Klimawandelmodelle sagen für Deutschland sowohl höhere Jahresdurchschnittstemperaturen als auch eine Abnahme von Niederschlägen in den Sommermonaten voraus. Mögliche Konsequenzen für Bäume sind eine verlängerte Vegetationsperiode, ein erhöhtes Spätfrostrisiko und mehr Trockenstress während des Sommers. Diese veränderten Umweltbedingungen könnten zu Veränderungen der Konkurrenzverhältnisse zwischen Baumarten führen. Die Rotbuche (Fagus sylvatica L.) ist eine der wichtigsten Laubbaumarten Mitteleuropas. Daher ist das genetische Anpassungspotential dieser Baumart an den Klimawandel von großem Interesse. In dieser Studie wurden sowohl die neutrale als auch die adaptive genetische Variation der Buche untersucht. Dafür wurde ein Translokationsexperiment mit Nachkommen von Buchenpopulationen, die unter verschiedenen Umweltbedingungen in Norddeutschland wachsen, etabliert. Wiederholte Aufnahmen wichtiger phänotypischer Merkmale (Höhe, Austrieb, Trockenstresssensitivität, Sterblichkeit) zeigten signifikante Unterschiede zwischen den Populationen. Interessanterweise zeigten Populationen mit einer größeren geographischen Distanz teilweise ähnlichere Phänotypen als benachbarte Populationen. Die neutrale genetische Variation der untersuchten Sämlingspopulationen wurde anhand neun verschiedener Mikrosatellitenmarker analysiert. Zwischen den analysierten Buchenpopulationen wurde nur eine geringe genetische Differenzierung ermittelt. Die genetische Diversität war hoch und statistisch nicht signifikant unterschiedlich von den Altbeständen, aus denen sie stammten. Die hohe genetische Diversität ist eine gute Basis für Adaption, allerdings könnte sie wahrscheinlich nur eine kurzfristige Anpassung an den Klimawandel ermöglichen. Daher ist es wichtig, Einblicke in die genetische Basis von klimawandelrelevanten Merkmalen zu gewinnen. Deshalb wurden in dieser Studie Kandidatengene für das Austriebsverhalten untersucht. Bei der Analyse von Fragmenten von zehn verschiedenen Kandidatengenen wurden 20 Indels und 116 SNPs identifiziert. Insgesamt wurden 46 SNPs erfolgreich zur Genotypisierung von über 1.400 Individuen, die aufgrund ihres Austriebsverhaltens ausgewählt wurden, verwendet. Assoziationsanalysen wurden durchgeführt, um potentiell adaptive SNP-Marker zu identifizieren. Diese ergaben unter einem „generalisierten linearen Modell“ 23 signifikant mit dem Austrieb assoziierte SNPs. Ein zusätzlich verwendetes „gemischtes lineares Modell“ ergab nahezu gleiche Ergebnisse. Die phänotypische Variation, die durch signifikant mit dem Austrieb assoziierte SNPs erklärt wird, war niedrig (R2 < 2,2), aber in Übereinstimmung mit anderen Studien mit Waldbaumarten. Zusätzlich zu den Assoziationsanalysen wurden auch FST-Outlier-Analysen durchgeführt. Diese ergaben sieben verschiedene SNPs, die potentiell unter ausgleichender oder gerichteter Selektion stehen. Insgesamt wurden vier potentiell adaptive SNPs gleichzeitig durch Assoziations- und Outlier-Analysen identifiziert. Diese könnten die höchste Wahrscheinlichkeit aufweisen, an der Ausprägung des Austriebsverhaltens beteiligt zu sein. Allerdings sind viele potentiell adaptive SNPs, die in dieser Studie identifiziert wurden, nicht-kodierend oder synonym und somit nicht die kausativen SNPs, sondern eher gelinkt mit ihnen. Allerdings wurde in dieser Studie ein geringes Kopplungsungleichgewicht (linkage disequilibrium) gefunden. Somit könnten die kausativen SNPs in naher Umgebung liegen. Die in dieser Studie identifizierten potentiell adaptiven SNPs sollten in weiteren Studien mit zusätzlichen Populationen bestätigt werden.

The metanephric kidney consists of two types of epithelia; the Wolffian duct-derived ureteric bud and the nephrogenic components that originate from mesenchymal-toepithelial transitions in the metanephric mesenchyme. The ureteric bud forms when inductive signals from the metanephric mesenchyme stimulates the evagination of an epithelial tube from the Wolffian duct into the mesenchyme. Reciprocal signalling between the ureteric bud and the metanephric mesenchyme regulates the branching of the ureteric bud and the induction of nephron formation. Inductive and inhibitory signalling of ureteric bud growth and branching has been shown by several protein families, however, the mechanical aspects of ureteric bud branching and nephrogenesis are largely unknown. I investigated the roles of Rac1-GTPase and Rho-kinase during kidney development. These proteins are important regulators of the cytoskeleton where Rac1 is a promoter of actin filament polymerisation and Rho-kinase directly stimulates the formation and contraction of actin-myosin stress fibres. Using a cell-permeable inhibitor, Rac1 was inhibited with no effects on nephron formation or subsequent segmentation and patterning. Inhibition of active Rac1 significantly reduced the level of ureteric bud branching and also resulted in lower proliferation rates. Rho-kinase was similarly targeted using two inhibitors. Rho-kinase inhibition had important effects on nephron formation and nephron maturation. Inhibition of Rhokinase resulted in decreased levels of nephron formation and severely morphologically abnormal nephrons. The formation of apical-basal polarity was disturbed as was the development of the visceral and parietal epithelia; precursors of the renal corpuscle. Inhibition of Rho-kinase led to abnormal formation of the proximal-distal axis and abnormal segmentation of the nephron. The effects of Rho-kinase inhibition were partially mimicked by direct targeting of actin-myosin contractions using a myosin-ATPase inhibitor. This demonstrated that Rho-kinase is necessary during multiple stages of nephrogenesis and maturation, at least in part, as a result of its ability to regulate actin-myosin contraction. These results show that Rac1 and Rho-kinase play important roles during several aspects of kidney development and highlights the significance of further investigating the mechanisms involved during kidney organogenesis.

Doctor of Philosophy / Department of Biology / David C. Hartnett / Grazing and drought are instrumental in the development and maintenance of perennial grasslands. In this research I tested the belowground bud bank contribution to tallgrass prairie resistance and resilience when perturbed by grazing and drought. First, I tested the bud bank role in vegetation response to and recovery from severe drought (Chapter 2). I compared above- and belowground responses of experimentally droughted plots to ambient controls and irrigated plots during two years of severe drought and two years of recovery. I found that although aboveground net primary productivity declined 30-60% during drought, bud bank density and demography were insensitive to drought. These results suggest that grassland resistance and resilience when perturbed by drought may be mediated by stability of belowground bud banks. Second, I investigated vegetation and soil nutrient legacies following release from long-term grazing (Chapter 3). I documented a relatively rapid shift in aboveground vegetation within four years of grazer exclusion, with productivity, stem density, and diversity becoming relatively more similar to ungrazed than grazed prairie. The density and composition of the belowground bud bank and soil seed bank shifted more slowly, remaining more similar to grazed than ungrazed prairie. Responses of soil nutrients to removal of grazers varied, and in some cases was affected by recent fire history. These results demonstrate the contribution of belowground propagules to the maintenance of a diverse plant community both during grazing and after grazers are removed. Finally, I examined short-term vegetation responses to both drought and grazing (Chapter 4). Despite extreme drought and simulated grazing that reduced productivity and increased mortality of individual stems, the dominant C4 grasses maintained a stable bud bank. Aboveground net primary productivity and bud bank density of sedges and forbs, however, were reduced by both drought and grazing. This differential response of species to extreme drought and grazing led to shifts in community composition and species diversity over one growing season. Across drought and grazing treatments, live rhizome biomass was highly correlated with bud bank density and may be a useful, more easily measured index of bud bank density.

As células derivadas de fígado embrionário tanto de animais quanto de humanos tem sido cada vez mais estudas devido ao seu potencial antiinflamatório, imunomodulador e regenerativo, demonstrado as mesmas bipotencial de diferenciação em hepatócitos e colangiocitos. Na presente pesquisa utilizou-se células derivadas de fígados embrionários de ratos com 14,5 dias de gestação. As células apresentaram marcadores de células progenitoras hepáticas, bem como marcadores de células hepáticas e biliares diferenciadas confirmando, seu bipotencial. A terapia celular utilizando as células supracitadas, reduziu significativamente a progressão da fibrose hepática, diminuindo a inflamação e ainda estimulando a regeneração hepática de ratos submetidos à cirrose por ligadura do ducto biliar. As análises realizadas mediante avaliação quantitativa pela técnica de morfometria, demonstraram redução da deposição de fibras de colágeno, bem como menor proliferação de ductos biliares nos animais tratados. Os resultados foram ainda complementados por analise semiquantitativa, a qual avaliou a intensidade da necroinflamação dos tecidos hepáticos analisados, apontando menor escore de inflamação dos animais tratados. As células poderão ter efeito benéfico para o tratamento de doenças hepáticas crônicas, que estimulam a formação de fibrose. A cirrose é o estágio final comum à doenças hepáticas crônicas por causadas por fatores de diversas etiologias. Esta ocupa a decima quarta causa mundial de mortalidade em humanos, sendo que o único tratamento definitivo atualmente é transplante do órgão. Entretanto o número de transplantes está longe de suprir a demanda atual, visto que há um déficit de doadores do órgão. Terapias que possam oferecer uma alternativa de tratamento confiável, segura e acessível são bastante oportunas. Nossos resultados sugerem que as células utilizadas neste trabalho podem modular a fibrogênese, e consequentemente retardar o estabelecimento da cirrose em doenças hepáticas crônicas. / Studies on human and animal embryonic liver stem cells have been growing due to its anti-inflammatory, immunomodulatory and regenerative potential. These cells show also a bipotential do differentiate into hepatocytes and cholangiocytes. In the present study, it was used rodent embryonic liver with 14.5 of gestation. The cells presented hepatic progenitor, as well adult hepatic and biliary cells markers, confirming their bipotential. Previous studies with these cells in therapy decreased hepatic fibrosis progression in rat models submitted to cirrhosis by biliary duct ligation. Quantitative analysis was performed by morphometry showed decreased collagen fibers deposition and lower proliferation of biliary ducts in treated animals. Results were complemented with semiquantitative analysis with evaluation of necroinflammation of the analyzed hepatic tissues, in which a decreased inflammation score was observed. Cirrhosis is a common final stage for chronic hepatic diseases caused by different factors in several etiologies. It occupies the 14th world cause of mortality in human. However, the number of liver transplants is insufficient for current demand, caused by deficit in organs donors. Therapies that could offer an alternative for a reliable, safe and accessible treatment is opportune. Our results suggest that cells used in this study can modulate fibrogenesis and consequently delay the establishment of cirrhosis in chronic liver diseases.

A área de Cerrado escolhida para o estudo está em regeneração na Estação Ecológica de Santa Bárbara, localizada no município de Águas de Santa Bárbara, SP. Nesta área o Pinus, que estava sob cultivo desde os anos 70, foi retirado em 2012 e, em 2014, foi realizada uma queimada. As espécies Eugenia dysenterica (Mart.) DC. e E. punicifolia (Kunth) DC. foram escolhidas devido ao elevado número de indivíduos que rebrotaram na área. O objetivo geral do projeto foi conduzir análises morfoanatômicas e químicas das estruturas relacionadas ao potencial de rebrotamento. Foram realizadas: a contagem das gemas subterrâneas nos primeiros 10 centímetros abaixo do nível do solo, as análises químicas das raízes e análises anatômicas na região terminal de ramos aéreos e no sistema subterrâneo. Nas duas espécies, além da gema apical dos ramos aéreos, nas três regiões nodais subsequentes, acima da gema axilar ocorre uma gema acessória. Em todas as gemas, observam-se estruturas de proteção, pois junto ao meristema apical caulinar (MAC) ocorrem coléteres e cristais. Além disso, o MAC é protegido por primórdios foliares com cavidades de óleo, muitos cristais e tricomas unicelulares não glandulares que possuem espessamento parietal em celulose e podem acumular compostos fenólicos. Nos sistemas subterrâneos, o número de gemas, localizadas preferencialmente nos primeiros cinco centímetros do solo, variou entre os três indivíduos da mesma espécie: E. dysenterica 162, 17 e 253, E. punicifolia 24, 40 e 109. Esta variação deveu-se ao grau de desenvolvimento das estruturas subterrâneas as quais certamente formaram-se antes do período da retirada do Pinus. O sistema subterrâneo das espécies é formado por um eixo lenhoso cuja porção superior é caulinar e emite vários ramos aéreos enquanto a porção inferior é constituída por uma raiz axial espessada que pode atingir até um metro de profundidade em E. dysenterica. Em E. punicifolia o sistema subterrâneo ocupa uma região mais superficial do solo e, assim como o caule, as raízes axial e adventícias são distribuídas num plano mais horizontal. O tecido de revestimento nas raízes de E. punicifolia e no caule e raízes de E. dysenterica, apresenta camadas alternadas de células de paredes suberizadas e de células com espessamentos parietais em pectina; no caule de E. punicifolia há esclereides que se alternam com as células suberizadas. Em E. punicifolia o espessamento resulta de divisões anticlinais e expansão tangencial das células do parênquima floemático. Há floema interno e parênquima medular nos caules subterrâneos de ambas as espécies. Compostos fenólicos e grãos de amido estão presentes nas células parenquimáticas de regiões mais espessadas dos caules e raízes. Os teores de carboidratos totais, hemicelulose, celulose e lignina não diferem significativamente nas duas espécies. No entanto, as concentrações de flavonoides totais e de compostos fenólicos totais foram superiores nas raízes de E. punicifolia. O grau de proteção das gemas aéreas, o elevado número de gemas e o acúmulo de compostos de reserva (amido) e de proteção (fenóis, flavonoides, lignina) nas estruturas subterrâneas podem ter favorecido o seu rebrotamento após a retirada do Pinus e a sua permanência das espécies na área. / The Cerrado area studied is under regeneration at the Estação Ecológica de Santa Bárbara, located in Águas de Santa Bárbara (São Paulo, Brazil). In this area, the Pinus, cultivated since the 1970s, was removed in 2012 and then, in 2014, the area was burnt. The species Eugenia dysenterica (Mart.) DC. and E. punicifolia (Kunth) DC. were studied due to their high resprouting after the burnt event. The aim was to carry out morphoanatomical and chemical analyses of the structures related to the resprouting capacity. The number of belowground buds was counted in the first 10 cm below the soil surface. The chemical analyses of the roots and anatomical analyses of the end portion of the aerial branches and the underground system were performed. Besides the apical bud, both species showed an accessory bud above the axillary one at the three subsequent nodal regions. In all studied buds, protection structures were observed, as colleters and crystals occur close to the shoot apical meristem (SAM). Moreover, SAM is protected by leaf primordia with oil cavities, many crystals and non-glandular unicellular trichomes. These trichomes present parietal cellulose thickening and are able to accumulate phenolic compounds. In underground systems, the number of buds, mostly in the first five centimeters below the soil surface, varied between individuals per species: E. dysenterica 162, 17 and 253; E. punicifolia 24, 40 and 109. This variation was caused by the development degree of the underground system, whose structures were certainly developed before Pinus removal. The underground system of the species display a woody axis, whose upper portion is a stem structure and produces several aerial branches. Its lower part is formed by an axial thickened root that can reach one metre deep in E. dysenterica. In E. punicifolia, the subterranean system occupies a more superficial region of the soil and, similar to the stem, the axial and adventitious roots are distributed in a more horizontal plane. The covering tissue of underground systems in E. punicifolia and E. dysenterica displays alternate layers of cells with suberized walls and pectin-thickened walls. In E. punicifolia stem, the covering tissue exhibits sclereids alternate with cells of suberized walls. The thickening in E. punicifolia is a result of anticlinal divisions and tangential expansion of phloematic parenchyma cells. Both species show internal phloem and medullary parenchyma in underground stems. Phenolic compounds and starch grains are present in parenchyma cells of thickened regions in stems and roots. The contents of total carbohydrates, hemicellulose, cellulose and lignin do not differ significantly in the two species. However, concentrations of total flavonoids and total phenolic compounds are higher in roots of E. punicifolia. The protection level of aerial buds, large number of underground buds, and accumulation of storage (starch) and protection (phenols, flavonoids, lignin) compounds in the underground structures may have favoured resprouting of the species after Pinus removal and their maintenance in the area.

Magister Chirurgiae Dentium - MChD (Oral Medicine and Periodontics) / Candida species are frequently isolated from oral mucosal surfaces of healthy individuals and is the most common genus responsible for up to 75% of all candidal infections. The most common problems associated management of oral candidiasis are antifungal drug resistance and side effects Natural medicine is an emerging field and is being explored to overcome drug resistance and to reduce side effects. Gymnemagenin (will be known as Gymnemic acid; GA) is a purified extract from Gymnema sylvestre, a slow growing, perennial, medicinal plant found in Central and Western India, Tropical Africa and Australia is regarded as one of the plants with potent antimicrobial and antifungal activity.

Doenças envolvendo órgãos endodermicamente derivados afetam milhares de pessoas no mundo. O sucesso da terapia celular para o tratamento de doenças de órgãos oriundos da mesoderme e ectoderme gera ótimas perspectivas para o uso do mesmo em tratamento de doenças de órgãos de origem endodérmica (tireóide, pulmões, fígado, vesícula biliar e pâncreas). Particularmente, no fígado, ainda que sejam atribuídas propriedades regenerativas em muitas lesões o mecanismo de reparação é insuficiente, tornando o transplante hepático à única opção definitiva. Dentre as células-tronco embrionárias, as células de linhagens hepáticas fetais podem estabelecer-se como fonte importante para a terapia celular em indivíduos com doenças hepáticas, pois possuem um alto índice de diferenciação de hepatócitos e células do ducto biliar in vitro. Evidências apontam a progranulina como um fator de crescimento de grande habilidade para a indução de proliferação celular, uma vez que está envolvida no desenvolvimento embrionário e neonatal. Sendo assim, neste trabalho foram utilizados embriões de ratos Fischer 344 com idades gestacionais 12,5; 13,5; 14,5; 15,5; 16;5 para caracterizar o papel do novo fator de crescimento progranulina na hepatogênese. Foram realizadas análises histológicas, de microscopia eletrônica de transmissão e imuno-histoquímicas nos embriões. Houve expressão de progranulina e Oct-4 (marcador de célula tronco indiferenciada) principalmente nas idades gestacionais de 13,5 a 16,6 dias e 12,5 a 16,5 dias para PCNA (marcador de proliferação celular). Dessa forma acredita-se que, a progranulina atua nos processos de proliferação celular e diferenciação das células-tronco do broto hepático, podendo ser usada como um fator de diferenciação em culturas in vitro visando à diferenciação de células-tronco do broto hepático em hepatócitos funcionais para a terapia celular. / Diseases involving endodermal-derivated organs affect thousands of people all over the world. The cell therapy success for treating diseases of organs originated from mesoderm and ectoderm generates excellent perspectives for its use in treating diseases of organs of endodermal origin (thyroid, lungs, liver, gallbladder and pancreas). Particularly in the liver, although regenerative proprieties are attributed in many lesions, the repairing mechanism is insufficient, making the hepatic transplant the only definitive option. Among the embryo stem cells, the fetal hepatic lineage cells may establish themselves as important sources for cell therapy in individuals with hepatic diseases, since they have high ability in hepatocytes and billiar duct cells differentiation in vitro. However, for the use of hepatic lineage embryo stem cells as a bi-potential source of differentiation, it is necessary the establishment of efficient proliferation methods in this type of cells. Evidences point to progranuline as a growth factor with high capability for the induction of cell proliferation, since it is involved in the embryo and neo-natal development. Thus, this study used embryos of Fischer 344 rats with gestational ages 12.5, 13.5, 14.5, 15.5 and 16.5 to characterize the role of the new growth factor progranuline in hepatogenesis. We conducted histological, transmission electron microscopy and immunohistochemical in embryos. There was expression of progranuline and Oct-4 (undifferentiated stem cell marker), especially at gestational ages 13.5 to 16.5 days and 12.5 to 16.5 days for PCNA (proliferation marker). So it is believed that progranuline acts on cell proliferation and differentiation of stem cells from the liver bud, which can be used as a differentiating factor in order to cultures in vitro differentiation of stem cells from the liver bud into functional hepatocytes for cell therapy.

A área de Cerrado escolhida para o estudo está em regeneração na Estação Ecológica de Santa Bárbara, localizada no município de Águas de Santa Bárbara, SP. Nesta área o Pinus, que estava sob cultivo desde os anos 70, foi retirado em 2012 e, em 2014, foi realizada uma queimada. As espécies Eugenia dysenterica (Mart.) DC. e E. punicifolia (Kunth) DC. foram escolhidas devido ao elevado número de indivíduos que rebrotaram na área. O objetivo geral do projeto foi conduzir análises morfoanatômicas e químicas das estruturas relacionadas ao potencial de rebrotamento. Foram realizadas: a contagem das gemas subterrâneas nos primeiros 10 centímetros abaixo do nível do solo, as análises químicas das raízes e análises anatômicas na região terminal de ramos aéreos e no sistema subterrâneo. Nas duas espécies, além da gema apical dos ramos aéreos, nas três regiões nodais subsequentes, acima da gema axilar ocorre uma gema acessória. Em todas as gemas, observam-se estruturas de proteção, pois junto ao meristema apical caulinar (MAC) ocorrem coléteres e cristais. Além disso, o MAC é protegido por primórdios foliares com cavidades de óleo, muitos cristais e tricomas unicelulares não glandulares que possuem espessamento parietal em celulose e podem acumular compostos fenólicos. Nos sistemas subterrâneos, o número de gemas, localizadas preferencialmente nos primeiros cinco centímetros do solo, variou entre os três indivíduos da mesma espécie: E. dysenterica 162, 17 e 253, E. punicifolia 24, 40 e 109. Esta variação deveu-se ao grau de desenvolvimento das estruturas subterrâneas as quais certamente formaram-se antes do período da retirada do Pinus. O sistema subterrâneo das espécies é formado por um eixo lenhoso cuja porção superior é caulinar e emite vários ramos aéreos enquanto a porção inferior é constituída por uma raiz axial espessada que pode atingir até um metro de profundidade em E. dysenterica. Em E. punicifolia o sistema subterrâneo ocupa uma região mais superficial do solo e, assim como o caule, as raízes axial e adventícias são distribuídas num plano mais horizontal. O tecido de revestimento nas raízes de E. punicifolia e no caule e raízes de E. dysenterica, apresenta camadas alternadas de células de paredes suberizadas e de células com espessamentos parietais em pectina; no caule de E. punicifolia há esclereides que se alternam com as células suberizadas. Em E. punicifolia o espessamento resulta de divisões anticlinais e expansão tangencial das células do parênquima floemático. Há floema interno e parênquima medular nos caules subterrâneos de ambas as espécies. Compostos fenólicos e grãos de amido estão presentes nas células parenquimáticas de regiões mais espessadas dos caules e raízes. Os teores de carboidratos totais, hemicelulose, celulose e lignina não diferem significativamente nas duas espécies. No entanto, as concentrações de flavonoides totais e de compostos fenólicos totais foram superiores nas raízes de E. punicifolia. O grau de proteção das gemas aéreas, o elevado número de gemas e o acúmulo de compostos de reserva (amido) e de proteção (fenóis, flavonoides, lignina) nas estruturas subterrâneas podem ter favorecido o seu rebrotamento após a retirada do Pinus e a sua permanência das espécies na área. / The Cerrado area studied is under regeneration at the Estação Ecológica de Santa Bárbara, located in Águas de Santa Bárbara (São Paulo, Brazil). In this area, the Pinus, cultivated since the 1970s, was removed in 2012 and then, in 2014, the area was burnt. The species Eugenia dysenterica (Mart.) DC. and E. punicifolia (Kunth) DC. were studied due to their high resprouting after the burnt event. The aim was to carry out morphoanatomical and chemical analyses of the structures related to the resprouting capacity. The number of belowground buds was counted in the first 10 cm below the soil surface. The chemical analyses of the roots and anatomical analyses of the end portion of the aerial branches and the underground system were performed. Besides the apical bud, both species showed an accessory bud above the axillary one at the three subsequent nodal regions. In all studied buds, protection structures were observed, as colleters and crystals occur close to the shoot apical meristem (SAM). Moreover, SAM is protected by leaf primordia with oil cavities, many crystals and non-glandular unicellular trichomes. These trichomes present parietal cellulose thickening and are able to accumulate phenolic compounds. In underground systems, the number of buds, mostly in the first five centimeters below the soil surface, varied between individuals per species: E. dysenterica 162, 17 and 253; E. punicifolia 24, 40 and 109. This variation was caused by the development degree of the underground system, whose structures were certainly developed before Pinus removal. The underground system of the species display a woody axis, whose upper portion is a stem structure and produces several aerial branches. Its lower part is formed by an axial thickened root that can reach one metre deep in E. dysenterica. In E. punicifolia, the subterranean system occupies a more superficial region of the soil and, similar to the stem, the axial and adventitious roots are distributed in a more horizontal plane. The covering tissue of underground systems in E. punicifolia and E. dysenterica displays alternate layers of cells with suberized walls and pectin-thickened walls. In E. punicifolia stem, the covering tissue exhibits sclereids alternate with cells of suberized walls. The thickening in E. punicifolia is a result of anticlinal divisions and tangential expansion of phloematic parenchyma cells. Both species show internal phloem and medullary parenchyma in underground stems. Phenolic compounds and starch grains are present in parenchyma cells of thickened regions in stems and roots. The contents of total carbohydrates, hemicellulose, cellulose and lignin do not differ significantly in the two species. However, concentrations of total flavonoids and total phenolic compounds are higher in roots of E. punicifolia. The protection level of aerial buds, large number of underground buds, and accumulation of storage (starch) and protection (phenols, flavonoids, lignin) compounds in the underground structures may have favoured resprouting of the species after Pinus removal and their maintenance in the area.

Norway spruce (Picea abies L. Karst) is a conifer belonging to the group gymnosperms and is an ecologically and economically important species in several parts of Europe. It is crucial for trees like Norway spruce to adapt timing of events such as bud set and growth cessation to the local environment in order to maximize the growth period while avoiding frost damage. This thesis aims at widening the knowledge about genetic control of annual growth rhythm in Norway spruce and particularly the control of bud set. Using spruce transformants ectopically expressing PaFT/TFL1-LIKE 2 (PaFTL2) the prior hypothesis that PaFTL2 induces bud set is confirmed. This is further supported by spatial and temporal expression patterns in seedlings and adult trees. It is further shown that gymnosperms possess at least two FLOWERING LOCUS T/TERMINAL FLOWER 1 (FT/TFL1)-like genes with TFL1-like function, suggesting the ancestor of FT and TFL1 to be more TFL1-like. PaFTL1 appears to have complementary expression patterns to that of PaFTL2 both spatially and temporally indicating they may act together to control growth in Norway spruce. Since bud set is controlled by photoperiod and circadian clock genes are implicated in this process, putative clock homologs were studied to gain insight into the circadian clock in gymnosperms. Several clock homologs were identified and their expression showed a diurnal pattern but the expression was rapidly damped in constant conditions. Transgenic Arabidopsis expressing putative core clock genes from spruce indicate that at least three genes, PaCCA1, PaGI and PaZTL, appear to have a conserved function between angiosperms and gymnosperms. Taken together these results suggest that gymnosperms have a similar core clock structure as angiosperms even though fundamental differences might exist since the cycling of the clock genes were rapidly damped in free-running conditions. The studies presented in this thesis support substantial conservation of pathway components controlling photoperiodic responses in angiosperms and gymnosperms and identify PaFTL2 as a component of growth rhythm control. However, important changes in these processes are also evident. The results provide a solid basis for future research on molecular mechanisms controlling an adaptive trait in an important non-model organism.

Branching is a highly plastic trait, enabling plants to adapt their growth form in response to environmental stimuli. In flowering plants, shoot branching is regulated through the activity of axillary buds, which grow into branches. Several classes of plant hormones have been shown to play pivotal roles in regulating bud outgrowth. Auxin derived from the primary shoot apex and active branches inhibits bud outgrowth, whereas cytokinin promotes it. Strigolactones also inhibit bud outgrowth, by changing properties of the auxin transport network, increasing the competition between buds. This occurs by modulating access to the polar auxin transport stream (PATS) in the main stem. The PATS provides directional, long distance transport of auxin down the stem, involving basal localisation of the auxin transporter PIN-FORMED1 (PIN1). Buds need to export their auxin across the stem towards the PATS in order to activate, but since PIN1 is mainly expressed in narrow files of cells associated with the stem vasculature, PIN1 itself it is unlikely to facilitate this connectivity. This thesis re-examines the role of auxin transport in the stem, showing that, besides the PIN1-mediated PATS, other auxin transport proteins constitute a more widespread and less polar auxin transport stream, allowing auxin exchange between the PATS and surrounding tissues. Disruption of this transport stream is shown to reduce bud-bud communication and to partially rescue the increased branching observed in strigolactone mutants. Furthermore, it is shown that distinct classes of auxin transport proteins within this stream can differentially affect bud outgrowth mediated by BRANCHED1 (BRC1). BRC1 is a transcription factor proposed to determine bud activation potential. Taken together, the data presented here provide a more comprehensive understanding of the shoot auxin transport network and its role in shoot branching regulation.