Philosophiae Doctor - PhD / Water availability and climate related issues are some of the greatest crop production risks to irrigated agriculture in arid regions. In South Africa, for example, the increasing frequency and severity of droughts related to climate change and the growing competition for limited water resources among different users threaten the sustainability and growth of irrigated agriculture, especially the water-intensive fruit industry. Major fruit such as apples (Malus domestica Borkh) are produced entirely under irrigation in South Africa. As a result, there has been considerable research to accurately quantify the water requirements of fruit tree orchards in order to maximize water productivity.

A rearing methodology for dogwood borer was developed, using standardized procedures at each developmental stage. These methods enabled the establishment of a laboratory colony and efficient production of synchronized cohorts of each of its lifestages throughout the year for specific experimental needs. The behavioral repertoire shown by mated female dogwood borer in an apple orchard was characterized and quantified and the diel periodicity with which those behaviors occurred was determined. Mated females were easily recognized, based on their characteristic casting flight directed toward areas below the graft union of apple trees, and were observed more frequently during the late afternoon and evening. Casting flight, probing with the ovipositor, and oviposition were the most frequent behaviors observed, but the duration of those behaviors was relatively short compared with the much lengthier periods of resting behavior that typically occurred within the canopy. Data from a previous, three-year study in two newly planted apple orchards were subjected to geostatistical analyses to examine the temporal and spatial patterns of infestation by larval dogwood borer and to gain further information about the spatial scales at which oviposition occurs. There were moderate to high degrees of aggregation of dogwood borer infestations on neighboring apple trees, with ranges of spatial dependence from 7.50–19.87 m. No directionality was observed in the spatial autocorrelation of infestation and it appears that females utilized oviposition sites equally along and across orchard rows. The aggregated nature of infestations requires that random, independent samples must be taken from a number of sample pairs at distances greater than the range of spatial dependence to ensure that sample data are not autocorrelated. Alternatively, an efficient sampling program for mapping dogwood borer infestation can be achieved by limiting sample points to distances within the range of spatial dependence. These sample points can be used in interpolating algorithms, such as kriging, to predict infestation at unsampled locations in space for use in site-specific pest management programs. The external morphology of male and female dogwood borer antennae and their sensilla were examined using light and scanning electron microscopy to characterize, measure and compare the types, number, and distribution of sensilla. Although the general shape and size of male and female antennae were similar, those from females possessed a greater number of generally smaller antennal flagellomeres. The flagellum of both male and female antennae contained seven sensillum types including auricillica, basiconica, chaetica, coeloconica, squamiformia, styloconica, and three subtypes of sensilla trichoidea. With the exception of sensilla basiconica, which were present in roughly equal numbers on male and female antennae, all other sensillum types were significantly more abundant on female antennae. The antennae of female dogwood borer appear well equipped to perceive olfactory stimuli, based on the types and number of sensilla present. Coupled gas chromatography and electroantennogram detection (GC-EAD) analyses of headspace collections from damaged and undamaged tissues from apple and dogwood trees were conducted to examine and compare the antennal responsiveness of female dogwood borer to host plant volatiles. A total of 16 and 9 compounds from apple and dogwood tissues, respectively, consistently elicited an antennal response in females. There were no differences in the response of antennae from virgin and mated females, and the amplitude of the female response to host odors was greater than that of males. Six compounds were identified from the headspace collections from apple trees, four of which (octanal, nonanal, decanal, and methyl salicylate) were identified from all apple tissues sampled. A novel compound,α-bergamotene, was identified from injured apple bark, from apple burr knots infested with dogwood borer larvae and from larval dogwood borer frass, and appears to be produced by apple trees in response to injury. Another novel compound, methyl-2,4-decadienoate, was identified from infested burr knot tissue and appears to be produced in response to an insect-plant interaction. Two compounds, hexanoic and nonanoic acid, were identified from headspace collections from dogwood trees. Numerous approaches were used to examine the behavioral response of mated female dogwood borer to host plant headspace collections and to individual compounds from those collections that elicited a strong and repeatable antennal response. Under both natural and semi-natural conditions in the field and in laboratory bioassays, neither attraction/orientation or consistent oviposition were documented and it is apparent that correlating the electrophysiological and behavioral responses of mated female dogwood borer to olfactory stimuli from their host plants will require further research on bioassay development. / Ph. D.

En raison de leur rôle central dans la physiologie et le développement des plantes, les phythormones ont depuis longtemps été considérées comme des médiateurs chimiques déterminants dans la capacité des insectes à contrôler leur environnement végétal. Les mécanismes permettant aux insectes de manipuler la balance phytohormonale permettant ainsi la régulation des apports nutritifs et la modulation des défenses végétales demeurent néanmoins pour la plupart inconnus, en particulier pour les insectes galligènes et mineurs de feuilles. L’objectif de ma thèse visait à caractériser les capacités de modulation phytohormonale par les insectes manipulateurs de plantes avec un accent particulier sur le lépidoptère mineur de feuille Phyllonorycter blancardella. Nous avons ainsi développés une caractérisation spatio-temporelle de la réponse des plantes aux attaques des larves mineuses au niveau moléculaire et biochimique. Une comparaison entre différents systèmes biologiques nous a permis d’évaluer les similitudes entre les stratégies adoptées par les les insectes galligènes et les insectes mineurs de feuilles, d’identifier l’origine possible des phytohormones impliquées dans la manipulation de la plante et le rôle des bactéries endosymbiotiques d’insectes dans ces interactions. / Because phytohormones lie at the very core of molecular mechanisms controlling the plant physiology and development, they have long been hypothesized to be involved in insect-induced plant manipulations. Insects are using phytohormones to manipulate their host plants for their own benefit, regulating nutrient provisioning and plant defenses. However, a mechanistic understanding of how phytohormones operate in plant reconfigurations by plant-manipulating insects, especially by gall-inducing and leaf-minging insects, is lacking. The objective of my Ph.D. was to provide an extensive characterization of how plant-manipulating insects modulate the plant global hormonal balance with a specific focus on the leaf-mining moth Phyllonorycter blancardella. We thus developed a time course characterization of plant transcriptomic and biochemical responses following attack by leaf-mining larvae. A comparative analysis between different biological systems allowed us to estimate similarities in strategies developed leaf-mining and gall-inducing insects, to identify the possible origin of phytohormones involved in the plant manipulation and to estimate the role of insect endosymbiotic bacteria in these interactions.

Made available in DSpace on 2016-12-08T16:44:38Z (GMT). No. of bitstreams: 1 PGPV11MA076.pdf: 421773 bytes, checksum: 6fbf2a3579e5da5aa39372d361a40b76 (MD5) Previous issue date: 2011-07-22 / The aim of this study was to evaluate effects of spraying apple trees with prohexadione-calcium (ProCa) (na inhibitor of gibberellins synthesis) and gibberellin (GA₃) on vegetative growth, yield potential, fruit quality, and occurrence of bitter pit in the fruit. The experimente was conducted in na orchard located in São Joaquim, Santa catarina State (Southern Brazil), in 2009/2010. catarina and Fuji apple trees were sprayed with water (control), ProCa and gibberellin (both products at the dose of 319 mg L¯¹), at the petal fall stage (October 15, 2009), when shoots were 5-10 cm long, with treatments repeated after 20 days. Leaves were assessed (in termsmof chlorophyll contente, área, dry mater and specific área) in January/2010, and length of current season shoots and weight of shoots removed by winter pruning were assessed in May/2010. The number of flower buds ando f fruit per flower bud were assessed in October/2009 and November/2010, respectively. Fruit were harvest at comercial maturity and then assessed for percentage of red skin área, skin color, density, weight, skin and pulp textures, flesh firmness, titratable acidity (TA), solids soluble contente (SSC) and starch index. Fruit were cold stored (0±0.5°C/90-95% RH) for four months, followed seven days for simulate marketing (20±4°C/60-70% RH) for four months, same evaluation carried out at harvest, as well as for incidence (%) and index of bitter pit. Trees treated with ProCa had higher leaf chlorophyll contente and lower specific leaf área in Fuji , lower leaf área in Catarina , lower shoot growth in both cultivars, and lower weight of shoots removed by winter pruning in Catarina , in the year following the treatment of the trees, both cultivars sprayed with ProCa had lower fruit set. the ProCa incresead the red color in Catarina apples. At harvest, the force for pulp penetratoon in the red side of the fruit was incresead in apple trees of both cultivars sprayed with ProCa. After cold stored, Fuji apples of trees sprayed with GA₃ had lower flesh firmness, while Catarina apples of trees sprayed with ProCa had higher firmness. Fruit Ca contente was not diferente between ProCa and GA₃ and between ProCa e the control in both cultivars. Fuji apples from trees treated with ProCa had lower N/Ca ratio in the skin tissue. Teh treatment with ProCa incresead the flesh firmness in the reddish fruit side and reduced the index of bitter pit during cold storage in both cultuivars. The use of ProCa in apples orchards might represent a new technology to reduce trees vegetative growth and the occurrence of bitter pit in the fruit. The GA₃ sprayed after full bloom increases trees vegetative growth and might cause changes in fruit quality atributes, leading to advanced maturiry, as well as increases the development of bitter pit / Este trabalho foi desenvolvido com o objetivo avaliar os efeitos da pulverização de macieira com um inibidor da síntese de giberelinas, o prohexadiona-cálcio (ProCa), e com giberelina (GA₃), no crescimento vegetativo e potencial produtivo das plantas, e na qualidade e ocorrência de bitter pit nos frutos. O experimento foi conduzido em um pomar localizado no município de São Joaquim, SC, na safra 2009/2010. Macieiras Catarina e Fuji foram pulverizadas com água (tratamento controle), ProCa e GA₃ (ambos os produtos na dose de 319 mg L¯¹), na queda das pétalas (15/10/2009), quando as brotações do ano estavam com 5-10 cm de comprimento, sendo repetidas após 20 dias. Foram feitas avaliações foliares (teor de clorofila, área, matéria seca e área específica), em janeiro/2010, e de comprimento dos ramos do ano e de peso dos ramos podados, em maio/2010. A contagem dos cachos florais e do números de frutos por cacho floral ocorreram em outubro/2009 e novembro/2010, respectivamente. A colheita foi realizada na maturação comercial, ocasião em que foram feitas análises nos frutos do índice de cor vermelha, cor da epiderme, densidade, peso, atributos de textura (força para a penetração da casca e força para a penetração da polpa), firmeza de polpa, acidez titulável, sólidos solúveis totais (SS) e índice de iodo-amido. Os frutos foram armazenados em câmara fria convencional (0±0,5°C/90-95% UR), durante quatro meses, seguido de sete dias de comercialização simulada (20±4°C/60-70 UR), e após submetido as mesmas avaliações feitas na colheita, bem como de incidência (%) e índice de bitter pit . O tratamento com ProCa aumentou o teor de clorofila e reduziu a área foliar específica em macieira Fuji , reduziu a área foliar em Catarina , reduziu o comprimento dos ramos do ano nas duas cultivares e o peso dos ramos podados em Catarina . No ano subsequente ao da aplicação dos tratamentos, macieiras pulverizadas com o ProCa, de ambas as cultivares, apresentaram menor frutificação. O tratamento com ProCa proporcionou maior coloração vermelha em maçãs Catarina . No momento da colheita, maçãs Fuji e Catarina provenientes de plantas pulverizadas com ProCa apresentaram maior força para a penetração da polpa no lado mais vermelho dos frutos. Após o armazenamento, maçãs Fuji de plantas pulverizadas com GA₃ apresentaram menor firmeza de polpa e maçãs Catarina de plantas pulverizadas com ProCa apresentaram maior firmeza de polpa. O teor de Ca nos frutos não diferiu entre os tratamentos ProCa e GA₃ e entre ProCa e controle, em ambas as cultivares. Maçãs Fuji apresentaram menor relação N/Ca no tecido da casca quando pulverizadas com ProCa. O tratamento com ProCa proporcionou aumento na firmeza no lado mais vermelho do fruto e reduziu o índice de bitter pit durante o armazenamento refrigerado em ambas as cultivares. A utilização do ProCa na cultura da macieira pode ser uma nova alternativa para o controle do crescimento vegetativo de da manifestação do bitter pit nos frutos. O GA₃, pulverizado em pós-floração, em macieiras aumenta o crescimento vegetativo e pode ocasionar algumas modificações nos atributos de qualidade nos frutos, indicando um avanço na maturação, e na manifestação do bitter pit

Made available in DSpace on 2014-08-20T14:22:08Z (GMT). No. of bitstreams: 1 Tese_Fernando_Jose_Hawerroth.pdf: 6659971 bytes, checksum: e37f7fea29ee58245d356204894a8ce6 (MD5) Previous issue date: 2010-11-23 / The proper balance between vegetative growth and frutification in pome fruit species such as apple and pear, it is essential to increase production efficiency and improving fruit quality. In this sense, control of vegetative growth and frutification increase are needed in the management of these species, which may be obtained by use of growth regulators. The objective of this study was to evaluate the control of vegetative growth and frutification increase of apple and pear orchards in the Southern Brazil climatic conditions in response to the use of growth regulators. For this, three experiments were carried out in this research. In the first experiment, different concentrations of prohexadione calcium were evaluated in 'Imperial Gala' and 'Fuji Suprema' apple trees, grafted on Marubakaido rootstock with M9 interstock, in an orchard located in Fraiburgo/SC. The treatments (0, 165, 330, 495, 660, and 990 g ha-1 prohexadione calcium) were applied during the vegetative growth in 2008/2009 and 2009/2010 growing seasons. The concentrations corresponding to each treatment were applied split into three parts. The first application was done when the shoots of the control treatment showed growth of 10 cm. The second and third applications were made at 30 and 60 days after the first application, respectively. The prohexadione calcium was effective in controlling vegetative growth of apple trees 'Imperial Gala' and 'Fuji Suprema', reducing the total weight and average weight of pruned shoots, as well as the average shoot length, in the Southern Brazil climatic conditions. The use of prohexadione calcium at concentrations ranging from 165 to 330 g ha-1 increased the fruit production of 'Imperial Gala' apples, but high concentrations of this growth regulator tends to reduce the fruit production, especially in 'Fuji Suprema' apples. The reduction of vegetative growth by the use of prohexadione calcium contributed to increase calcium content in fruits of 'Fuji Suprema'. The second experiment was carried out in Pelotas/RS, using 'Hosui' pears grafted on Pyrus calleryana rootstock. Different 8 concentrations of prohexadione calcium were evaluated (0, 275, 550, and 825 g ha- 1), being half the concentration corresponding to each treatment applied when the shoots had between 5 to 10 cm in length, and the remainder applied 30 days after the first application. The use of prohexadione calcium was effective in controlling the vegetative growth of 'Hosui' pears, decreasing the need for winter pruning by reducing the total weight and the number of shoots pruned. The control of vegetative growth by use of prohexadione calcium determined increase the productive capacity of 'Hosui pears, especially at concentrations ranging from 450 to 750 g ha-1. The aim of the third experiment was to evaluate the effectiveness of thidiazuron, gibberellic acid, prohexadione calcium and combination of these substances on frutification increase of 'Shinseiki' asian pears. The following treatments were applied at full bloom: 1. control (no application); 2. thidiazuron (TDZ) 20 mg L-1; 3. gibberellic acid (GA) 20 mg L-1; 4. prohexadione calcium (PCa) 600 mg L-1; 5. PCa 600 mg L-1 + TDZ 20 mg L-1; 6. PCa 600 mg L-1 + AG 20 mg L-1; e 7. AG 20 mg L-1 + TDZ 20 mg L-1. The application thidiazuron at 20 mg L-1, gibberellic acid at 20 mg L-1 and the combination of these substances during the full bloom increased significantly the frutification and the fruit production of 'Shinseiki' pears. The prohexadione calcium, when sprayed at flowering alone or in combination to thidiazuron and gibberellic acid, was not effective to increase the fruit set and fruit production. The use of growth regulators on flowering decreased the number of seeds per fruit. / O adequado balanço entre o desenvolvimento vegetativo e a frutificação em espécies pomáceas, como a macieira e a pereira, é fundamental ao aumento da eficiência produtiva e a melhoria da qualidade dos frutos. Neste sentido, o controle do desenvolvimento vegetativo e o aumento da frutificação são necessários no manejo de tais espécies, podendo ser obtidos pelo uso de fitorreguladores. Objetivou-se com este estudo avaliar o controle do desenvolvimento vegetativo e o aumento da frutificação de macieiras e de pereiras nas condições climáticas do Sul do Brasil em resposta ao uso de fitorreguladores. Para tanto, foram realizados três experimentos. No primeiro experimento, foram avaliadas diferentes concentrações de proexadione cálcio em macieiras Imperial Gala e Fuji Suprema , enxertadas no porta-enxerto Marubakaido com interenxerto de M9, em pomar localizado em Fraiburgo/SC. Os tratamentos (0; 165; 330; 495; 660; e 990 g ha-1 de proexadione cálcio) foram aplicados durante o período de desenvolvimento vegetativo nos ciclos 2008/2009 e 2009/2010. As concentrações respectivas a cada tratamento foram aplicadas parceladamente em três momentos. A primeira aplicação foi realizada quando as brotações do tratamento testemunha apresentavam crescimento de 10 cm. A segunda e a terceira aplicação foram realizadas aos 30 e 60 dias após a primeira aplicação, respectivamente. O proexadione cálcio foi eficiente no controle do desenvolvimento vegetativo de macieiras Imperial Gala e Fuji Suprema , reduzindo a massa total e a massa média de ramos, assim como o comprimento médio dos ramos, nas condições climáticas do Sul do Brasil. O uso de proexadione cálcio em concentrações variando de 165 a 330 g ha-1 aumentou a produção de maçãs Imperial Gala , porém altas concentrações deste fitorregulador tendem a reduzir a produção de frutos por planta, sobretudo em macieiras Fuji Suprema . A redução do desenvolvimento vegetativo pelo uso do proexadione cálcio contribuiu 6 para o aumento dos teores de cálcio em maçãs Fuji Suprema . O segundo experimento foi realizado no município de Pelotas/RS, utilizando pereiras Hosui enxertadas no porta-enxerto Pyrus calleryana. Foram avaliadas diferentes concentrações de proexadione cálcio (0; 275; 550; e 825 g ha-1), sendo metade da concentração respectiva a cada tratamento aplicada quando as brotações apresentavam entre 5 a 10 cm de comprimento, e o restante aplicado 30 dias após a primeira aplicação. O uso de proexadione cálcio foi efetivo no controle do desenvolvimento vegetativo de pereiras Hosui , minimizando a necessidade de poda hibernal pela redução da massa total e do número de ramos podados. O controle do desenvolvimento vegetativo pelo uso de proexadione cálcio determinou aumento da capacidade produtiva de pereiras Hosui , sobretudo em concentrações de 450 a 750 g ha-1. O objetivo do terceiro experimento foi avaliar a efetividade do thidiazuron, ácido giberélico, proexadione cálcio e a combinação destas substâncias no aumento da frutificação de pereiras asiáticas Shinseiki . Os seguintes tratamentos foram aplicados na plena floração: 1. testemunha (sem aplicação); 2. thidiazuron (TDZ) 20 mg L-1; 3. ácido giberélico (AG) 20 mg L-1; 4. proexadione cálcio (PCa) 600 mg L-1; 5. PCa 600 mg L-1 + TDZ 20 mg L-1; 6. PCa 600 mg L-1 + AG 20 mg L-1; e 7. AG 20 mg L-1 + TDZ 20 mg L-1. A aplicação de thidiazuron 20 mg L-1, ácido giberélico 20 mg L-1 e a combinação destas substâncias durante a plena floração aumentaram significativamente a frutificação e a produção de pereiras Shinseiki . O proexadione cálcio, quando aplicado na floração isoladamente ou em combinação ao thidiazuron e ao ácido giberélico, não se mostrou efetivo no aumento da frutificação e na produção de frutos. A utilização dos fitorreguladores na floração diminuiu o número médio de sementes por fruto.

Dans un contexte de changements climatiques, le stress hydrique et la gestion de l'eau sont considérés comme une contrainte importante pour le secteur agricole. Ainsi la sélection pour la tolérance à la sécheresse est devenue un objectif majeur pour de nombreux programmes de sélection. La vulnérabilité à la cavitation est considérée comme un trait d'intérêt pour la sélection à une sécheresse extrême, en particulier pour les plantes ligneuses. Pourtant, l'étendue de sa variabilité et sa relation avec la tolérance à la sécheresse sont mal documentées à l’échelle intra-spécifique, et en particulier dans les espèces cultivées. Dans cette étude, la variabilité génétique de la vulnérabilité à la cavitation a été étudiée sur trois dispositifs expérimentaux différents avec trois arbres d’intérêt agronomique qui sont menacés par le risque de sécheresse: noyer, hévéa et pommier. Une faible ou aucune, variation de la vulnérabilité à la cavitation a été trouvée dans les espèces étudiées ainsi qu'entre deux espèces de noyers malgré les différences de traits précédemment rapportés. Ces résultats suggèrent une canalisation de la résistance à la cavitation dans les organes critiques (branches, tiges). Chez le pommier, le porte-greffe est soupçonné de provoquer de la plus étroite résistance à la cavitation sur le greffon. Par contre, des différences ont bien étés trouvées sur d’autres traits de réponse à la sécheresse comme la régulation stomatique, la chute des feuilles ou encore la vulnérabilité à la cavitation dans le pétiole. Ainsi, la vulnérabilité à la cavitation des organes critiques n’est pas un paramètre pertinent pour la sélection de la tolérance à la sécheresse, et les programmes de sélection sur les espèces étudiées ne semblent pas avoir affecté ce paramètre. / In a context of climatic changes, drought stress and water management are regarded as one of the most important constraints for agricultural sector. Thus the selection for drought tolerance became a main objective for many breeding programs. Vulnerability to cavitation is considered a trait of interest for the selection for extreme drought stress, especially for woody species. However, the extent of its variability and its relation to drought tolerance are poorly documented on intraspecific level, particularly for cultivated species. In this study the genetic variability of vulnerability to cavitation was studied on three different experimental devices with three trees of agronomic interest that are threatened by the risk of drought: walnut, rubber and apple trees. Low or no variation in xylem vulnerability to cavitation was found in the studied species and between two species of walnuts despite differences previously reported features. These results suggested a canalization of cavitation resistance on critical organs (branches and stems). In apple tree, the rootstock was suspected to cause the narrow resistance to cavitation on the scion. On the contrary, differences on other traits in response to drought stress such as stomatal regulation, leaf shedding or vulnerability to cavitation on petiole were found. Therefore, vulnerability to cavitation of critical organs was not a relevant parameter for the selection of drought tolerance and breeding programs on the species studied did not appear to affect this parameter.

Esta tese alia dois enfoques principais dentro da Química Farmacêutica. Por um lado, busca explorar uma nova fonte de insumos naturais, cascas de Malus  domestica, com vistas a obtenção dos triterpenos ácidos ursólico (AU) e betulínico (AB); e por outro lado, estuda a relação dos derivados triterpênicos semissintéticos obtidos com a proteína-alvo (PfATP6) destacada atualmente na literatura da terapia da malária com vistas ao planejamento de novos antimaláricos. O primeiro Capítulo inclui o desenvolvimento de um método eficiente, fácil e extremamente rápido onde são combinadas as técnicas de extração por ultrassom (UAE) e análise por Cromatografia Líquida de Alta Eficiência Acoplada a Detector de Espectroscopia de Massas (LC-MS), para identificação e doseamento dos ácidos ursólico (AU) e betulínico (BA) em extratos de cascas frescas de maçã de cinco clones das cultivares Gala e Fuji (“Baigent”, “Fuji Mishima”, “Fuji Suprema”, “Fuji Select” and “Maxi Gala”) oriundas da Região Sul do Brasil. Os parâmetros cromatográficos do método analítico incluem: ionização por eletro spray em modo positivo (ESI+), fluxo de 1,0 mL/min, em modo de eluição isocrático, consistindo de 80% acetonitrila e 20% acetato de amônio 10 mM em pH 6,0 e temperatura ambiente. O método desenvolvido foi validado e mostrou ser seletivo, sensível (LOD e LOQ de 0,087 e 0,266 μg/mL para BA, e 0,398 e 2,117 μg/ mL para UA), coeficiente de regressão linear (r >0.99), preciso, exato e robusto para os analitos de interesse. A otimização do método combinado de UAE com LC-MS permitiu concluir os procedimentos de extração e análise em tempo inferior a 4 h, uma vez que o método não requer a secagem da amostra, etapa que demanda longos tempos de processamento. Este método foi aplicado e forneceu a primeira caracterização fitoquímica dos cinco clones de maçã estudados. Os resultados demonstraram que o método combinado de UAE-LC-MS é adequado às práticas do controle de qualidade. O segundo Capítulo apresenta o estudo da interação dos ligantes semissintéticos derivados dos AU e AB, sintetizados pelo nosso grupo de pesquisa, com a proteína Ca2+-ATPase do Plasmodium falciparum (PfATP6), através do emprego da técnica de Docking Molecular. A PfATP6 é descrita como um importante alvo para novos antimaláricos como Artemisinina (ART), cujo mecanismo de ação inclui, dentre outros, a modulação da homeostasia do cálcio intracelular. Investigações conduziram à hipótese do extravasamento do cálcio, do interior do retículo sarco-endoplasmático como mecanismo plausível para ação dos derivados triterpênicos do AU e AB. Os escores de energia determinados no Docking, de cada um dos nove ligantes (derivados triterpênicos) e quatro compostos de controle (ácidos ursólico e betulínico, artemisinina (ART) e tapsigargina (TPG) foram determinados (análises realizadas em triplicata) e correlacionados com os valores de IC50, para a atividade antimalárica. Os resultados mostraram excelente correlação entre os escores de energia (com a PfATP6) e os valores de IC50, superior a 80% (r > 0,83360). O estudo fornece fortes evidências de que a PfATP6 pode constituir um alvo para os derivados pentacíclicos do estudo, bem como permitiu identificar o perfil conformacional dos ligantes e os principais resíduos do sítio de ligação (SL) da PfATP6 envolvidos nas interações; bem como, contribuiu para a melhor compreensão das propriedades envolvidas na interação do ligante com o receptor e mecanismo de ação. O terceiro Capítulo faz uso de Métodos Clássicos e Quânticos para descrever as mudanças conformacionais da proteína PfATP6. As simulações de Dinâmica Molecular do receptor na forma isolada e complexada com os ligantes foram realizadas durante 10 ns. As conformações finais obtidas para os receptores foram avaliadas em termos RMSD, efeito da presença dos ligantes no sítio de ligação e tipos de interações estabelecidas entre o ligante e o receptor. Os resultados mostraram que as proteínas PfATP6 e SERCA tendem a manter sua conformação nativa e que o modelo utilizado é adequado aos propósitos do estudo. O monitoramento dos resíduos da região citoplasmática das proteínas permitiu evidenciar o efeito alostérico da presença dos ligantes AU e ART no SL, sobre os domínios A e N, da PfATP6. Esse efeito reproduz as conformações E1 e E2, bem estabelecidas para PfATP6, na presença e ausência de Ca2+. As análises de Dinâmica Molecular corroboram os achados do Capítulo II ao evidenciarem o estabelecimento de interações de hidrogênio com os resíduos importantes do SL da PfATP6. Esses resultados fundamentam os indícios de que as bombas de Ca2+-ATPase (SERCA), possam ser de fato, um alvo para os derivados triterpênicos dos AU e AB. / This thesis combines two main focuses within Pharmaceutical Chemistry. On the one hand, it seeks to explore a new source of natural inputs, bark of Malus domestica, in order to obtain ursolic (UA) and betulinic acid (BA) triterpenes. On the other hand, it studies the relation of the semi-synthetic triterpenic derivatives obtained with the target proteins (PfATP6), currently highlighted in the literature on malaria therapy with a view to planning new antimalarial drugs. The first chapter includes the development of an efficient, easy and extremely fast method where ultrasonic extraction techniques (UAE) and high-performance liquid chromatography coupled to mass spectroscopy (LC-MS) are combined for identification and assay of ursolic acid (UA) and betulinic acid (BA) in fresh apple peel extracts from five clones of the Gala and Fuji cultivars (Baigent, Fuji Mishima, Fuji Suprema, Fuji Select and Maxi Gala ") in the Southern Region of Brazil. Chromatographic parameters of the analytical method include: electrospray ionization (ESI +), flow rate of 1.0 mL/min in isocratic elution mode, consisting of 80% acetonitrile and 20% 10 mM ammonium acetate at pH 6.0 and room temperature. The method was validated and proved to be selective, sensitive (LOD and LOQ of 0.087 and 0.266 μg/mL for BA, and 0.398 and 2.117 μg/mL for UA), linear regression coefficient (r> 0.99), accurate, robust for analytes of interest. The optimization of the combined method of UAE with LC-MS allowed to complete the procedures of extraction and analysis in less than 4 h, since the method does not require drying the sample, a stage that demands long processing times. This method was applied and provided the first phytochemical characterization of the five apple clones studied. The results demonstrated that the combined UAE-LC-MS method is suitable for quality control practices. The second chapter presents the study of the interaction of the semi-synthetic ligands derived from the UA and BA, synthesized by our research group, with the Plasmodium falciparum Ca2+-ATPase protein (PfATP6), using the Molecular Docking technique. PfATP6 is described as an important target for new antimalarials such as Artemisinin (ART), whose action mechanism includes, among others, the modulation of intracellular calcium homeostasis. Investigations led to the hypothesis of extravasation of calcium from the interior of the sarco-endoplasmic reticulum as a plausible mechanism for the action of the triterpenic derivatives of UA and BA. The Docking energy scores (binding energy) of each of the nine ligands (triterpenic derivatives) and four control compounds (ursolic and betulinic acids, artemisinin (ART) and tapsigargine (TPG)) with PfATP6, were calculated (analyses performed in triplicate) and correlated with its antimalarial IC50 value. The results showed an excellent correlation between energy scores (with PfATP6) and IC50 values, higher than 80% (r > 0.83360). The study supplies strong evidence that PfATP6 may be a target for the pentacyclic derivatives of the study, and also allowed identifying the conformational profile of the ligands and the main residues of the PfATP6 binding site (BS) involved in the interactions. It further contributed to a better understanding of the properties involved in the interaction of the ligand with the receptor and its action mechanism. The third chapter uses Classical and Quantum Methods to describe the conformational changes of the PfATP6 protein. The Molecular Dynamics simulations of the receptor in the isolated and complexed form with the ligands were performed for 10 ns. The final conformations obtained for the receptors were evaluated in RMSD terms, effect of the presence of ligands at the binding site and types of interactions established between the ligand and the receptor. The results showed that the PfATP6 and SERCA proteins tend to maintain their native conformations and that the model used is adequate for the purposes of the study. The monitoring of the residues of the cytoplasmic region of the proteins allowed evidencing the allosteric effect of the presence of UA and ART ligands in BS, on the A- and N-domains of PfATP6. This effect reproduces the well-established E1 and E2 conformations for PfATP6, in the presence and absence of Ca2+, respectively. Molecular Dynamics analyses corroborate the findings of Chapter II, by showing the possibility of establishing hydrogen interactions with the important residues of PfATP6 BS. These results support the evidence that Ca2+-ATPase (SERCA) pumps may be a target for the triterpenic derivatives of UA and BA.

Depuis la domestication du pommier, l’homme a progressivement sélectionné des variétés plus performantes, notamment pour la qualité du fruit, la productivité ou la résistance aux pathogènes. Les bases génétiques de ces caractères ont été explorées par cartographie en descendances F1 ne permettant d’explorer qu’une infime partie de la diversité génétique disponible.L’objectif de la thèse portait sur l’analyse des bases génétiques de caractères de qualité du fruit et de résistance du pommier à la tavelure et au feu bactérien dans des collections représentant une diversité plus large. Le génotypage de core collections de variétés anciennes s’est fait à l’aide de deux puces 8k et 480kSNPs ou grâce à du ré-séquençage de gènes. Des traces de différenciation génétique entre pommes à cidre et à couteau ont été identifiées et partiellement reliées à la voie des polyphénols. Après analyse de l’étendue du déséquilibre de liaison à large et fine échelle, une approche de génétique d’association a permis l’identification de régions génomiques associées à la variation de plusieurs caractères de qualité du fruit, dont le haut du groupe de liaison 16 rassemblant l’acidité (locus Ma), la fermeté, la jutosité et l’amertume (gène LAR). Pour la résistance au feu bactérien, une région contenant un homologue du gèneNPR1 (activateur de défenses) a été identifiée.Cette thèse a ainsi permis de préciser la localisation potentielle de QTLs identifiés préalablement par cartographie génétique et d’identifier de nouvelles ressources utiles dans de futurs programmes de sélection assistée par marqueurs. / Since apple domestication, humans have progressively selected improved varieties, especially for traits linked with fruit quality, productivity or resistance to pathogens. The genetic bases underlying these traits have been explored thanks to genetic mapping in F1 segregating populations that only allows the study of a small part of the available genetic diversity. The aim of this work was to analyze the genetic bases of fruit quality and disease resistance against apple scab and fire blight, in collections of old apple varieties representing a much larger diversity. Genotyping of core collections was performed either with arrays of 8k and 480k SNPs or by resequencing of chosen genes. Signs of genetic differentiation were identified between cider and dessert apples and were partially linked to the polyphenols pathway. After studying linkage disequilibrium, both on a large and a small scale, an association genetics approach allowed the identification of genomic regions associated with the variation of several fruit quality traits. Especially, the top of linkage group 16 was found to be linked with acidity (locus Ma), firmness, juiciness and bitterness (LAR gene). Concerning the resistance of apple to fire blight, a region containing a homolog of the NPR1 gene (defense activator) was identified. This thesis allowed the refining of the putative localization of previously identified QTLs and the identification of new genetic resources that could be useful in future selection programs using marker assisted selection.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2017-03-24T15:50:08Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO_GPZ_versãofinal.pdf: 1750560 bytes, checksum: 45861d58aabcf6100696f8b44e555dea (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-28T20:23:47Z (GMT) No. of bitstreams: 2 DISSERTAÇÃO_GPZ_versãofinal.pdf: 1750560 bytes, checksum: 45861d58aabcf6100696f8b44e555dea (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-28T20:23:47Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO_GPZ_versãofinal.pdf: 1750560 bytes, checksum: 45861d58aabcf6100696f8b44e555dea (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A demanda por produtos com características semelhantes àquelas das frutas in natura vem sendo uma tendência atual, devido à divulgação dos benefícios trazidos por uma dieta rica em compostos bioativos. Nesse contexto, os sucos ganham espaço no mercado, especialmente quando se amplia a disponibilidade de compostos bioativos. Assim uma das possibilidades é associar aos sucos de maçã pequenos frutos como amora, framboesa e morango, que são ricas fontes de compostos com propriedades antioxidantes, além de fornecer diferenciadas características sensoriais. Com isso, objetivou-se desenvolver suco integral de maçã e suco de maçã com adição de pequenos frutos, avaliando as características físicoquímicas e sensoriais. O trabalho foi realizado em três etapas: 1) desenvolvimento de suco integral de maçã das cultivares Fuji, Gala, Granny Smith e Pink Lady; 2) adição individual de 5, 10, 15 e 20 % amora, framboesa e morango ao suco de maçã ‘Gala’; 3) produção de suco integral de maçã ‘Gala’ com adição individual de 5 % de cada pequeno fruto. Na primeira etapa, observou-se que o suco de maçã ‘Granny Smith’ apresentou maior acidez e maior presença de compostos bioativos. A menor acidez foi evidenciada para o suco da cv. Gala. O suco da cv. Fuji, apresentou maior quantidade de açúcares totais. Em relação à aceitabilidade o suco de maçã ‘Gala’ e ‘Fuji’ tiveram as melhores notas. Com isso, escolheu-se o suco de maçã ‘Gala’ para aplicação de diferentes concentrações de pequenos frutos. Na segunda etapa, podese perceber que as maiores concentrações de pequenos frutos adicionados ao sucos de maçã resultaram em menores notas na avaliação sensorial, demonstrando que a adição de mais do que 10 % de pequenos frutos pode causar depreciação sensorial dos sucos. Na terceira etapa observou que a adição de amora e de morango ao suco de maçã proporcionou o incremento de 18,5 % e 16,2 %, respectivamente, no teor de compostos fenólicos totais. Os principais compostos fenólicos individuais detectados nos sucos de maçã e nos sucos adicionados de pequenos frutos foram ácido cafeico, ácido ferúlico, ácido cumárico e ácido gálico, os quais são importantes antioxidantes. A adição de pequenos frutos proporcionou o incremento de antocianinas ao suco de maçã, a amora forneceu o acréscimo de pelargonidina, cianidinas e keracianina. Quanto aos aspectos sensoriais, a adição de amora e framboesa proporcionaram maior aceitação para o atributo cor, e para o sabor o suco de maçã com morango, o suco de maçã integral e suco de maçã com amora tiveram melhor aceitabilidade. De forma geral, foi observado que a adição de pequenos frutos ao suco de maçã obteve boa aceitação e contribuiu para o aumento do teor de compostos bioativos, sendo uma boa alternativa para o enriquecimento do suco de maçã em compostos potencialmente benéficos à saúde, além de promover maior diversificação de produtos derivados de maçã. / The demand for products with characteristics similar to fresh fruits has been a current trend, due to the announcing of the benefits brought by a diet rich in bioactive compounds. In this context, juices gain market, especially when they expand the availability of bioactive compounds. Thus, one of the possibilities is to associate green apple juice with blackberry, raspberry, and strawberry, which are rich sources of compounds with antioxidant properties, and additionally provide unique sensory characteristics. The objective of this study was to develop integral apple juice and apple juice with the addition of small fruits, and to characterize their physicochemical and sensory characteristics. The work was developed in three steps: 1) apple integral juice development using cultivars Fuji, Gala, Granny Smith and Pink Lady; 2) individual addition of 5, 10, 15, and 20% blackberry, raspberry, and strawberry pulp to apple juice ‘Gala’; 3) production of whole apple juice 'Gala' with individuality of 5 % of each small fruit. In the first step, it was observed that 'Granny Smith' juice presented the highest acidity and greatest bioactive content. The lowest acidity was observed for the cv. Gala and the highest sugar content was found in cv. Fuji juice. 'Gala' and 'Fuji' juices had the highest acceptability rates. Therefore 'Gala' was chosen for the application of different concentrations of small fruits. In the second step of the study, it could be noticed that the higher the concentration of small fruits added to the apple juice the lower the sensory evaluation scores, demonstrating that the addition of more than 10 % of small fruits can cause sensorial depreciation of the juices. In the third step, it was observed that the addition of blackberry and strawberry to apple juice contributed to an increase of total phenolic content of 18.5% and 16.2%, respectively. The main individual phenolic compounds detected in apple juices and juices added of small fruits were caffeic acid, ferulic acid, coumaric acid, and gallic acid, important antioxidant compounds. The addition of small fruits contributed to an increase in anthocyanin content in the apple juice; blackberry contributed with increases in pelargonidin, cyanidins, and keracyanin contents. As for the sensory, the addition of blackberry and raspberry contributed to a greater acceptance for the color attribute. Apple juice with strawberry, whole apple juice, and apple juice with blackberry had the best taste acceptability. In general, it was observed that addition of small fruits to apple juice promoted good acceptance and contributed to the increase bioactive compound content. These blends constitute a good alternative for the enrichment of apple juice in health beneficial compounds and promote a greater diversification of apple products.

Cette thèse est mue par la volonté de son auteur (et de son promoteur) de mettre en évidence combien le concept d'ingéniérie actuarielle est non seulement un concept actuel mais également porteur d'avenir pour l'actuariat. Dans ingéniérie, on entend ingénieur, soit un individu formé à l'application des sciences, dans le but de résoudre des problèmes technologiques concrets et complexes. Ces compétences, traditionnellement plutôt utilisées par l'industrie, sont ici mises au service de l'Actuariat. Nous espérons montrer combien un actuaire ouvert aux techniques récentes de la Statistique peut enrichir sa panoplie d'outils pour répondre aux questions toujours plus variées que pose la pratique. Car là est aussi un des messages de ce travail: montrer que ces développements récents sont loin d'être de pures gymnastiques intellectuelles mais offrent de réelles solutions ou alternatives valables à des problèmes connus. Avec pour bagage les modèles de régression non linéaires, nous nous promenons dans les différents domaines de l'Actuariat, abordant tout d'abord un aspect méthodologique. Ensuite, nous traitons de deux problèmes liés à la branche Non Vie : tarification géographique et échelles bonus-malus. Enfin, nous voyons comment des perspectives nouvelles peuvent également s'inscrire dans la branche Vie, à travers la problématique de la modélisation de la mortalité future. Il ne s'agit en aucun cas d'un inventaire exhaustif des possibilités récentes offertes par la Statistique à l'Actuariat, mais bien d'un tour d'horizon qui entend ouvrir des portes dans des domaines variés. Cette thèse est composée d'articles (rédigés en anglais) publiés dans des revues nationales et internationales.