocótilos) ha sido posible por medio de la poliploidización, lo que favorece el procedimiento de realización del injerto y la producción de la planta injertada. Los resultados presentados en esta tesis doctoral han sido posibles gracias a la colaboración de grupos de investigación interdisciplinarios de la Universidad Politécnica de Valencia, la Universidad Jaime I de Castellón y la Politécnica de Cataluña, en el marco del proyecto AGL2014-53398-C2-2-R APROXIMACIONES BIOTECNOLOGICAS Y CULTURALES PARA LA MEJORA DE LAS RESISTENCIAS Y EL CONTROL DE ENFERMEDADES EN MELON Y SANDIA y con apoyo de los proyectos AGL2017- 85563-C2-1-R de FEDER/Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación, y proyecto para Grupos de Excelencia PROMETEO 2017/078 además del apoyo de la SECRETARIA DE EDUCACIÓN SUPERIOR, CIENCIA, TECNOLOGÍA E INNOVACIÓN (SENECYT-ECUADOR) que por medio del Instituto de Fomento al Talento Humano (IFTH), proporcionó una beca de estudio al doctorando. / [EN] The melon (Cucumis melo L.) is one of the most demanded horticultural species in the world and as such, plant improvement has the constant challenge of thinking up alternatives for the farmer through the development of new varieties with tolerance / resistance to biotic (pests and diseases) and abiotic (temperature, humidity, soil) stress factors and quality attributes appreciated by the consumer. Conventional breeding in melon through crossings between selected lines is a widely used strategy, but it has some limitations, such as cross barriers between commercial varieties and wild germplasm, in addition to the extended periods of time required to evaluate the progeny. In this context, grafting and the development of new rootstocks are being adopted worldwide as alternative breeding strategies in horticultural crops, which allows to take advantage of relevant characteristics present in non-cultivated or non-commercial species without extensive and complex breeding programs (crossing and backcrossing). Development of rootstocks for watermelon crop derived from intra and interspecific diversity have been successful and well documented in a wide scientific bibliography. However, advantages and benefits from grafting show inconsistent data in melon assays. These results can be explained since different cultivation methods have been evaluated and because of the large number of commercial varieties of melon; this fact motivates a higher investment and strengthening of research in this area of knowledge. The present Doctoral Thesis has as objective the generation of information related with melon grafting, through the development, characterization and study under stress conditions of novel rootstocks used for two commercial cultivars consumed in Spain and Europe, like ‘Piel de Sapo’ and ‘Cantaloupe’, besides a traditional variety as the ‘Meló d’Or d’Ontinyent’. Recovery of traditional cultivars is a challenge for modern agriculture and thanks to grafting we have validated the potential of this technique to crop ‘Meló d’Or d’Ontinyent’ in soil stressed by Monosporascus cannonballus Pollack et Uecker. The experimental rootstock UPV-PRMc, developed by Institute for the Preservation and Improvement of Valencian Agro-diversity (COMAV) of the Polytechnic University of Valencia, from an hybridization between one ‘Piel de Sapo’ melon and one accession of wild agrestis melon, provided resistance and vigor to the traditional variety, which is not able to complete the crop cycle without be grafted onto a resistant rootstock. Complementary field trials allowed to test the behavior of experimental and commercial rootstocks of Cucurbita and Cucumis under stress conditions in conventional production systems. Regarding vigor and growth of grafted plants, Cucurbita rootstocks showed a delay in early vigor of plants and Ma x Mo EF and Pe x Pe ZS had the lowest yields. The Ma x Ec hybrid highlights as a good alternative to the C. maxima x C. moschata hybrids, commonly used in commercial grafting. On the other hand, C. melo rootstocks showed better affinity and higher vigor consistently along these 3 years, as well as the interspecific hybrids Fi x My and Fi x An. Additionally to the compatibility behavior between ‘Piel de Sapo’ melon and different experimental and commercial rootstocks, also the impact on fruit quality was studied. This issue is one of the most controversial about grafting because of the use of rootstocks phylogenetically distant that belong to other species or genera than melon. Our data showed an effect over shape and size of seed cavity when using Cucurbita rootstocks, while Cucumis melo ones showed a lower impact on fruit quality traits. It is important to note that rootstocks derived from wild or non-cultivated Cucumis species did not alter significantly the quality, despite of that C. metuliferus E. Meyer ex Naudin showed vine decline symptoms in the second year and consistently the smallest fruits compared to non-grafted plants. The complementary analysis of the aromatic and metabolic profile from fruits have allowed to increase the selection criteria for melon rootstocks. Variation in acids and sugars was observed mainly due to maturation degree and to a lesser extent to a specific rootstock-scion combination. The modification in volatile compounds levels displayed an effect of the Cucurbita rootstocks on melon cultivar, increasing aromatic compounds related to pumpkin and decreasing other key compounds in ‘Piel de Sapo’ aroma melon. With the prospect to take advantage from wild germplasm to develop new rootstocks, in the Chapter 2 in this Doctoral Thesis, we showed the studies focused on the characterization of wild or non-cultivated species in the Cucumis genus, which are a resistance resource against pathogens and diseases of economic importance like nematodes, powdery mildew or Fusarium, and that are absent in commercial melon. The standardization of methodologies to optimize the germination of wild Cucumis accessions has been the first step to maximize the seed yield as a started material, since the dormancy in non-cultivated germplasm is usually a limiting factor. Based on preliminary field studies, where the differences in vigor between C. metuliferus and ‘Piel de Sapo’ melon were observed, the development of interspecific hybrids was approached as an option to increase the vigor and combine characteristics of interest in one same plant able to bear the grafting with a commercial variety of melon. The incompatibility barriers prevented viable crosses with C. metuliferus (phylogenetically distant of the rest of African Cucumis), although two interspecific hybrids were viable from crosses between Cucumis ficifolius A. Rich. x Cucumis anguria L. and Cucumis ficifolius x Cucumis myriocarpus Naudin. These F1 plants had higher vigor, hypocotyl diameter and good compatibility with ‘Cantaloupe’ and ‘Piel de Sapo’ melons under field and greenhouse conditions. High level of resistance to Fusarium oxysporum f. sp. melonis as result of the combination of their parental genomes, as well as a good tolerance to osmotic stress under in vitro conditions, highlight as desirable characteristics in these two new interspecific hybrids. With the preliminary results on the improvement of traits in wild Cucumis germplasm from the hybridization, some methodologies were evaluated to obtain polyploids, in order to enhance the desirable features in these hybrids (vigor, hypocotyl size and diameter). Colchicine was the most effective antimitotic agent applied over pregerminated seeds of two Cucumis interspecific hybrids. In homozygous lines of C. anguria, C. ficifolius, C. myriocarpus, or C. metuliferus, genome duplication was not stable. The polyploidy level confirmed that allopolyploids obtained were hexaploids (6n=72). The novel materials obtained are synthetic allopolyploids that result from the duplication of a hybrid genome. Unlike their triploid ancestors, allopolyploids showed the recovery of their fertility, possibly associated with the presence of specific SNPs related to genes that encode reproductive processes. The improvement of characteristics of interest in wild species has been possible through polyploidization, which favors the development of new improved materials capable of adapting to the grafting process. The results presented in this Doctoral Thesis have been possible thanks to the collaboration of interdisciplinary research groups from Polytechnic University of Valencia, Jaime I University of Castellón and Polytechnic University of Catalonia, within the framework of the AGL2014-53398-C2-2 project. -R BIOTECHNOLOGICAL AND CULTURAL APPROACHES FOR THE IMPROVEMENT OF RESISTANCES AND THE CONTROL OF DISEASES IN MELON AND WATERMELON and with the support of the projects AGL2017-85563-C2-1-R of FEDER / Ministry of Science, Innovation and Universities-State Agency of Research and project for Groups of Excellence PROMETEO 2017/078, in addition to the support of the SECRETARIA DE EDUCACIÓN SUPERIOR, CIENCIA, TECNOLOGÍA E INNOVACIÓN (SENECYT-ECUADOR) which through the Institute for the Promotion of Human Talent (IFTH), provided a scholarship for the doctoral candidate. / [CA] El meló (Cucumis melo L.) és una de les especies hortícoles més demandades en el món, i consegüentment el millorament vegetal té el desafiament continu de generar alternatives per a l’agricultor per mitjà del desenvolupament de noves varietats que presenten, tolerància/resistència a factors d’estrès biòtic (plagues i malalties) i abiòtic (temperatura, humitat, sòl) i al seu torn atributs de qualitat apreciats pel consumidor. El millorament convencional en meló mitjançant creuaments entre línies seleccionades és una estratègia àmpliament utilitzada, però que presenta algunes limitacions, com la impossibilitat de creuar-se d’algunes varietats comercials i determinat germoplasma silvestre d’interès, a més dels períodes extensos de temps requerits per a avaluar les progènies. En aquest context, l’empelt i el desenvolupament de nous patrons estan sent adoptats a nivell mundial com a estratègies alternatives de millora en cultius hortícoles. L’empelt permet aprofitar caràcters d’interès presents en espècies no cultivades o no comercials sense necessitat de realitzar llargs i complexos plans de millora (creuament i retrocruzamiento). L’ús de la diversitat intra i interespecífica com a portaempelts de meló d’Alger ha donat molt bons resultats, documentats en una àmplia bibliografia científica. No obstant això, els avantatges i beneficis de l’empelt presenten inconsistències en els estudis realitzats en meló. Això s’explica pels diferents mètodes de cultius avaluats, així com per l’àmplia gamma de varietats i tipus comercials de meló existents; això exigeix al seu torn, major inversió i enfortiment de la investigació en aquesta àrea del coneixement. La present Tesi Doctoral té per objectiu la generació d’informació relacionada amb l’empelt en meló, mitjançant l’avaluació de nous portaempelts que s’han obtingut en els grups de millora del COMAV (Instituto de Conservación y Mejora de la Agrodiversidad Valenciana) on s’ha dut a terme aquest treballs fent servir diferents condicions de cultiu i per tant de estressos i dos tipus de meló molt consumits a Espanya i Europa com el ‘Piel de Sapo’ i el ‘Cantalupo’ a més d’una varietat tradicional reconeguda per la seua excel·lent qualitat, el ‘Meló d’Or d’Ontinyent’. La recuperació de varietats tradicionals és un repte per a l’agricultura moderna i mijançant l’empelt validem el potencial d’aquesta tècnica per a produir ‘Meló d’Or d’Ontinyent’ sota condicions d’estrès degudes a la presència en la terra d’un fong patogen com es el Monosporascus cannonballus Pollack et Uecker. El patró experimental UPV-PRMc, desenvolupat pel grup de millora de cucurbitàcies del COMAV, mitjançant la hibridació d’un meló tipus ‘Piel de Sapo’ i una accessió d’un meló silvestre del tipus agrestis, va mostrar la seua capacitat per a proporcionar resistència i vigor a la varietat tradicional, que sense l’ús d’un portaempelt resistent no completaria el cicle de cultiu sota aquestes condicions. Assajos de camp complementaris van permetre avaluar el comportament de diferents portaempelts experimentals i comercials de Cucurbita i Cucumis, en condicions d’estrès en sistemes de producció convencional a camp obert. En relació amb el vigor i al desenvolupament de les plantes empeltades, els patrons de Cucurbita van mostrar un retard en el vigor a l’inici del cultiu i tant Ma x Mo EF com Pe x Pe ZS van tindre els rendiments més baixos. Destaca l’ús de l’híbrid Ma x Ec com una bona alternativa als híbrids comercials normalment utilitzats que deriven de creuaments C. maxima x C. moschata. Per la seua part els portaempelts de C. melo van mostrar una millor afinitat i major vigor consistentment al llarg dels 3 anys d’estudi, igual que els híbrids interespecífics Fi x My i Fi x An. Addicionalment a l’estudi de la compatibilitat entre el meló ‘Piel de Sapo’ i els diferents patrons experimentals i comercials, també es va estudiar l’impacte sobre la qualitat del fruits. Aquest aspecte és potser el que més controvèrsia crea al voltant de l’ús de l’emplet on s’utilitzen portampelts filogenèticament distants que poden pertànyer a una altra espècie o gènere diferent al meló. Les nostres dades van reflectir que els patrons de Cucurbita van tindre efecte sobre la forma del fruit i la cavitat seminal, mentre que els patrons de Cucumis melo van presentar un menor impacte sobre els paràmetres de qualitat del fruit. També és important destacar que els patrons d’espècies silvestres o no cultivades de Cucumis no van afectar significativament la qualitat, a pesar que C. metuliferus E. Meyer ex Naudin va mostrar signes de col·lapse en el segon any d’avaluació i consistentment va produir els fruits més xicotets en comparació a les plantes sense empeltar. L’estudi complementari sobre el perfil metabòlic i aromàtic dels fruits ha permès ampliar els criteris de selecció de portaempelts de meló. Es va observar la variació en el contingut d’àcids i sucres principalment com a consequència del grau de maduració i en menor mesura a una combinació de patró-varietat específica. La modificació del perfil de compostos volàtils va mostrar un efecte clar dels portaempelts de Cucurbita sobre la varietat de meló, amb l’increment de compostos aromàtics relacionats amb la carabassa i la reducció d’altres compostos claus en l’aroma del meló tipus ‘Piel de Sapo’. Amb la perspectiva d’aprofitar el germoplasma silvestre per al desenvolupament de nous portaempelts, al Capítol 2 de la present tesi doctoral, presentem els estudis enfocats a la caracterització d’espècies silvestres o no cultivades del gènere Cucumis, que són una font de resistències a patògens i malalties d’importància econòmica com a nematodes, oïdi o Fusarium, i que no tenen en les varietats comercials de meló. L’estandardització de metodologies per a optimitzar la germinació d’accessions de Cucumis silvestres ha sigut un primer pas per a maximitzar el rendiment de la llavor com a material de partida, ja que la dormancia present en el germoplasma no cultivat sol ser un factor limitant. Basats en els estudis preliminars de camp en els quals es va observar la diferència de vigor entre una accessió de C. metuliferus i el meló ‘Piel de Sapo’, es van avaluar els híbrids interespecífics desenvolupats als grups de millora com una opció per incrementar el vigor i combinar característiques desitjables en una mateixa planta que a l’hora fora capaç de suportar l’empelt d’una varietat comercial de meló. Les barreas d’incompatibilitat, no fan posible la hibridació amb C. metuliferus (filogenèticament separat de la resta de Cucumis Africans) però els híbrids interespecífics viables dels creuaments entre Cucumis ficifolius A. Rich. x Cucumis anguria L. i Cucumis ficifolius x Cucumis myriocarpus Naudin són viables. Aquests materials F1, van mostrar un major vigor, un increment en el diàmetre del seu hipocòtil i una bona compatibilitat amb melons de tipus ‘Cantalupo’ i ‘Piel de Sapo’, en condicions d’hivernacle i a camp obert. També aquests materials han mostrat un alt grau de resistència a Fusarium oxysporum f. sp. melonis i una bona tolerància a l’estrès osmòtic (avaluada en condicions de cultiu in vitro), característiques desitjables en aquestos dos nous híbrids interespecífics. Partint del germoplasma de Cucumis silvestre i els híbrids obtinguts, es va avaluar diferents metodologies per a l’obtenció de poliploides, amb l’objectiu de potenciar el vigor de les plàntules (grandària i diàmetre dels hipocòtil) i estudiar els efectes de la poliploidització. La colxicina va ser el compost antimitòtic més efectiu al aplicar-lo a llavors pregerminadas dels dos híbrids interespecífics de Cucumis. En línies homozigòtiques de C. anguria, C. ficifolius, C. myriocarpus, o C. metuliferus, la duplicació del genoma no va ser estable en cap cas. L’avaluació del nivell de ploidía va confirmar que els al·lopoliploides obtinguts van ser hexaploids (6n=72). Els nous materials obtinguts, són al·lopoliploides sintètics que van resultar de la duplicació d’un genoma híbrid. A diferència dels seus antecessors triploides, els al·lopoliploides van mostrar la recuperació de la seua fertilitat, associat possiblement a la presència de SNPs concrets relacionats a gens que codifiquen processos reproductius. La millora de les característiques d’interès (major diàmetre dels hipocòtils) ha sigut possible mitjançant l’obtenció de poliploides, la qual cosa facilita el procediment per a dur a terme l’empelt i per tant la producció de planta empeltada. Els resultats presentats en aquesta Tesi Doctoral han sigut possibles gràcies a la col·laboració de grups d’investigació interdisciplinaris de la Universitat Politècnica de València, la Universitat Jaume I de Castelló i la Politècnica de Catalunya, en el marc del projecte AGL2014-53398-C2-2-R APROXIMACIONS BIOTECNOLOGICAS Y CULTURALES PARA LA MEJORA DE LAS RESISTENCIAS Y EL CONTROL DE ENFERMEDADES EN MELON Y SANDIA i amb suport dels projectes AGL2017- 85563-C2-1-R de FEDER/Ministeri de Ciència, Innovació i Universitats–Agència Estatal d’Investigació, i projecte per a Grups d’Excel·lència PROMETEO 2017/078 a més del suport de la SECRETARIA DE EDUCACIÓN SUPERIOR, CIENCIA, TECNOLOGIA E INNOVACIÓN (SENECYT-L’Equador) que per mitjà del ‘Instituto de Fomento del Talento Humano’ (IFTH), va proporcionar una beca d’estudi al doctorand. / Finalmente agradecer a mi país Ecuador y a la Secretaria De Educación Superior, Ciencia, Tecnología e Innovación (SENECYT) y al Instituto Nacional De Fomento Al Talento Humano (IFTH) que me apoyaron con una beca de estudios para realizar mi tesis Doctoral, con la que espero aportar además de conocimiento científico, alternativas y soluciones para la realidad agrícola del país. / Cáceres Burbano, AE. (2020). Desarrollo de nuevos portainjertos para melón mediante el uso de estrategias y herramientas biotecnológicas [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/147863 / TESIS