Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Sabendo da importÃncia de cÃlulas de mamÃferos em cultura para avaliar a citotoxicidade de novas substÃncias com aÃÃo terapÃutica, o presente trabalho determinou, inicialmente, a atividade citotÃxica por MTT e hemolÃtica de 7 diterpenos clerodanos (Ãcido hardiwickiico, casearinas L, O, U e sua forma degradada, e casearinas X e Y) isolados a partir das folhas de Casearia sylvestris frente a um painel de 9 linhagens de cÃlulas tumorais e a eritrÃcitos de camundongos. Na ausÃncia de hemÃlise de todos os diterpenos, a Casearina U (Cas U) mostrou ser o mais ativo contra cÃlulas tumorais. A atividade citotÃxica da Cas U parece depender do anel formado pelos carbonos C-18 e C-19, uma vez que a hidrÃlise Ãcida e sua abertura levam à diminuiÃÃo ou perda total da bioatividade. Posteriormente, os estudos de mecanismo de aÃÃo com a Cas U (0,4; 0,8 e 1,6 Âg/mL) revelaram reduÃÃo concentraÃÃo-dependente na viabilidade celular por azul de tripan e na sÃntese de DNA por incorporaÃÃo de BrdU, sendo o mecanismo antiproliferativo da Cas U independente de aÃÃo inibitÃria sobre a topoisomerase I. As anÃlises morfolÃgicas feitas por hematoxilina/eosina e por incorporaÃÃo de brometo de etÃdio/laranja de acridina mostraram alteraÃÃo no padrÃo de morte em favorecimento da necrose proporcional à concentraÃÃo, como desintegraÃÃo membranar e picnose nuclear (1,6 Âg/mL), embora tenha ocorrido tambÃm retraÃÃo celular, condensaÃÃo e fragmentaÃÃo da cromatina nucleares (0,4 e 0,8 Âg/mL), sinais condizentes com apoptose. Nos ensaios de fragmentaÃÃo do DNA por citometria de fluxo e de genotoxicidade por cometa, a atividade da Cas U foi concentraÃÃo-dependente e sem diferenciaÃÃo entre cÃlulas normais (linfÃcitos perifÃricos humanos) e cancerosas (HL-60). A avaliaÃÃo antitumoral (10 e 25 mg/kg/dia, intraperitoneal; 25 mg/kg/dia, oral) em camundongos transplantados com Sarcoma 180 revelou atividade apenas na maior dose via intraperitoneal, causando reduÃÃo de 90 % do crescimento tumoral e alteraÃÃes renais incipientes e reversÃveis, enfatizando a potencialidade da Cas U como molÃcula modelo para a sÃntese de novos compostos com propriedades anti-cÃncer. / Knowing the importance of mammalian cell culture in evaluating the cytotoxicity of new substances with therapeutic action, this work initially examined the cytotoxicity (by MTT assay) and hemolytic activity of 7 clerodane diterpenoids (acid hardiwickiico, casearins L, O and U and its degradation product, and casearins X and Y) isolated from leaves of Casearia sylvestris against a panel of 9 tumor cell lines and on mouse erythrocytes. All diterpenes studied showed no hemolytic effect, while casearin U (Cas U) was found to be the most active against tumor cells. Cytotoxic activity of Cas U seems to depend on the ring structure formed by carbons C-18 and C-19, since acid hydrolysis and ring opening led to a decrease or total loss of bioactivity. Subsequently, studies on the mechanism of action of Cas U (0.4, 0.8 and 1.6 Âg/mL) revealed a concentration-dependent decrease in cell viability as determined by trypan blue dye exclusion and in DNA synthesis assayed by BrdU incorporation, where this antiproliferative mechanism of Cas U was not found to be dependent on an inhibitory action on topoisomerase I. Morphological analysis assessed by hematoxylin/eosin and ethidium bromide/acridine orange staining showed alterations in the pattern of cell death toward necrosis according to concentration, as seen by membrane disintegration and pyknotic nuclei (1.6 Âg/mL). However, there also were cell volume reduction, and condensation and fragmentation of nuclear chromatin, consistent signs with apoptosis. DNA fragmentation was examined by flow cytometry and genotoxicity determined with the comet assay, and Cas U activity was found to be dependent on concentration but did not differ between normal (human peripheral lymphocytes) and malignant (HL-60) cells. Antitumor activity of Cas U was tested in mice transplanted with sarcoma 180 (10 and 25 mg/kg/day, intraperitoneally; 25 mg/kg/day, orally), and only the highest intraperitoneal dose was found to effective, leading to 90 % inhibition of tumor growth, with reversible changes in the kidneys. These findings point to the potential of Cas U as a model molecule to synthesize new compounds with anticancer properties.

Resumo: O presente trabalho teve como objetivo produzir ferramentas e informações úteis para a conservação e exploração racional de Casearia sylvestris Sw. (Salicaceae), uma espécie que produz diterpenos clerodânicos de grande importância farmacológica (casearinas), e que é explorada por extrativismo. Tal objetivo foi alcançado através do desenvolvimento de marcadores microssatélites específicos para C. sylvestris e de um estudo da diversidade genética e química existente entre e dentro de populações do Estado de São Paulo. Tradicionalmente são reconhecidas duas variedades em C. sylvestris (var. sylvestris e var. lingua), o que é motivo de debate devido à existência de formas intermediárias. Este trabalho objetivou, adicionalmente, contribuir com argumentos genéticos para esta discussão. Foi construída uma biblioteca enriquecida em microssatélites, a partir da qual obtiveram-se e validaram-se dez pares de iniciadores (primers) microssatélites específicos para C. sylvestris. Estes pares de iniciadores foram utilizados para o estudo da estrutura genética de populações de C. sylvestris através da amostragem de 376 indivíduos em nove populações distribuídas em quatro ecossistemas (Floresta Ombrófila Densa, Floresta Estacional Semidecidual, Cerrado e ecótonos). As duas variedades foram amostradas de acordo com sua distribuição nestes ecossistemas. A genotipagem dos indivíduos para os locos amostrados foi realizada através de eletroforese em gel de acrilamida lido a 700 e 800 nm por um seqüenciador IR2-DNA Analyser (LI-COR). Os dados foram analisados através de abordagens frequentistas, bayesianas e baseadas na teoria de coalescência, utilizando-se diversos programas computacionais. Para o estudo da diversidade química, as mesmas populações foram amostradas, selecionando-se 12 indivíduos por população, totalizando 108 indivíduos. Adicionalmente, foram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work aimed obtaining tools and information for the conservation and rational exploitation of Casearia sylvestris Sw. (Salicaceae), a tree species which produces casearins, pharmacologically important clerodane diterpenes. This goal was achieved through the development of a set of polymorphic microsatellite markers, and through the study of chemical and genetic diversity in populations of C. sylvestris from São Paulo State. Also, we aimed contributing for the debate on the existence of two varieties within this species (var. sylvestris e var. lingua). A genomic library was constructed and 10 primer pairs were obtained. Those primers were utilized for a population genetic structure analysis, in which 376 individuals from nine populations distributed on four different ecosystems (Evergreen Atlantic Forest, Semideciduous Atlantic Forest, Cerrado and ecotones) were sampled. The two varieties were sampled according to its distribution among these populations. Genotyping was performed at 700 and 800 nm by electrophoresis on an IR2-DNA Analyser (LI-COR). The data were analyzed through frequentist, Bayesian and coalescence-based approaches, through the use of several softwares. Chemical diversity was studied by sampling in the same populations (12 individuals per population, i.e. 108 individuals). Also, cuttings of these individuals were prepared, aiming to verify its' chemical compounds after a year of green-house cultivation. Cuttings' rooting was problematic and a methodology was developed. Only 46 cuttings survived. Casearins from these 154 individuals (108 + 46) were extracted and analyzed by HPLC. Genetic analysis results suggests a partial genome duplication, as more than two alleles for the same locus were observed in 8% of var. sylvestris individuals and in 70% of var. lingua individuals. Additional studies are necessary to verify the hypothesis of partial genome duplication... (Complete abstract click electronic access below) / Orientador: Marcos Aparecido Gimenes / Coorientador: Alberto José Cavalheiro / Banca: Roseli B. Torres / Banca: Maria Imaculada Zucchi / Banca: Giancarlo C. X. Oliveira / Doutor

A planta Casearia sylvestris é catalogada como planta medicinal de interesse ao SUS, indicada para o tratamento de gastrite e como cicatrizante, para ser utilizada na forma de infusão ou compressas. Assim, foi objetivo deste estudo avaliar o potencial terapêutico antiulcerogênico do extrato fluido (EF), extrato metanólico (EM) e fração diclorometano (FDM), pelo modelo de indução de úlcera por etanol-acidificado. Realizou-se também a análise fitoquímica objetivando identificar os compostos ativos presentes nos extratos, através de técnicas de cromatografia, espectrometria de massas e ressonância magnética nuclear. Os resultados obtidos mostraram que tanto o EF, quanto o EM e FDM foram eficazes na prevenção de úlceras gástricas. Além disso, foram avaliados os possíveis efeitos do EF no estresse oxidativo hepático e na indução ou inibição do complexo enzimático citocromo P450. Os resultados obtidos mostraram alteração apenas do ciclo-redox da glutationa com a razão GHS/GSSG diminuída nos animais tratados, sugerindo que deve ser ter cautela ao utilizá-lo concomitantemente a medicamentos pelo risco de interações medicamentosas. Adicionalmente, foi realizado um estudo preliminar do potencial antitumoral do EM e frações através de ensaio bioguiado de citoxicidade (ensaio sulforrodamina B), no qual foi observado pronunciada atividade citotóxica (IC50 ≤ 5µ/ml) em diferentes linhagens tumorais. A análise fitoquímica identificou flavonóides (quercetina, rutina, kaempferol) e terpenos (espatulenol, diterpeno clerodânico). Portanto, pode-se sugerir que os efeitos terapêuticos ocorrem da sinergia destes princípios ativos / The Casearia sylvestris plant is cataloged as a medicinal plant of interest to the Unified Health System in Brazil indicated for the treatment of gastritis and healing, to be used in the form of infusion or compresses. Thus, the aim of this study was to evaluate the antiulcerogenic therapeutic potential of fluid extract (EF), methanol extract (EM) and dichloromethane fraction (FDM) on ethanol/HCl induced gastric model ulcer. The results obtained showed that EF, as EM and FDM, have been effective in preventing gastric ulcers. Also, a phytochemical analysis by chromatographic techniques, mass spectrometry and nuclear magnetic resonance was carried out to identify the active constituents present in the extracts. Moreover the possible effects of EF on hepatic oxidative stress and on induction or inhibition of cytochrome P450 enzime complex were evaluated. The results obtained showed only an alteration on the redox-cycle of glutathione with the GHS/GSSG ratio decreased in treated animals, suggesting caution in the use of EF concomitantly with other drugs as there might be a risk of drug interactions. In addition, a preliminary study was carried out to evaluate through bio-guided citotocixity assay (sulforhodamine B) the antitumor potential of the EM and fractions, in which pronounced cytotoxic activity was observed (IC50 ≤ 5µg/ml) in different tumor cell lines. Therefore, it can be suggested that the therapeutic effects occur in consequence of the synergy of these active ingredients

Pour étudier le rôle de la mitochondrie dans la tolérance à la sécheresse, la réponse à la contrainte hydrique a été comparée entre une lignée sauvage (WT) et un mutant CMSII (Cytoplasmic Male Sterile) de Nicotiana sylvestris. Chez le mutant CMSII, le complexe I mitochondrial est absent et la respiration est assurée par les NAD(P)H déshydrogénases alternes et elle est maintenue à un niveau supérieur de l’ordre de 20 à 30% à celui du WT. La différence observée entre les plantes WT et CMSII met en jeu non seulement le fonctionnement mitochondrial, mais également le fonctionnement des chloroplastes. En effet, l’activité photosynthétique du mutant est plus faible que celui du WT et elle est corrélée avec une plus faible conductance stomatique (gs) et mésophyllienne (gm).Après l’arrêt de l’arrosage, on observe que le contenu relatif en eau (RWC) diminue plus lentement chez les feuilles du CMSII. Ceci n’était pas le résultat d’une plus petite surface de transpiration ou d’une masse racinaire d’absorption plus élevé puisque le rapport partie aérienne/racine et la surface foliaire totale ont été similaires au début de l’expérience chez les deux génotypes. De plus la mutation n’a pas induit des changements au niveau des paramètres hydriques (P0, PTLP, RWCTLP) ni au niveau de la densité stomatique. La tolérance des plantes CMSII a été le plus probablement la conséquence de sa plus faible transpiration en conditions bien hydratées et aux premiers jours de déshydratation et non pas d’une meilleure efficacité d’absorption de l’eau puisque le contenu en eau du sol reste plus élevé chez CMSII après l’arrêt de l’arrosage. La plus faible conductance stomatique chez le CMSII bien hydraté a été expliquée par sa plus faible conductance hydraulique. De plus, contrairement au WT, le niveau des acides aminés totaux diminue au cours de la déshydratation lorsque le contenu en protéines solubles augmente chez les feuilles du CMSII, suggérant une accélération de la remobilisation des acides aminés. D’autre part, il a été aussi montré que le mutant CMSII est capable de s’acclimater mieux à la sécheresse que le WT lorsqu’ils ont été maintenus à un RWC de 80 % sur plusieurs jours. Sous ces conditions, la photosynthèse reste plus élevée chez le mutant que chez le WT. Cette meilleure acclimatation corrèle avec une plus forte photorespiration du CMSII sous conditions bien hydratées et sous conditions d’acclimatation. La photorespiration chez CMSII et le WT a été estimée par le transport électronique dévolu à l’oxygénation de RuBP et en plus par l’accumulation des métabolites impliqués dans la photorespiration. D’une part, l’acclimatation à la sécheresse diminue gm plus fortement chez le WT que chez le CMSII. D’autre part, le WT accumule la glycine ce qui laisse supposer que le glycine décarboxylase mitochondrial est plus affectée chez le WT que chez le CMS et inhibe ainsi la photorespiration. En effet, cette plus faible photorespiration chez le WT affecte les réactions primaires de la photosynthèse par une accumulation d’un gradient de protons estimé par le quenching non-photochimique (NPQ) de la fluorescence chlorophyllienne ce qui induit une diminution du transport électronique des réactions primaires de la photosynthèse. / To investigate the role of mitochondria in drought stress, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant. In CMSII, alternative NAD(P)H-dehydrogenases bypassing complex 1 allow respiration.. The difference of mitochondrial function between WT and CMSII plants affect also photosynthesis. The CMSII has lower photosynthetic actitvity than the WT and lower stomatal (gs) and internal (gm) conductances to CO2. When watering of plants with similar leaf surface and similar shoot/root ratio was stopped the relative water content (RWC) declined faster in WT as compared to CMSII leaves. Furthermore, CMSII and WT leaves had the same osmotic potential at leaf saturation (P0) and at leaf turgor lost pressure (PTLP) and the same stomatal density. The slower decline of RWC in CMSII, compared to WT leaves, was most likely the consequence of the lower stomatal conductance (gs) under well-watered conditions and during the first days after withholding watering, The lower stomatal conductance of well-watered CMSII leaves correlated with a lower hydraulic conductance of leaves. Remarkably, total free amino acid levels declined and total soluble protein content increased in CMSII leaves, while the opposite was observed in WT leaves. This suggests protein synthesis in CMSII but protein degradation in WT leaves during drought stress. We also show that CMSII leaves better acclimate to drought stress than the WT leaves. After several days at 80 % RWC , photosynthesis is higher in the mutant than in WT. As compared to the WT, the mutant shows higher rates of photorespiration before and after acclimation to drought.The strong accumulation of glycine in the WT suggests that photorespiration may be limited at the level of glycine decarboxylase. In addition, after acclimation to drought gm declined markedly in WT but not in CMSII leaves, thus further limiting CO2 supply for photosynthesis in the WT. The resulting lower photosynthesis and photorespiration in WT leaves affect also the primary reaction of photosynthesis by increasing the non-photochemical fluorescence quenching (NPQ) and decreasing linear electron transport.

A planta Casearia sylvestris é catalogada como planta medicinal de interesse ao SUS, indicada para o tratamento de gastrite e como cicatrizante, para ser utilizada na forma de infusão ou compressas. Assim, foi objetivo deste estudo avaliar o potencial terapêutico antiulcerogênico do extrato fluido (EF), extrato metanólico (EM) e fração diclorometano (FDM), pelo modelo de indução de úlcera por etanol-acidificado. Realizou-se também a análise fitoquímica objetivando identificar os compostos ativos presentes nos extratos, através de técnicas de cromatografia, espectrometria de massas e ressonância magnética nuclear. Os resultados obtidos mostraram que tanto o EF, quanto o EM e FDM foram eficazes na prevenção de úlceras gástricas. Além disso, foram avaliados os possíveis efeitos do EF no estresse oxidativo hepático e na indução ou inibição do complexo enzimático citocromo P450. Os resultados obtidos mostraram alteração apenas do ciclo-redox da glutationa com a razão GHS/GSSG diminuída nos animais tratados, sugerindo que deve ser ter cautela ao utilizá-lo concomitantemente a medicamentos pelo risco de interações medicamentosas. Adicionalmente, foi realizado um estudo preliminar do potencial antitumoral do EM e frações através de ensaio bioguiado de citoxicidade (ensaio sulforrodamina B), no qual foi observado pronunciada atividade citotóxica (IC50 ≤ 5µ/ml) em diferentes linhagens tumorais. A análise fitoquímica identificou flavonóides (quercetina, rutina, kaempferol) e terpenos (espatulenol, diterpeno clerodânico). Portanto, pode-se sugerir que os efeitos terapêuticos ocorrem da sinergia destes princípios ativos / The Casearia sylvestris plant is cataloged as a medicinal plant of interest to the Unified Health System in Brazil indicated for the treatment of gastritis and healing, to be used in the form of infusion or compresses. Thus, the aim of this study was to evaluate the antiulcerogenic therapeutic potential of fluid extract (EF), methanol extract (EM) and dichloromethane fraction (FDM) on ethanol/HCl induced gastric model ulcer. The results obtained showed that EF, as EM and FDM, have been effective in preventing gastric ulcers. Also, a phytochemical analysis by chromatographic techniques, mass spectrometry and nuclear magnetic resonance was carried out to identify the active constituents present in the extracts. Moreover the possible effects of EF on hepatic oxidative stress and on induction or inhibition of cytochrome P450 enzime complex were evaluated. The results obtained showed only an alteration on the redox-cycle of glutathione with the GHS/GSSG ratio decreased in treated animals, suggesting caution in the use of EF concomitantly with other drugs as there might be a risk of drug interactions. In addition, a preliminary study was carried out to evaluate through bio-guided citotocixity assay (sulforhodamine B) the antitumor potential of the EM and fractions, in which pronounced cytotoxic activity was observed (IC50 ≤ 5µg/ml) in different tumor cell lines. Therefore, it can be suggested that the therapeutic effects occur in consequence of the synergy of these active ingredients

Treelines in high-mountain regions are constrained by heat deficiency, although the working mechanisms are still not entirely understood. Observational and paleoecological studies on treeline performance may contribute to increased understanding of the treeline phenomenon in general. The present thesis addresses elevational shifts of alpine treelines in the Swedish Scandes. By various analytical tools, the studies embrace widely different temporal scales. The concept treeline refers to the elevation (m a.s.l.) at a specific site of the upper individual tree of a certain tree species, at least 2 m tall. All the principal tree species in the Scandes are concerned, i.e. mountain birch (Betula pubescens ssp. czerepanovii), Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). Paper I deals with regional treeline dynamics at more than 100 sites over the past 100 years. Concurrent with temperature rise by c. 1.4 °C over the same period, maximum treeline advances of all species amount to about 200 m. Thus, under ideal conditions, treelines respond in close equilibrium with air temperature evolution. However, over most parts of the landscape, treeline upshifts have been much smaller than 200 m, which relates to the combined action of geomorphology, wind, snow distribution and soil depth. After 1975, the birch has lost its role as the most rapidly advancing tree species, being superseded by pine and spruce. Paper II is a short-term (2005/2007-2010/2011) study of mountain birch treeline performance along a regional maritimity-continentality gradient. Upshift by 3.0 yr-1 in the maritime part of the gradient contrasts to retreat by 0.4 m yr-1 in the continental part. In the latter area, earlier and more complete melting of late-lying snow patches has seemingly progressed to a state when soil drought sets back the vigour of existing birches and precludes sexual regeneration and upslope advance of the treeline. In the maritime area, extensive and deep snow packs still exist above the treeline and constrain its position, although some release is taking place in the current warm climate. Paper III explores treeline change by phenotypic transformation of old-established stunted and prostrate spruce individuals (krummholz) growing high above the treeline and is based on analyses of radiocarbon-dated megafossils, preserved in the soil underneath clonal groups of spruce. Living spruce clones, which in some cases may date back to the early Holocene (9500 cal. yr BP), suggests that spruce immigrated from “cryptic” ice age refugia much closer to Scandinavia than conventionally thought. As the krummholz form presupposes open and windy habitats, it is inferred that permanently open spots prevailed in the high-mountain landscape even during periods when treelines in general were much higher than today. Paper IV reports radiocarbon dates of wood samples, retrieved from newly exposed glacier forefields at three main sites, located high above the modern treelines and embracing the entire Swedish Scandes. It appears that pine colonized early emerging nunataks already during the Late Glacial. Around 9600-9500 cal. yr BP a first massive wave of tree establishment, birch and pine, took place in “empty” glacier cirques. Both species grew 400-600 m above their present day treeline position and accordingly, the summer temperatures may have been 3.5 °C warmer than present (uncorrected for land uplift). During the entire interval 9600 to 4400 cal. yr BP, birch prospered 100-150 m above the uppermost pines. In response to Neoglacial cooling, treelines of both birch and pine descended until their final disappearance from the record 4400 and 5900 cal. yr BP, respectively. Thereafter, these habitats experienced increased snow accumulation and glacier inception. / Avhandlingen belyser förskjutningar i olika tidsskalor av den alpina trädgränsens läge i de svenska Skanderna. Trädgränsen definieras som den högsta nivån (m ö.h.) för minst 2 m höga individer av en viss art i en definierad del av en fjällsluttning. Avhandlingen består av fyra separata uppsatser, publicerade i olika välrenommerade vetenskapliga tidskrifter.  I Paper I analyseras förändringar av trädgränserna för fjällens vanligaste trädarter, fjällbjörk, gran och tall (Betula pubescens ssp. czerepanovii, Picea abies och Pinus sylvestris) mellan 1915 och 2007. Undersökningen omfattar ett 8000 km2 stort, naturgeografiskt heterogent område, med mer än 100 lokaler. De maximala trädgränsförskjutningarna för samtliga trädarter uppgår till omkring 200 m. Resultaten motsvarar de trädgränsförskjutningar som teoretiskt kan förutsägas utifrån den temperaturhöjning med cirka 1,4 °C som skett under samma tidsperiod, förutsatt ideala förhållanden. För större delen av området råder emellertid andra, icke-ideala förhållanden, varför trädgränsernas uppflyttning i allmänhet blivit avsevärt mindre än 200 m. Den främsta anledningen till detta är lokala topoklimatiska begränsningar, d.v.s. kombinerade effekter av geomorfologi, vind, snöfördelning, jorddjup, etc., som i stora delar av det starkt brutna fjällandskapet mer eller mindre effektivt hindrar träden från att nå sina potentiellt högst belägna växtplatser betingade av temperaturen. Efter 1975 har björken förlorat sin roll som arten med den snabbast expanderande trädgränsen. I stället har tall och gran avancerat med större hastighet. Det innebär att även om klimatets uppvärmning fortsätter, så kommer det subalpina björkbältet att expandera i avsevärt mindre omfattning än vad som ofta förebådats. Möjligtvis kommer det att ersättas av tall. Paper II behandlar björkens trädgränsdynamik under perioden 2005/2007-2010/2011 längs en regional klimatgradient med avseende på maritimitet/kontientalitet. Trädgränsen har under den aktuella perioden avancerat 3,0 m/år i den maritimt präglade delen av gradienten, vilket kontrasterar signifikant mot en sänkning med 0,4 m/år i området med mer kontinentalt klimat. Skillnaderna diskuteras i termer av klimatförändringens varierande effekter på snötäckets utbredning och varaktighet och dess inverkan på markfuktigheten. En allt tidigare total utsmältning av snölegorna i de kontinentala områdena har av allt att döma resulterat i vattenbrist under sommaren. Torka medför reducerad vitalitet för existerande björkar och förhindrar både sexuell förökning och uppflyttning av trädgränsen. I de maritima delarna kvarligger alltjämt mycket snö under en stor del av sommaren. Trädgränsens position har därför kunnat bibehållas eller flyttas upp. Vissa omständigheter tyder på att trädgränsens stigning i högre grad har varit baserad på fröföryngring efter 1975, jämfört med perioden 1915-1975. Utgångspunkten för Paper III är erfarenheter från Paper I, som visar att trädgränsens uppflyttning för gran och björk huvudsakligen är resultatet av ökad höjdtillväxt av äldre, i vissa fall flertusenåriga, mer eller mindre buskformiga individer (krummholz), som vuxit på nivåer långt ovanför trädgränsen. Som ett svar på de senaste hundra årens varmare klimat har dessa antagit trädform, varigenom trädgränsen höjts. För en fördjupad förståelse av den här mekanismen har megafossil, d.v.s. grova vedrester bevarade i marken under gamla grankloner i trädgränsekotonen, 14C-daterats. Resultaten tyder på att granar i exponerad fjällmiljö kan uppnå i det närmaste ”evigt” liv genom sin förmåga till vegetativ förökning och möjligheten att växla mellan busk- och trädform i takt med klimatets växlingar. Vissa nu levande kloner existerade av allt att döma redan för 9500 år sedan. Den nu dokumenterat tidiga förekomsten av gran, bekräftar den på senare tid allt tydligare bilden av granen som en tidig invandrare till fjällkedjan. Möjligtvis har granen ”övervintrat” den senaste istiden närmare Skandinavien än vad som till helt nyligen varit den gängse uppfattningen. Paper IV behandlar en för Skandinavien ny metod för historisk trädgränsrekonstruktion. I uppsatsen analyseras 14C-dateringar av totalt 78 större veddelar (megafossil) som nyligen exponerats i anslutning till smältande glaciäris och ”perenna” snölegor i tre huvudområden, Helags-Sylarna, Tärna och Abisko, högt ovanför dagens trädgräns. Det framkommer att tall (Pinus sylvestris) koloniserade tidigt framsmälta nunatakker redan under senglacial tid. För omkring 9600-9500 år sedan inträffade en första massiv våg av björk- och talletablering i isfria glaciärnischer. Båda arterna växte 400-600 m ovanför sina nuvarande trädgränspositioner, i ett klimat som kan ha varit 3,5 °C varmare än idag. Under intervallet 9600 till 4400 BP uppträdde björken i ett 100-150 m brett bälte ovanför de översta tallarna. Som ett svar på klimatets successiva avkylning under senare delen av Holocen sänktes både björkens och tallens trädgränser i de aktuella miljöerna, till dess de för 4400 respektive 5900 år sedan helt försvann från lokaler där glaciärer och perenna snöfält började bildas. De analyserade trädresterna, som länge bevarats av glaciäris och perenn snö representerar en period med ett klimat långt varmare än under det senaste århundradet. Med denna analogi från det förflutna kan det därför antas att i en framtid där sommartemperaturerna rent hypotetiskt är 3,5 °C högre än i nutiden, skulle trädgränserna lokalt kunna flyttas upp med ungefär 600 m.

Darbo objektas - paprastoji pušis (Pinus sylvestris L.) – Lietuvoje labiausiai paplitusi, jautri užterštam orui medžių rūšis. Paprastosios pušies tyrimai buvo atliekami Lietuvos įmonių AB “Achema” apylinkėse 25 km ruože nuo gamyklos, AB „Akmenės cementas“ 10 km ruože ir AB „Mažeikių nafta“ – 5.4 km ruože nuo gamyklos. Darbo tikslas – Įvertinti paprastosios pušies (Pinus sylvestris L.) spyglių pigmentinės ir antioksidantinės sistemų priklausomybę nuo medynų atstumo iki taršos šaltinio. Darbo metodas. Tyrimai atlikti 14-oje pušyn����������, nutolusių skirtingu atstumu nuo gamyklų: JonA apylinkėse 5 medynai 25 km ruože, šalia AkmC – 4 medynai 10,0 km ruože, šalia MažN – 5 medynai 5,4 km ruože. Šakos, specialiais kirtikliais pjautos 6-8 metrų virš žemės aukštyje. Spygliai buvo atšaldomi ledu ir transportuojami i laboratoriją. Atskirai tiriami pirmamečiai ir antramečiai spygliai. Pigmentų ir askorbo rūgšties koncentracijų nustatymui naudota žalioji spyglių masė. Pigmentams tirti ėminiai buvo ekstrahuojami nedideliu 100 % acetono kiekiu. Spektrofotometru (Genesys 5) išmatuota pigmentų ekstrakto absorbcija 662 nm (chlorofilas a), 644 nm (chlorofilas b), 440,5 nm (karotinoidai) bangos ilgyje. Askorbo rūgštis spygliuose buvo ekstrahuojama su 0.001 N 2,6 dichlorfenolindofenoliu ir titruojama. Darbo rezultatai. Tyrimai AB “Akmenės cementas” transektoje parodė, kad, fotosintezės pigmentų kiekiai reikšmingai mažesni prie gamyklos 0,5 km atstumu augančiose pušyse nei tolimiausiame (10 km... [toliau žr. visą tekstą] / Object of the research – Scots pine (Pinus sylvestris L.) – common tree in Lithuania, sensitive to air pollution. Research of Scots pine were conducted in the industrial areas of Lithuania companies JSC – (25 km transect) near the factory JSC (10 km sector) and an oil refinery (in 2 – 5,4 km distance). Goal of the research – to evaluate quality of photosynthetic pigments and ascorbic acid in the needles of Scots pine growing around main sources of industrial pollution. Methodology of the research. Research was performed in 17 pine forests, at different distance from the factories. In the surroundings of the nitrogen fertilizer factory 8 forests were examined in 25 km distance, near the cement factory – 4 forests in 10 km distance and near the oil refinery – 5 forests in 5.4 km distance. In each site, samples of current-year and 1-year-old needles were taken from 4 pines at the height of 6-8 m aboveground. The needles were cooled and transported to laboratory. Concentrations of the pigments and ascorbic acid were determined in the fresh needle material. The needles were grinded and in 20ml of 100% acetone. The absorbance at 662 nm (chlorophyll a), 644 nm (chlorophyll b) and 440.5 nm (carotenoids) was measured by spectrophotometer (Genesys 5, US). The ascorbic acid from the needles was extracted with 0.001 N 2.6-dichlorophenolindophenol and estimated titrimetrically. Results of the research. Research results showed that number of photosynthesis pigments is meaningly... [to full text]

Tyrimų pagrindimas. Dubravos eksperimentinės mokomosios miškų urėdijos įsigyto šlapio sėklų nusparninimo įrenginio „Nomeco Dewinger B1086” naudojimo instrukcijoje nėra nurodytas optimalus paprastosios pušies ir paprastosios eglės sėklų nusparninimo laikas. Tam, kad optimizuoti šlapio nusparninimo laiką aukščiau minėtame įrenginyje reikalingi tyrimai kaip sėklų nusparninimo kokybė priklauso nuo nusparninimo trukmės. Tyrimo tikslas buvo nustatyti optimalią paprastosios pušies ir paprastosios eglės sėklų nusparninimo trukmę šlapiojo nusparninimo metodu įrenginyje „Nomeco Dewinger B1086”. Tyrimo uždaviniai: (1) atlikti 15, 30, 45, 60 ir 120 minučių trukmės paprastosios pušies ir paprastosios eglės sėklų nusparninimo testus sėklų nusparninimo įrenginyje „Nomeco Dewinger B1086”, kiekvienai rūšiai naudojant tris skirtingas sėklų siuntas, (2) nustatyti paprastosios eglės ir paprastosios pušies sėklų nusparninimo kokybę priklausomai nuo sėklų nusparninimo trukmės. Tyrimo objektas yra sėklų nusparninimo kokybės testai, atlikti sėklų šlapiojo nusparninimo principu veikiančiame nusparninimo įrenginyje „Nomeco Dewinger B1086”. Tyrimo metodai. Kiekvienam iš 15, 30, 45, 60 ir 120 minučių trukmės nusparninimo bandymų buvo naudotos 3 skirtingos paprastosios eglės ir 3 skirtingos paprastosios pušies sėklų siuntos. Kiekvienam tam tikros trukmės ir sėklų siuntos testui buvo analizuoti 4 sėklų bandiniai po 100 sėklų. Viso buvo įvertinta 12 000 sėklų nusparninimo kokybė. Tai rodo aukštą gautų... [toliau žr. visą tekstą] / The Essence of Master Thesis: Dubrava experimental and teaching forestry owns „Nomeco Dewinger B1086” which is a dewinger for the wet seedlots. However, in its manual, there is no indication how much time it takes to dewing the seeds of pine and spruce. In order to optimize the time of the wet dewinging it is necessary to explore whether the quality of dewinging is influenced by the period of time that the process of dewinging takes. The main aim of the research is to determine the optimal time that a method of a wet dewinging takes with „Nomeco Dewinger B1086”. The Tasks of the Thesis. To perform 15, 30, 45, 60, and 120 min long dewinging tests for the seeds of pine and spruce with a dewinger for the wet seedlots „Nomeco Dewinger B1086”, using three different kinds of seeds of each tree. To detect the quality of dewinging of a pine and a spruce depending on the time the process takes. The objects of the thesis are the tests of the seeds dewinging, carried out with the device „Nomeco Dewinger B1086”. The methods of the Thesis. In each of 15, 30, 45, 60, and 120 minutes long tests of dewinging were used three different kinds seeds of pine and spruce. For each of the time period test were used four samples of the seeds, each consisting of one hundred seeds. All in all, there were 12 000 seeds that were evaluated. It shows great reliability of the research. The quality of the seeds dewinging were classified into four main categories. The data that was achieved during the... [to full text]

Baigiamajame darbe 2012–2013 m. tirta netikrosios miltligės (Peronospora viciae (Berk) Caspary) įtaka skirtingos geografinės kilmės miškinio pelėžirnio (Lathyrus sylvestris L.) cenopopuliacijų morfologiniams ir sėklų produktyvumo rodikliams. Darbo objektas – iš skirtingų Lietuvos rajonų surinktos dvylika miškinio pelėžirnio cenopopuliacijų, augančių vienodos agrotechnikos sąlygomis. Darbo tikslas – įvertinti netikrosios miltligės įtaką skirtingų cenopopuliacijų miškinio pelėžirnio morfologinias ir sėklų produktyvumo rodikliams. Darbo metodai – įvertinti skirtingų miškiniopelėžirnio cenopopuliacijų stiebo aukštis, žiedynų, ankščių, sėklapradžių ankštyje skaičius, ankšties produktyvumo, vieno stiebo sėklų produktyvumas. Atsparumas netikrajai miltligei įvertintas 5 balų sistema, atsparumo įtaka ankščiau išvardintiems rodikliams įvertinta naudojantis programa STATISTIKA 8. Rezultatai. Tyrimo metų duomenimis, miškinio pelėžirnio stiebo aukščiui, žiedynų skaičiui ir ankščių produktyvumui lemiamą įtaką turėjo cenopopuliacijos genotipas ir tyrimo metų meteorologinės sąlygos. Didesnis miškinio pelėžirnio stiebo aukštis teigiamai įtakojo ankščių (r=0,7539) ir sėklų skaičių (r = 0,6223). Stabiliausias iš tirtų rodiklių tyrimo metais buvo sėklapradžių (vid. 12,4 vnt.) skaičius ankštyje (V = 4,7), labiliausi – ankščių (vid. 21,6 vnt.; V = 36,5) ir subrandintų sėklų skaičiaus (vid. 151,1 vnt., V = 39,8) rodikliai. Miškinio pelėžirnio atsparumas netikrajai miltligei įtakojo... [toliau žr. visą tekstą] / Influence of downy mildew (Peronospora viciae (Berk) Caspary) on morfological and seed productivity parameters of different geographical origins flat pea (Lathyrus sylvestris L.) cenopopulations have been investigated in 2012-2013. Subject of the research – 12 flat pea (Lathyrus sylvestris L.) cenopopulations from different geographical origins of Lithuania, grown on equal agrotechnic conditions. Aim of the research – estimate an influence of downy mildew on flat pea morfological and seed produktivity parameters. Methodology – to evaluate stem height, number of inflorescence, pods, ovules per pod and pod productivity, one stalk of seed productivity of different flat pea coenopopulations. Resistance to downy mildew is rated by 5-point scale, impact of resistance on above listed parameters estimated using the statistics program STATISTICA 8. Results. The research data shows, that genotype of coenopopulation and meteorological conditions had a decisive influence on flat pea stem height, number of inflorescences and pod productivity. Height of stem influenced positively pod (r = 0,7539 ) and seed number (r = 0,6223 ). The most stabile from the investigated parameters were number of ovules (mean 12,4 pcs.) per pod (V = 4,7 ), most labile – number of pods (mean 21,6 pcs., V = 36,5 ) and the number of mature seeds (mean 151,1 pcs., V = 39.8). The flat pea resistance to downy mildew influenced such parameters as the number of pods, number ovules in pod and especially stem seeds... [to full text]

Made available in DSpace on 2016-06-02T20:36:45Z (GMT). No. of bitstreams: 1 5223.pdf: 14772341 bytes, checksum: f9288ce2d5f0b661781b49aa3722146b (MD5) Previous issue date: 2013-03-27 / Financiadora de Estudos e Projetos / Diabrotica speciosa is a pest insect which occurs in most Brazilian states, standing out as one of the most important polyphagous pest of in grain crops. The understand of the role of the microbiota present in the digestive tract and of this insect the identification of ways to interfer or even to eliminate this microbial flora they are knowlegdes that, may lead mode of action to control of the pest insect. In this context, this study aimed to evaluate the microbial diversity present in the digestive tract of larvae and adults of D. speciosa and in the sometime, to investigate the potential of C. sylvestris as an antimicrobial agent. As results, they were obtained 21 bacterial isolates and 8 fungal isolates. The proteic profile of these microrganisms were analyzed by using MALDI-TOF/MS. Afterwards, these data were grouped identifying clusters of isolates. A bacteria each groups was identified by rDNA sequencing, 16S region. Identified bacteria were: Pantoea agglomerans, Luteibacter sp., Serratia marcescens, and Pseudomonas temperans Acidovorax sp. Two isolated fungi were identified through morphological comparison with literature data, and identified as Cladosporium sp. and Penicillium sp. A further study was carried out with Luteibacter sp bacteria, which were isolated from D. speciosa larva. In this study was evaluated growth profile and the parameters to carry out the antimicrobial assay. Several kind of microbial assays were investigated such as agar diffusion and microdilution plate ELISA®. The second method showed the best results. Thus, a microdiluition methodology using fluorometric assay, was developed and validated, highlighting parameter such as linearity, specificity, accuracy, precision, robustness, recovery, limites of detection, quantification and detection, etc. This method was applied evaluating extracts, fraction and secondary molecules of cerrado plants. Phytochemical study of C. sylvestris leaves resulted in the isolation of an umpublished tricyclic sesquiterpene, the flavonoid catechin and a clerodane diterpene, which was used as a reference marker in chromatographic method to quality control of the extracts and fractions of C. sylvestris that were used in assays. / A Diabrotica speciosa é uma espécie que ocorre na maioria dos estados brasileiros, destacando-se como uma das principais pragas polífagas nas culturas de grãos. O entendimento da função da microbiota presente no trato digestivo deste inseto e a identificação de meios de interferir ou até mesmo destruir essa flora microbiana são conhecimentos que podem levar a um novo modo de ação para o controle de insetos praga. Neste contexto, este trabalho teve como objetivo avaliar a diversidade microbiana presente no trato digestivo de larvas e insetos adultos de D. speciosa e avaliar o potencial de Casearia sylvestris e extratos de plantas do cerrado como agente antimicrobiano. Como resultados, foram obtidos 19 isolados bacterianos e 8 fúngicos. Estes foram avaliados em termos de perfis protéico por MALDI-TOF/MS, e após a obtenção de agrupamentos estatísticos alguns microrganismos foram identificadas através do seqüenciamento de rDNA região 16S. As bactérias identificadas foram: Pantoea agglomerans, Luteibacter sp., Serratia marcescens, Acidovorax temperans e Pseudomonas sp. Dos isolados fúngicos, dois foram identificados através de comparação morfológica como dados da literatura, pertencendo aos gêneros; Cladosporium sp. e Penicillium sp. Um estudo mais aprofundado foi realizado com a bactéria Luteibacter sp isolada da larva, que teve seu perfil de crescimento avaliado, possibilitando o ajuste de parâmetros para realização de ensaios antimicrobiano. Para estes ensaios foram testadas metodologias de difusão em ágar e por microdiluição em placa de ELISA®, sendo na segunda metodologia obtido os melhores resultados. Assim, uma metodologia por microdiluição foi validada através da utilização do fluorímetro, analisando figuras de mérito como faixa linear de trabalho, precisão, exatidão, limites de quantificação e detecção, etc. O método por microdiluição foi aplicado ensaiando extratos, frações e metabólitos de plantas do cerrado. O estudo fitoquímico das folhas de C. sylvestris propiciou o isolamento de um sesquiterpeno tricíclico inédito na literatura, um flavonóide (catequina) e um diterpeno clerodânico que foi utilizado como marcador de referência para controle cromatográfico de qualidade de extratos e frações C sylvestris que foram submetidos a ensaios antimicrobianos.