Silverleaf nightshade (Solanum elaeagnifolium), a perennial broadleaf weed, has become a serious pest in many semi-arid areas of the world. Control of silverleaf nightshade is confounded by its ability to produce thousands of viable seeds from a single mother plant, a deep and well-developed root system and the ability to propagate vegetatively from creeping lateral roots, root fragments and from rhizomes. Exacerbating factors are longevity of its propagules in soil, particularly under adverse environmental conditions. Currently, in South Africa, it is one of the more serious weeds in cotton (Gossypium hirsutum L.) production, where its interference results in remarkable loss of product quantity and quality. Research has been done on the competition aspect of silverleaf nightshade with cotton but there is a serious lack of information on the allelopathic aspect. In this study, a series of experiments that included laboratory and glasshouse experiments were conducted to evaluate the allelopathic interference of silverleaf nightshade on early growth of cotton. Cotton seeds were exposed to silverleaf nightshade extract solutions or planted in soil into which silverleaf nightshade leaf material or ripe berries were incorporated. In all the experiments attempts were made to avoid or to reduce, at least, the influence of factors that could be potentially confounding. In this regard, PEG-6000 was used to evaluate the sensitivity of crop parameters to the osmolality of test solutions in order to ensure that osmotic inhibition was not a confounding factor in the bioassays where the biological activity (phytotoxicity) of plant extracts were assessed. Competition for growth factors was the other major potentially confounding factor that was considered throughout, and steps were taken to negate its influence. Germination and early seedling growth of cotton cultivars Sicala, CA 223, Siokra V15, Tetra and Delta Opal were inhibited by test solutions (silverleaf nightshade extracts) and by soil-incorporated residues of silverleaf nightshade. Inhibitory effects of silverleaf nightshade solutions were observed when either a layer of filter paper or a thin layer of soil or quartz sand was used as substrate. In both laboratory and pot experiments, it appeared that cotton cultivar Sicala was the most sensitive to allelochemicals contained in extracts and residues of silverleaf nightshade. This finding of differential tolerance of crop cultivars towards allelochemicals contained in a weed is a rare occurrence in allelopathy research. Berries of silverleaf nightshade were generally more inhibitory to cotton than leaf material. For both types of plant material used, residues lost their inhibitory effect over time, probably as decomposition of allelopathic compounds in soil progressed. Information obtained from this study can be viewed as knowledge that contributes to the bridging of the gap between identification and isolation of allelochemicals from silverleaf nightshade, and confirmation of silverleaf nightshade allelopathy under natural conditions. Experiments involving soil as growth medium, in particular fieldwork, are needed to verify the validity of these findings under natural conditions. / Dissertation (MSc(Agric) Agronomy (Weed Science))--University of Pretoria, 2007. / Plant Production and Soil Science / MSc(Agric) / unrestricted

Potato (Solanum tuberosum L) is an outcrossing, highly heterozygous cultivated in which the elucidation of the genetic basis of quantitative traits, is more complex than in self-pollinated crops. Both a candidate gene approach and a whole genome SNP genotyping analysis were used to assess allelic variation and to identify loci associated with biosynthesis and accumulation of steroidal glycoalkaloids (SGAs). SGAs are secondary metabolites produced in Solanum species as defense against insects and pathogens. Fragments of genomic DNA coding for regions of five SGA biosynthetic candidate genes were amplified, cloned and sequenced [3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2); 2,3-squalene epoxidase (SQE); solanidine galactosyltransferase (SGT1); and solanidine glucosyltransferase (SGT2)]. A germplasm panel of six wild potato species [Solanum chacoense (chc 80-1), S. commersonii subsp. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, and S. stoloniferum] and a cultivated clone S. tuberosum Group Phureja (phu DH) was used in an allelic variation analysis. A segregating interspecific F2 population phu DH �" chc 80-1 was screened to assess association with SGAs. Sequence diversity analysis showed a tendency of purifying selection and increased frequency of rare alleles in most of the candidate genes. Genes of primary metabolism (HMG1, HMG2 and SQE) had stronger selection constraints than those in secondary metabolism (SGT1 and SGT2). Sequence polymorphism in HMG2, SQE, SGT1 and SGT2 separated either the phu DH clone which produced no SGAs, or chc 80-1, the greatest SGA accumulator, from other accessions in the panel. Segregation analysis of the F2 population revealed that allelic sequences of HMG2 and SGT2 derived from chc 80-1 were significantly associated with the greatest SGA accumulation. In the whole genome analysis, SNP genotyping and cluster analysis based on putative association with SGA accumulation in the germplasm panel, allowed identification of eight informative SNPs that can be used in future studies. In the segregating F2 population, loci located on five pseudochromosomes were associated with SGA synthesis. Loci on pseudochromosomes 1 and 6 explained segregation ratios of synthesis for α-solanine and α-chaconine, the most common SGAs in most potato species. In addition, loci on seven pseudochromosomes were associated with accumulation. New candidate genes, putatively affecting synthesis and accumulation of SGAs, were identified in adjacent genomic regions of significant SNPs. This research demonstrates how the newly available genome sequence of potato and associated biotechnological tools accelerates the identification of genetic factors underling complex traits in a species with a difficult breeding structure. / Ph. D.

Tomato serves as an important model organism for Solanaceae in both molecular and agronomic research. With whole genome sequencing in progress, there is a need to study functional genetics through mutant lines that exceed the practical limitations imposed by the popular research cultivar, Micro-Tom. This project utilized Agrobacterium transformation and the transposon tagging construct, Ac-DsATag-Bar_gosGFP, to produce activation tagged and knockout mutants in the processing tomato variety, M82. The construct contained hygromycin resistance (hyg), green fluorescent protein (GFP), and maize transposase (TPase) on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 10,568 selfed and M82 backcross progeny was produced from the functional T0 line. This population was screened by spraying with 0.05% Liberty® herbicide, followed by a 100 mg/L hygromycin leaf painting procedure to select for Ds only (herbicide tolerant and hygromycin sensitive) individuals. The T-DNA genotype of Ds only plants was confirmed through multiplex PCR and the location of insertions within the genome was determined through TAIL-PCR. Resulting product sequences were blasted against the pre-publication tomato genome browser to determine insertion sites. A population of 309 independent transposants dispersed to all twelve chromosomes from the original insertion site on chromosome five has been developed. The transposon tagged lines are currently being immortalized in seed stocks. / Master of Science

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This project involved the analysis of genes in Solanaceae species that have previously been shown to be involved in the phosphorylation of starch or its subsequent dephosphorylation. Both these processes are essential for normal starch mobilization. A tomato conditional mutant lacking the starch phosphorylating enzyme glucan water dikinase was analyzed. It is known that starch accumulates transiently in tomato fruit and is degraded throughout the ripening process. The study aimed to determine the effect of inhibited starch degradation on fruit development. Unfortunately no effect on starch mobilisation was found in the fruit of the mutant. Immunoblot analysis revealed expression of Glucan Water Dikinase (GWD) within the fruit of the tomato mutant indicating that the conditionality of the mutation was compromised. The second set of experiments analyzed the roles of Starch Excess4 (SEX4), Like Sex Four-1 and Like Sex Four-2 (LSF1 and LSF2) in starch degradation in potato and Nicotiana benthamiana. These enzymes have, thus far, only been studied in Arabidopsis, with the proposed role for SEX4 and LSF2 being that they are involved in dephosphorylation of the C-6 and C-3 positions of starch breakdown products. The role of LSF1 is unclear, although it is not thought to be a phosphatase. SEX4, LSF1 and LSF2 were repressed individually while the expression of SEX4 and LSF2 were also inhibited simultaneously. Using a transient repression system in N. benthamiana it was shown that all of the genes play a role in leaf starch degradation. The SEX4 and LSF2 enzymes were shown to influence the proportion of phosphate located on the starch which contained an altered ratio of C-3/C-6 phosphate. Stably transformed potato plants were produced where SEX4 and LSF2 were successfully repressed in potato leaves and tubers. Although AtLSF2 had been shown not to be essential for normal starch degradation on its own, in potato plants when LSF2 was repressed, the plants developed a starch-excess phenotype. Taken together with the N. benthamiana data this indicates that LSF2 plays a bigger role in leaf starch degradation in Solanaceae than in Arabidopsis. The ratio of C-3/C-6 phosphate was also altered in tuber starch from some of the silenced plants. Starch from SEX4 repressed potato plants contained increased amounts of glucose-6-phosphate and increased glucose-3-phosphate in the tuber when compared to the WT. An increase in the proportion of C-6 or C-3 phosphate is not surprising with SEX4 being characterized as a phosphatase specific for C-6 position and LSF2 for the C-3 position in Arabidopsis, however the combined increase in C-3 and C-6 amounts in StSEX4 silenced plants is interesting. The differences seen in the phosphate alteration in both N. benthamiana leaves and potato tubers indicates that in Solanaceae species these proteins may have a slightly altered specificity when compared with Arabidopsis, although they are undoubtedly involved in starch degradation. The effect of silencing SEX4 or LSF2 on cold-induced sweetening was also investigated, with no effect being found. This may be because of functional redundancy between the proteins and a better approach in terms of blocking cold sweetening would be to simultaneously repress SEX4 and LSF2. Overall, these enzymes seem to play similar roles in leaves of Solanum species as has been described in Arabidopsis. The starch from the engineered plants did have an altered phosphate ratio and further analysis is needed to determine if this leads to improved or additional functionality. / AFRIKAANSE OPSOMMING: Die projek omhels die ontleding van gene van die Solanaceae spesie wat voorheengetoon het dat hulle deel neem in fosforilering of defosforilering van stysel. Altwee van hierdie reaksies is belangrik vir normale stysel metabolisme. ‘n Tamatie konditionele mutant was geanaliseer waarin die stysel fosforilering ensiem glucan water dikinase nie teenwoordig was nie. Die doel van die studie was om te ondersoek watter effek het n gebrek in stysel afbraak op die rypwording en ontwokkeling vrugte. Ongelukkig was geen effek op stysel metabolism in die munant se vrugte gesien. Immunoklad analise het getoon dat GWD protein wel uitdruk word in die vrugte en dus die mutant nie heeltemal effektief was nie. Die tweede stel van experimente het in aartappels en tabak die rol van SEX4, LSF1 en LSF2 in stysel afbraak ondersoek. Hierdie ensieme was huidiglik nog net deeglik in Arabidopsis bestudeer, waar daar gewys was dat SEX4 and LSF2 in die defosforilering van stysel by die C-6 en C-3 posisie deel neem. Die rol van LSF1 is nog onbekend, maar daar word huiglik gelgo dat dit is nie ‘n fosfatase nie. SEX4, LSF1, en LSF2 was onderdruk op sy eie, waar SEX4 en LSF2 gelyktydig onderdruk was. Met behulp van n verbygaande onderdrukking in tabak, was dit getoon dat al die bogenoemde gene n gedeeltelike rol speel in die afbraak van stysel. Dit was getoon dat SEX4 and LSF2 ensiemedie verhouding van waar fosfaat op stysel gelee is beinvloed en het n verandering in die C-3/C-6 phosphaat verhouding ook gehad. Aardappels was stabiel getransformeer en daar was suksesfol plante waar SEX4 en LSF2 onderdruk was in blare en knolle geproduseer. Alhoewel daar getoon was dat AtLSF2 op sy eie nie n groot rol speel in stysel katabolisme nie was daar wel gesien dat in aardappel wanner hierdie geen afgeskakel was dat daar n stysel oorskot fenotiepe ontwikkel. As die tabak resultate saamgevat word met die aardappel wil dit voorkom asof LSF2 n groter rol binne die stysel katabolisme in Solanaceae speel as in Arabidopsis. Daar was gevind dat die verhouding van C-3/C-6 fosfaat was in die knolle verander in perty van die lyne waar geen afskakeling wel plaasgevind het. Die verhouding van C-3/C-6 fosfaat was verander in knolle stysel van sommige stilgemaak plante. Sysel van SEX4 stilgemaak plante het hoër vlakke glukose-6-fosfaat en glukose-3-fosfaat in die knolle gehad wanner dit met die WT vergelyk was. n Toename in die persentasie van C-6 fosfaat is nie verbasend nie, SEX4 word gekenmerk as die spesifieke fosfatase verantwoordelik vir die fosfaat by die C-6 posisie en LSF2 spesifiek vir die C-3 posisie in Arabidopsis. Die gekombineerde toename in beide C-6 en C-3 bedrae in StSEX4 stilgemaak plante is wel heel interesant. Verandering in beide tabak blare and aartapple knolle dui daarop dat in solanacea spesie hierdie proteiene, n effens verandering in spesifisiteit kan hê as dit met Arabidopsis vergelyk word. Daar kan wel nie getwyfel word dat hulle wel n rol speel in stysel afbraak nie. Die effect watSEX4 of LSF2 op koue-geinduseerde soetheid het is ook ondersoek maar daar was geen effek gevind nie. Dit mag wees asgevolg van die funksionele onslag tussen die twee proteien en better benadering on die koue-soetheids effek te verhoed sou wees om beide protein op die selfde stadium aft e skakel. As daar in gegeheel gekyk word lyk dit asof hierdie protein die selfde rolle het in die Solanum spesies as in Arabidopsis.Die stysel van hierdie die ontwerpte plante het ‘n veranderde fosfaat verhouding getoon en veder analise is nodig om te bepaal of dit lei tot verbeterde einskappe of bykommende funksies.

La domestication des plantes a débuté il y a quelques milliers d’années quand les hommes se sont sédentarisés. Ils ont sélectionné les plantes sauvages portant des caractères phénotypiques d’intérêt pour la consommation et production humaine. Ce processus évolutif a par conséquent modifié le patrimoine génétique des espèces domestiquées. Cette thèse se penche sur les traces génétiques induites par la domestication chez trois espèces de Solanacées : l’aubergine (Solanummelongena), le piment (Capsicum annuum) et la tomate (S. lycopersicum). En effet, si les caractères phénotypiques des plantes cultivées ont été sélectionnés depuis des milliers d’années, les conséquences moléculaires d’une telle sélection restent peu étudiées à l'échelle du génome. Cette étude est basée sur des données de diversité et d’expression de gènes (RNAseq). En utilisant des méthodes comparatives entre des variétés cultivées et leurs espèces sauvages apparentées, j’a iétudié, à l’échelle intra-spécifique, d’une part les histoires démographiques de chacune des espèces,et d’autre part les changements de diversité nucléotidique et d’expression des gènes dus à la domestication. La comparaison de ces trois événements indépendants de domestication, offre l’opportunité de décrypter les changements génétiques qui convergent chez ces trois espèces lors du processus de sélection humaine.Suite à une introduction qui pose le cadre de cette étude et présente l’état de l’art, le premier chapitre, s’inscrit dans un ouvrage portant sur la génomique des populations d’espèces modèles. Il propose une synthèse des connaissances accumulées en plus d’un siècle de recherche sur l’espèce modèle qu’est la tomate (S. lycopersicum). Ce chapitre permet également de compléter le contexte scientifique dans lequel cette thèse s’inscrit, notamment, en retraçant l’importance que les espèces sauvages apparentées ont eu dans l’amélioration de l’adaptabilité des variétés cultivées actuelles.L’hypothèse du deuxième chapitre révèle la convergence des changements démographiques entre les trois espèces malgré leurs événements indépendants de domestication. L’étude comparée d’inférences de scénarios démographiques a permis de reconstruire l’histoire démographique de chaque espèce cultivée. Ces inférences ont aussi facilité l’estimation des paramètres tels que les flux migratoires entre les espèces sauvages et cultivées, la force des goulots d’étranglement liés à l’intensité de la sélection humaine et la durée des événements de domestication. Ce chapitre permet de démontrer que les changements démographiques liés à la domestication dépendent de l’état de sympatrie ou d’allopatrie des variétés cultivées avec leurs sauvages apparentées. Les connaissances quant à la datation des événements de domestication de nos trois espèces restent très faibles, et les inférences ont permis d’établir des estimations de durée de domestication relativement précise. Ces nouvelles connaissances apportent une plus-value à cette étude pour nos trois espèces et nous invitent à s’interroger sur les différents compartiments du génome qui ont été sélectionnées et modifiées lors de la domestication.Le troisième chapitre teste l’hypothèse d’une convergence évolutive des changements moléculaires, notamment transcriptionnels, induits par la domestication et l’amélioration moderne.La comparaison des variétés cultivées à leurs espèces sauvages apparentées permet d’évaluer la convergence des mécanismes de régulation et d’adaptation liés à la domestication. C’est en testant la corrélation entre les traces génétiques (diversité nucléotidique) de sélection et les changements d’expression des gènes observés chez les variétés cultivées que l’hypothèse de départ a été validée.Cette analyse montre que la domestication, au-delà même de changements nucléotidiques, a modifié l’expression des gènes chez les trois espèces. / Domestication started thousand years ago when human shifted from hunter-gatherer to agrarian societies. They started selecting wild plants for phenotypes related to consumption andyield. This evolutionary process induced changes in the gene pool of domesticated plants. This thesis focuses on genetic footprints induced by domestication within a trio of Solanaceae species: the eggplant (Solanum melongena), the pepper (Capsicum annuum) and the tomato (S. lycopersicum).Crop plants have been selected for thousand years on phenotypic traits, but the molecularconsequences of such selection remain unknown at the genome-wide scale. The study was performed on a RNAseq data set; using comparative methods between crops and their wild relatives,I studied, at the intra-specific scale, the demographic history, and, both the nucleotide diversity and the gene expression changes due to domestication. Comparing these three independent events ofdomestication, is a great opportunity to decipher the interspecific genetic changes, converging for the three species, during the human selection process.The first chapter is a book chapter about population genomics in model species. It details thestate of art of hundred years of research on tomato as model species (S. lycopersicum). Tomato is amodel species in genetics, as well as in population genomics thanks to the important collection of genomic data that have been accumulating over years. Tomato has the strongest economic importance within the trio of studied species. By highlighting the importance of crop wild relative species for adaptability improvement of modern cultivars, this chapter describes the scientific context of this thesis work.The two next chapters are following these researches and show the importance to both conserve and study the crop wild relative species.In the second chapter, I hypothesize that demographic changes within the three species experience a convergence, despite their independent domestication events. The comparative studyof demographic inferences allows the reconstruction of each domesticated species demographichistory. Theses inferences facilitate the parameter estimations such as the migration rate between crop and wild, the bottleneck strength paired with the human selection and the duration of thedomestication events. This chapter reveals a common bottleneck phenomenon as well as migration rate dependent to the allopatric or sympatric state of the crops with their wild relatives. Knowledge concerning the domestication events dating, for each of the three species, remain poorly studied and this thesis work discloses relative domestication time durations.These new insights bring valuable knowledge to the three species and induce a questioning on thedifferent genome parts that are selected and modified through domestication.The third chapter, test the hypothesis of a convergent evolution of molecular changes,especially transcriptional, induced by domestication and modern breeding. The comparative analysis of crop plants and their wild relatives assesses the convergence of regulation and adaptation mechanisms due to domestication. By testing the correlation between the selection footprints on genes and the gene expression changes in crop compared to their wild relative species, the previous hypothesis was confirmed. This analysis implies that domestication modified gene expression in the three species beyond only nucleotide polymorphisms. The ortholog analysis of our species genes, confirmed that domestication facilitated the fruit development and plant growth but relaxed selective pressure on genes of plant defense and environmental stresses tolerance. Demonstrating demographic changes and molecular footprints of domestication, my PhD thesis highlights several proofs of convergence.

O tomateiro (Lycopersicon esculentum Mill.) é comumente atacado por diferentes pragas. Tetranychus evansi Baker e Pritchard é um dos principais ácaros praga do tomateiro em alguns países. No continente africano, T. evansi tem causado até 90% de perdas nos cultivos de tomateiro. Recentemente, o ácaro predador Phytoseiulus longipes Evans foi encontrado na área urbana de Uruguaiana - RS, desenvolvendo-se sobre tomateiros de ocorrência espontânea infestados com T. evansi. O objetivo deste presente trabalho foi avaliar o efeito da liberação inoculativa de P. longipes na densidade populacional de T. evansi em plantios de tomateiro realizados em telados, assim como avaliar a distribuição daquele predador e sua associação com T. evansi em plantas cultivadas e silvestres na região de Uruguaiana. O efeito da liberação inoculativa de P. longipes foi estimado através da comparação da densidade populacional de T. evansi em folíolos de tomateiro coletados em parcelas com e sem liberação do predador. Os níveis populacionais de T. evansi foram em média muito baixos nas parcelas em que foi feita a liberação de P. longipes. A ocorrência natural de T. evansi e P. longipes em tomateiros foi avaliada através da determinação de níveis destes organismos sobre plantas em 4 parcelas constituídas por 40 plantas cada. Nem T. evansi ou P. longipes foi observado nos 3 campos de tomateiros instalados na área rural de Uruguaiana, confirmando que T. evansi não é uma praga de tomateiro na região, mesmo quando nenhum controle químico é aplicado para seu controle. No entanto, P. longipes ocorreu naturalmente no campo infestado artificialmente com T. evansi reduzindo a população deste ácaro. A distribuição de T. evansi e P. longipes na região de Uruguaiana foi avaliada através de coletas quinzenais conduzidas em Uruguaiana e em 5 municípios vizinhos. T. evansi apresentou uma ampla distribuição, enquanto P. longipes foi encontrado quase que exclusivamente na zona urbana de Uruguaiana e em associação com T. evansi. No primeiro trabalho, conclui-se que a liberação inoculativa de P. longipes pode manter a população T. evansi em baixos níveis populacionais em tomateiros cultivados em telado. No segundo, conclui-se que a ocorrência natural de P. longipes pode contribuir para manter reduzidos os níveis populacionais de T. evansi na região de Uruguaiana. No terceiro, conclui-se que a ocorrência natural de P. longipes no Brasil aparentemente limita-se a área urbana do município de Uruguaiana. Uma possível razão para a sua distribuição restrita pode ser o fato de que este foi apenas recentemente introduzido na região e ainda não teve tempo suficiente para se dispensar. Outra possível razão seria a prevalências de condições climáticas adversas, especialmente no inverno, podendo restringir a distribuição à áreas protegidas dos efeitos de ventos fortes, chuvas intensas, ou baixas temperaturas. O efeito destes fatores pode ser tal que a cada ano, o processo de dispersão natural do predador pode ser restringido em áreas não protegidas. / Tomato (Lycopersicon esculentum Mill.) is commonly attacked by different pests. Tetranychus evansi Baker and Pritchard is one of the main tomato mite pests in some countries. In the African continent, T. evansi has caused up to 90% yield losses in tomato crops. The predator Phytoseiulus longipes was recently found in the urban area of Uruguaiana-RS developing on T. evansi infesting spontaneously growing tomato plants. The objective of the present work was to assess the effect of inoculative releases of P. longipes on the population density of T. evansi in screen-house tomato crops as well as to evaluate the distribution of that predator and its association with T. evansi on cultivated and wild plants in the region of Uruguaiana. The effect of the inoculative releases of P. longipes was estimated by comparing the population densities of T. evansi on tomato leaflets collected in plots with and without release of the predator. Population levels of T. evansi were on the average much lower in the plots where P. longipes was released. The natural occurrence of T. evansi and P. longipes on tomato plants was evaluated by determining the levels of those organisms on plants in 4 plots of 40 plants each. Neither T. evansi nor P. longipes were observed in any of the 3 tomato fields established in the rural area of Uruguaiana, confirming that T. evansi is not a pest of tomato in the region, even when no chemical is applied for its control. However, P. longipes occurred naturally in the field that had been artificially infested with T. evansi, reducing its population. The distribution of T. evansi and P. longipes in the region of Uruguaiana was assessed by bi-weekly samplings conducted in Uruguaiana and 5 neighboring counties. T. evansi had ample distribution, while P. longipes was found almost exclusively in the urban area of Uruguaiana in association with T. evansi. In the first work, it was concluded that inoculative releases of P. longipes can keep T. evansi at low population levels in screen-house tomato crops. In the second, it was concluded that the natural occurrence of P. longipes can contribute to maintain low population levels of T. evansi in the region of Uruguaiana. In the third, the conclusion was that the natural occurrence of P. longipes in Brazil seems almost restricted to the urban area of Uruguaiana. A possible reason for its restricted distribution may be that it was only recently introduced in the region and still did not have time to disperse. Another conceivable reason is that prevailing drastic weather, especially in the winter, may restrict its distribution to areas where it is protected from the effect of strong winds, heavy rainfall or low temperatures. The effect of those factors could be such that at each year the natural dispersal process of the predator could be halted in non protected areas.

Solanum lycocarpum A.St.-Hil. (Solanaceae) é uma espécie de ampla distribuição no bioma Cerrado. Popularmente conhecida com fruta-do-lobo, devido ao fato de o lobo-guará consumir frequentemente os frutos desta planta, sendo este seu principal agente dispersor de sementes. É utilizada pela população local para fabricação de doces, e empiricamente como medicinal. A espécie floresce e frutifica durante todo o ano, característica que permite constante fluxo de genes via pólen e sementes. Ocupa facilmente ambientes antropizados, o que permite que seja utilizada em projetos de restauração. O presente estudo teve como objetivo, caracterizar a estrutura genética populacional de S. lycocarpum em ambientes naturais e antropizados, utilizando dois marcadores moleculares: microssatélites (SSR) e isoenzimas. Foram estudadas quatro populações com SSR, e duas populações com Isoenzimas, formando pares de populações (uma natural e outra antropizada). As populações estudadas com marcadores SSR estavam situadas duas a Nordeste do Estado de Goiás e outras duas a Sul do estado. As duas populações estudadas com isoenzimas localizavam-se a Sul de Goiás. Coletou-se aleatoriamente amostras de 60 indivíduos em cada população, exceto na população antrópica estudada com marcador Isoenzimático, que foram amostrados 41 indivíduos. As amostras foram conduzidas ao LARGEA – ESALQ/USP, onde foram realizados os procedimentos laboratoriais. A análise dos dados consistiu em quantificar a diversidade genética e sua distribuição entre e dentro das populações. Embora os valores do índice de fixação tenham sido algumas vezes altos, não foram detectados valores significativos em nenhuma das populações para ambos os marcadores, sugerindo ausência de endogamia nas populações estudadas. As populações naturais estudadas com locos SSR apresentaram número de alelos por loco mais elevados que as populações antropizadas. Uma população situada em uma unidade de conservação foi a que apresentou maior número de alelos por locos e maior número de alelos exclusivos, o que permite sugerir que unidades de conservação abrigam maior diversidade genética que populações com influência antrópica. Utilizando o teste X2 foi possível confirmar que o número de alelos encontrados nas populações naturais é significativamente mais elevado que os encontrados no outro tipo de ambiente. O mesmo não foi verificado para as populações estudadas com locos isoenzimáticos. A divergência genética entre as populações foi substancial, significativamente diferentes de zero e semelhantes para ambos os marcadores (θp=0,095 e θp=0,081 para marcadores SSR e Isoenzimáticos, respectivamente). A divergência genética entre as populações medida pela estimativa GST(Hedrick), que considera tanto o tipo como a freqüência dos alelos, apresentou valores superiores para os dois marcadores (SSR=0,167 e Isoenzimáticos= 0,102), indicando maior restrição no fluxo gênico histórico se comparado com as estimativas obtidas da estatística FST. O presente estudo permite sugerir que existe diferença na estrutura genética entre áreas sob intervenção antrópica e aquelas com intervenção restrita, havendo nas áreas mais preservadas maior diversidade genética. Os resultados obtidos com a estimativa GST(Hedrick) possibilitam propor que mesmo em espécies de ampla distribuição e que frequentemente ocupam ambientes de ação antrópica, como a lobeira, pode estar ocorrendo restrição de fluxo gênico e apontam maior diferenciação entre populações de lobeira do que relatado em estudos anteriores. / Solanum lycocarpum A. St.-Hil. (Solanaceae) has a wide distribution in savanna biome. The species is common know as "fruta-do-lobo" due the "lobo-guará" frequently to eat your fruits. "Lobo-guará" is also the main seed disperser of the species. The species is used for local people to manufacture sweets and as medicine. The species flowering and produce fruits during all year, favoring pollen and seed gene flow. The species colonize easily anthropized environments, permitting your use in environmental restoration projects. The goal of this study was to characterize the genetic structure of S. lycocarpum in natural and anthropized populations, using two different kinds of genetic markers: microsatellites (SSR) and allozymes. Four populations were studied using SSR markers and two using allozyme markers, forming pair of populations (a natural and an anthropized). Two populations studied by SSR markers were located in Northeast of Goiás State and two in South of the State. The two South populations were also studied by allozyme markers. Samples were randomly collected from 60 individuals in each population, with exception in one anthropized population study by allozymes, where 41 individuals were sampled. The samples were envied to LARGEA – ESALQ/USP, where the Lab analyses were made. The genetic diversity and distribution among and within populations was quantified. Although fixation index values were highest in some populations, these values were not statistically different from zero for both used genetic markers, suggesting absence of inbreeding in the studied populations. For SSR markers, natural populations showed higher number of alleles per locus than anthropized populations. A population located in a conservation unity presents the highest number of alleles per locus and exclusive alleles, suggesting that this population have higher genetic diversity than anthropized populations. According to a X2 test, the number of SSR alleles was significantly higher that detected in anthropized population. The same was not observed for population studied by allozyme loci. The genetic divergence among populations was substantial and significant different from zero for both used genetic markers (θp=0.095 and θp=0.081 for SSR and allozymes, respectively). The genetic divergence measured by GST(Hedrick) statistic, that considerer simultaneously the kinds of alleles and gene frequency was higher for both genetic markers (SSR=0.167 and allozymes= 0.102), indicating lower historic gene flow than measured by FST statistic. The present study suggested that there are differences in genetic structure between natural and anthopized populations, where natural populations have more genetic diversity. The results from GST(Hedrick) statistic suggested that even species with wide geographic distribution and whish frequently colonizing anthropized environments, as lobeira, can experience restriction in gene flow. The present results also indicate higher genetic differentiation among population than has been related in previous population study with this species.

Solanum lycocarpum A.St.-Hil. (Solanaceae) é uma espécie de ampla distribuição no bioma Cerrado. Popularmente conhecida com fruta-do-lobo, devido ao fato de o lobo-guará consumir frequentemente os frutos desta planta, sendo este seu principal agente dispersor de sementes. É utilizada pela população local para fabricação de doces, e empiricamente como medicinal. A espécie floresce e frutifica durante todo o ano, característica que permite constante fluxo de genes via pólen e sementes. Ocupa facilmente ambientes antropizados, o que permite que seja utilizada em projetos de restauração. O presente estudo teve como objetivo, caracterizar a estrutura genética populacional de S. lycocarpum em ambientes naturais e antropizados, utilizando dois marcadores moleculares: microssatélites (SSR) e isoenzimas. Foram estudadas quatro populações com SSR, e duas populações com Isoenzimas, formando pares de populações (uma natural e outra antropizada). As populações estudadas com marcadores SSR estavam situadas duas a Nordeste do Estado de Goiás e outras duas a Sul do estado. As duas populações estudadas com isoenzimas localizavam-se a Sul de Goiás. Coletou-se aleatoriamente amostras de 60 indivíduos em cada população, exceto na população antrópica estudada com marcador Isoenzimático, que foram amostrados 41 indivíduos. As amostras foram conduzidas ao LARGEA – ESALQ/USP, onde foram realizados os procedimentos laboratoriais. A análise dos dados consistiu em quantificar a diversidade genética e sua distribuição entre e dentro das populações. Embora os valores do índice de fixação tenham sido algumas vezes altos, não foram detectados valores significativos em nenhuma das populações para ambos os marcadores, sugerindo ausência de endogamia nas populações estudadas. As populações naturais estudadas com locos SSR apresentaram número de alelos por loco mais elevados que as populações antropizadas. Uma população situada em uma unidade de conservação foi a que apresentou maior número de alelos por locos e maior número de alelos exclusivos, o que permite sugerir que unidades de conservação abrigam maior diversidade genética que populações com influência antrópica. Utilizando o teste X2 foi possível confirmar que o número de alelos encontrados nas populações naturais é significativamente mais elevado que os encontrados no outro tipo de ambiente. O mesmo não foi verificado para as populações estudadas com locos isoenzimáticos. A divergência genética entre as populações foi substancial, significativamente diferentes de zero e semelhantes para ambos os marcadores (θp=0,095 e θp=0,081 para marcadores SSR e Isoenzimáticos, respectivamente). A divergência genética entre as populações medida pela estimativa GST(Hedrick), que considera tanto o tipo como a freqüência dos alelos, apresentou valores superiores para os dois marcadores (SSR=0,167 e Isoenzimáticos= 0,102), indicando maior restrição no fluxo gênico histórico se comparado com as estimativas obtidas da estatística FST. O presente estudo permite sugerir que existe diferença na estrutura genética entre áreas sob intervenção antrópica e aquelas com intervenção restrita, havendo nas áreas mais preservadas maior diversidade genética. Os resultados obtidos com a estimativa GST(Hedrick) possibilitam propor que mesmo em espécies de ampla distribuição e que frequentemente ocupam ambientes de ação antrópica, como a lobeira, pode estar ocorrendo restrição de fluxo gênico e apontam maior diferenciação entre populações de lobeira do que relatado em estudos anteriores. / Solanum lycocarpum A. St.-Hil. (Solanaceae) has a wide distribution in savanna biome. The species is common know as "fruta-do-lobo" due the "lobo-guará" frequently to eat your fruits. "Lobo-guará" is also the main seed disperser of the species. The species is used for local people to manufacture sweets and as medicine. The species flowering and produce fruits during all year, favoring pollen and seed gene flow. The species colonize easily anthropized environments, permitting your use in environmental restoration projects. The goal of this study was to characterize the genetic structure of S. lycocarpum in natural and anthropized populations, using two different kinds of genetic markers: microsatellites (SSR) and allozymes. Four populations were studied using SSR markers and two using allozyme markers, forming pair of populations (a natural and an anthropized). Two populations studied by SSR markers were located in Northeast of Goiás State and two in South of the State. The two South populations were also studied by allozyme markers. Samples were randomly collected from 60 individuals in each population, with exception in one anthropized population study by allozymes, where 41 individuals were sampled. The samples were envied to LARGEA – ESALQ/USP, where the Lab analyses were made. The genetic diversity and distribution among and within populations was quantified. Although fixation index values were highest in some populations, these values were not statistically different from zero for both used genetic markers, suggesting absence of inbreeding in the studied populations. For SSR markers, natural populations showed higher number of alleles per locus than anthropized populations. A population located in a conservation unity presents the highest number of alleles per locus and exclusive alleles, suggesting that this population have higher genetic diversity than anthropized populations. According to a X2 test, the number of SSR alleles was significantly higher that detected in anthropized population. The same was not observed for population studied by allozyme loci. The genetic divergence among populations was substantial and significant different from zero for both used genetic markers (θp=0.095 and θp=0.081 for SSR and allozymes, respectively). The genetic divergence measured by GST(Hedrick) statistic, that considerer simultaneously the kinds of alleles and gene frequency was higher for both genetic markers (SSR=0.167 and allozymes= 0.102), indicating lower historic gene flow than measured by FST statistic. The present study suggested that there are differences in genetic structure between natural and anthopized populations, where natural populations have more genetic diversity. The results from GST(Hedrick) statistic suggested that even species with wide geographic distribution and whish frequently colonizing anthropized environments, as lobeira, can experience restriction in gene flow. The present results also indicate higher genetic differentiation among population than has been related in previous population study with this species.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This work describes the chemical study of Solanum buddleifolium (Solanaceae) aimed the isolation and structural characterization of its secondary metabolites. The chemical prospection was realized using chromatographic techniques such as chromatography over silica gel Sephadex LH-20 and solid phase extraction (SPE) besides High Performance Liquid Chromatography (HPLC) From EtOH were isolated the known compounds β-sitosterol and estigmasterol betulinic acid 13-hidroxysolavetrivone polistachiol N-trans-feruloyltiramine N-cis-Feruloyltiramine N-trans-feruloyl-3-methyldopamine N-trans-coumaroyltiramine N-trans-caffeoyltiramine N-trans-feruloyldopamine (+)-lioniresinol (-)-lioniresinol (+)-3-metoxisolariciresinol and the alkaloid solamargine alangilignoside C and ligalbumoside A (+)-alangilignoside D and (-)-alangilignoside D β-sitosterol glucoside and N-trans-caffeoyldopamine The structures of all compounds were determined by using spectrometric techniques (IR MS and 1H and 13C NMR) including 2D experiments (COSY HSQC HMBC and NOESy) and comparison with published data This is the first report about S. buddleifolium All lignans are been described for the first time in the genus Solanum and consequently represent an important contribution for the chemiotaxonomy of the genus / Este trabalho descreve o estudo quÃmico realizado com Solanum buddleifolium (Solanaceae) visando o isolamento e a caracterizaÃÃo estrutural de seus metabÃlitos secundÃrios A investigaÃÃo quÃmica do extrato etanÃlico dos talos da referida espÃcie foi realizada atravÃs de tÃcnicas cromatogrÃficas cromatografia em gel de sÃlica e por exclusÃo molecular (Sephadex L-20) cromatografia por extraÃÃo em fase sÃlida (SPE) e Cromatografia LÃquida de Alta EficiÃncia (CLAE) Do procedimento de extraÃÃo Ãcido/base da fraÃÃo hidroalcoÃlica do extrato etanÃlico foram isolados (+)-lioniresinol (SB-1) (-)-lioniresinol (SB-2) (+)-3-metoxisolariciresinol (SB-3) e o alcalÃide solamargina (SB-4) Da fraÃÃo diclorometano do extrato etanÃlico dos talos de S. buddleifolium foram isolados e caracterizados os seguintes compostos β-sitosterol (SB-5A) e estigmasterol (SB-5B) Ãcido betulÃnico (SB-6) 13-hidroxisolavetivona (SB-7) polistachiol (SB-8) N-trans-feruloiltiramina (SB-9A) N-cis-feruloiltiramina (SB-9B) N-trans-feruloil-3-metildopamina (SB-10) N-trans-coumaroiltiramina (SB-11) N-trans-caffeoiltiramina (SB-12) N-trans-feruloildopamina (SB-13) o glicosÃdeo do β-sitosterol (SB-14) alangilignoside C (SB-15A) e ligalbumoside A (SB-15B) (+)-alangilignoside D (SB-16A) e (-)-alangilignoside D (SB-16B) e N-trans-cafferoildopamina (SB-17) As estruturas de todos os compostos foram determinadas com base em tÃcnicas espectromÃtricas (IV EM-IES e RMN 1H e 13C 1D e 2D) alÃm de comparaÃÃo com dados jà registrados na literatura. Este à o primeiro estudo envolvendo S. buddleifolium e todas as lignanas caracterizadas estÃo sendo descritas pela primeira vez no gÃnero Solanum representando uma importante contribuiÃÃo para o conhecimento quÃmico deste

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-21T13:55:51Z No. of bitstreams: 2 Dissertacao_AltevirRossatoViana.pdf: 1076277 bytes, checksum: e699ace63c5cd785e56e303606eb4dd2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-21T13:55:51Z (GMT). No. of bitstreams: 2 Dissertacao_AltevirRossatoViana.pdf: 1076277 bytes, checksum: e699ace63c5cd785e56e303606eb4dd2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-02-20 / Cancer is the second largest cause of death in the world, only behind cardiovascular disease. This pathology is able to circumvent mechanisms of proliferative suppression, growing and surviving in hostile environments. Melanoma is a type of malignant tumor, has high metastatic potential and may be resistant to conventional anticancer agents. Although it does not correspond to the most frequent type of neoplasia, it represents a high mortality rate because of its capacity of metastases and as untreated difficulties. Due to these factors, the search for alternatives is highly encouraged. Many of the anticancer medicaments have their origin from natural, semi-synthetic and synthetic products. In Brazil, due to the richness of the national flora and the lack of knowledge about its bioactive products, the projects that seek this knowledge are important. The objective of the present study was to verify the antitumor activity in vitro and in vivo of a plant of the family Solanaceae, present in Rio Grande do Sul. Vassobia breviflora is known for its antitumor potential, mainly due to the presence of vitanolides, Withaferin A (WA). However, the studies found with the Brazilian plant are scarce. The vegetal species used in the work was extracted from a private property in the region of Boca do Monte, in the municipality of Santa Maria, RS. It was cataloged by the herbarium of the Federal University of Santa Maria and its aqueous extract was prepared with the dried leaves, extracted at room temperature. Controls were made using the commercially acquired WA active principle and the standard chemotherapy for melanoma, Temozolomide. Verification of the amount of WA was conducted by high performance liquid chromatography (HPLC). The tests for the detection of antioxidant compounds in the extract were made from the analysis of total phenols, diammonium salt - 2,20-azino-bis (acid 3-ethylbenzthiazoline-6-sulfônico) (ABTS) e 2,2-diphenyl-1-picrylhydrazil (DPPH). The cytotoxic potential was investigated from the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), in B16F10 cells. In vivo tests were conducted in the C57BL/6 line of isogenic mice, inoculated subcutaneously with the B16F10 line melanoma and treated for 30 days orally with the different treatments. The project was approved by the institution's animal ethics committee. During the treatment period, the weights of the animals were checked weekly with the aid of an analytical balance. The volume of the tumor by the measurement of the tumor mass, with a digital caliper and the survival time by the Kaplan Meyer curve. After the euthanasia of the animals, they had the tumors removed and weighed. In addition, other organs, such as spleen, lymph node, liver, lung, brain and kidney were removed and fixed for later histopathological analysis, and properly conditioned and frozen for biochemical and gene analyzes. The statistic of the results was performed by the statistical package for the social (SPSS) version 23.0. To compare the treatments, one-way analysis of variance (ANOVA) was performed, followed by the Tukey post hoc test. The results demonstrated a presence of WA in the aqueous extract of Brazilian Vassobia breviflora, as well as its antioxidant potential. The MTT assay in the B16F10 cell line showed cytotoxic ability of treatment with the aqueous extract of Vassobia breviflora, when compared to the controls. In the in vivo model the survival of the animals treated with the combination between the aqueous extract in the highest concentration and the chemotherapeutic Temozolomide was better in relation to the other treatments, compared to the control. The weight and volume of the tumors in the different treatments used did not vary significantly in relation to the control group. It is concluded that from the in vitro results, the plant species studied has a cytotoxic potential. However, for the in vivo assays, it is believed that the aqueous extract does not have a significant potential in the control of melanoma development, requiring models with the use of the ethanolic extract and other fractions of the constituents of the vegetal species for the best evaluation. As well as the use of melanoma induction by other routes of inoculation. Ex vivo tests will be needed to better elucidate the activity of the plant against this model of melanoma. / O câncer corresponde a segunda maior causa de mortes no mundo, atrás apenas das doenças cardiovasculares. Essa patologia faz com que as células adquiram a capacidade de driblar os mecanismos de supressão proliferativa, crescendo indefinidamente e sobrevivendo em ambientes hostis. O melanoma é um tipo de tumor maligno, apresenta alto potencial metastático e pode ser resistente a agentes anticancerígenos convencionais. Apesar de não corresponder ao tipo mais frequente de neoplasia, ele representa alta taxa de mortalidade, devido a sua capacidade de metástases e as dificuldades no tratamento. Por esses motivos, a busca por terapias preventivas e/ou adjuvantes é bastante incentivada. Muitos dos medicamentos anticancerígenos tiveram a sua origem a partir de produtos naturais, semissintéticos e sintéticos. No Brasil, devido a riqueza da flora nacional e o pouco conhecimento sobre os produtos bioativos da mesma, os projetos que buscam esse conhecimento são importantes. O objetivo do presente estudo foi o de verificar a atividade antitumoral in vitro e in vivo de uma planta da família Solanaceae, presente no Rio Grande do Sul. Vassobia breviflora é conhecida pelo seu potencial antitumoral, devido principalmente à presença de vitanolídeos, sendo o principal deles a Vitaferina A (VA). No entanto, os estudos encontrados com a planta brasileira são escassos. A espécie vegetal utilizada no trabalho foi extraída de uma propriedade privada na região de Boca do Monte, no município de Santa Maria, RS. Ela foi catalogada pelo herbário da Universidade Federal de Santa Maria e o seu extrato aquoso foi preparado com as folhas secas, extraídas a temperatura ambiente. Os controles foram feitos com a utilização do princípio ativo VA, adquirido comercialmente e do quimioterápico padrão para o melanoma, a Temozolomida. A verificação da quantidade de VA foi conduzida por cromatografia líquida de alta eficiência (CLAE). Os ensaios para a detecção de compostos antioxidantes no extrato foram feitos a partir da análise de fenóis totais, sal de diamônio 2,2'-Azino-bis (3-etilbenzotiazolina-6-sulfônico) (ABTS) e 2,2-Difenil-1- Picrilidrazilo (DPPH). O potencial citotóxico foi investigado a partir do ensaio de (3 (4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio brometo) (MTT), em células B16F10. Os testes in vivo foram conduzidos na linhagem C57BL/6, de camundongos isogênicos, inoculados pela via subcutânea, com a linhagem de melanoma B16F10 e tratados por um período de 30 dias, por via oral, com os diferentes tratamentos. O projeto teve aprovação do comitê de ética no uso de animais da Instituição. Durante o período de tratamento foram verificados os pesos dos animais, semanalmente, com o auxílio de balança analítica. O volume do tumor, pela medida do diâmetro da massa tumoral, com paquímetro digital e o tempo de sobrevida pela curva de Kaplan Meyer. Após a eutanásia dos animais, eles tiveram os tumores removidos e pesados. Além disso, outros órgãos como baço, linfonodo, fígado, pulmão, cérebro e rim foram retirados e fixados para posterior análise histopatológica, e devidamente acondicionados e congelados para as análises bioquímica e de expressão gênica. A estatística dos resultados foi realizada pelo statistical package for the social (SPSS) versão 23.0. Para comparar os tratamentos, foram utilizadas análises de variância (ANOVA) de uma via, seguido do teste post hoc de Tukey. Os resultados demonstraram a presença da VA no extrato aquoso da Vassobia breviflora brasileira, assim como o seu potencial antioxidante. O ensaio de MTT na linhagem celular B16F10 mostrou capacidade citotóxica do tratamento com o extrato aquoso de Vassobia breviflora, quando comparado aos controles. No modelo in vivo a sobrevida dos animais tratados com a associação entre o extrato aquoso na maior concentração e o quimioterápico Temozolomida foi melhor em relação aos demais tratamentos, comparados ao controle. O peso e o volume dos tumores, nos diferentes tratamentos utilizados, não variou significativamente em relação ao grupo controle. Conclui-se a partir dos resultados in vitro, que a espécie vegetal estudada apresenta um potencial citotóxico. No entanto, para os ensaios in vivo acredita-se que o extrato aquoso não tenha um potencial significativo no controle do desenvolvimento do melanoma, necessitando de modelos com a utilização do extrato etanólico e demais frações dos constituintes da espécie vegetal para a melhor avaliação. Bem como a utilização de indução do melanoma por outras vias de inoculação. Testes ex vivo serão necessários para a melhor elucidação da atividade da planta frente a esse modelo de melanoma.