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Abstract Natural seasonal variation and the effects of cold treatment and nitrogen fertilization on protein expression with special emphasis on dehydrin proteins, were studied using different aged Scots pine (Pinus sylvestris L.). Several different dehydrins were found and their expression depended on the tissue type, tree age or specific treatment. Their concentrations fluctuated seasonally and in response to nitrogen fertilization, but no effects of low temperature on the dehydrins of seedlings were observed. A 60-kDa dehydrin was associated with cold acclimation in the bud and bark tissues of mature trees and in the needles of seedlings. In the needles of mature trees, this dehydrin was associated with springtime desiccation, which was detected as a significant decrease in the osmotic potential of needles. The quantity and quality of soluble proteins altered seasonally in Scots pine tissues, but low temperature treatment alone did not have any effect on the proteins. Soluble protein concentration increased during autumn and decreased in spring in buds and bark, but not in the needles of mature trees. In needles of seedlings, however, protein concentrations altered seasonally. Several proteins, of varying molecular weights, were more abundant in winter in all the tissues studied and some increased in concentration in the nitrogen-fertilized seedlings. The role of these proteins as a storage reserve in Scots pine is discussed. The osmotic potential of needles showed seasonal fluctuation, being high in the summer and low during the winter. Low temperature treatment decreased the osmotic and water potential of needles and increased the concentrations of soluble sugars in seedlings. Based on carbohydrate analyses, the metabolism of seedlings acclimated to low temperature in less than ten days. Nitrogen fertilization increased the content of total nitrogen and the soluble protein concentrations in the needles of seedlings and the growth both in the mature trees and seedlings. Although the frost resistance showed no response to nitrogen-fertilization, the soluble proteins and dehydrins were affected in a manner that suggested an earlier growth resumption of spring in the fertilized trees.

Orientador: Gustavo Quevedo Romero / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T12:38:24Z (GMT). No. of bitstreams: 1 Goncalves_AnaZangirolame_M.pdf: 2619238 bytes, checksum: 7829d9724c33ae79b0974c3db972f845 (MD5) Previous issue date: 2011 / Resumo: Inúmeros organismos vivem associados a plantas da família Bromeliaceae e rejeitos derivados desses organismos (e.g., fezes e detritos vegetais) podem contribuir para a nutrição das bromélias. A aranha Psecas chapoda (Salticidae) habita Bromelia balansae, Ananas comosus e Aechmea distichantha (Bromelioideae) em uma grande extensão geográfica. Dependendo da estrutura da roseta e dos tricomas absorventes, estas bromélias podem absorver mais ou menos nitrogênio derivado da aranha. A obtenção de nitrogênio pode variar de acordo com o período do ano (e.g., seco vs. chuvoso) e também devido à presença de microorganismos associados às folhas das bromélias. No presente estudo utilizamos métodos isotópicos e fisiológicos para responder as seguintes questões: (1) a aranha contribui para a nutrição e crescimento de B. balansae, An. comosus e Ae. distichantha? (2) quais respostas fisiológicas (i.e., concentrações de clorofilas, carotenóides e proteínas solúveis) as plantas apresentam por ganharem nitrogênio derivado da aranha? (3) existe variação sazonal na absorção de nitrogênio proveniente das aranhas? (4) as bactérias associadas à filosfera de B. balansae facilitam a absorção de nutrientes por estas plantas? Nossos resultados mostraram que P. chapoda favorece nutricionalmente suas três bromélias hospedeiras. Entretanto, nossos resultados indicam que o mutualismo entre aranhas e bromélias é sazonalmente restrito gerando resultados condicionais. A variação interespecífica na obtenção de nitrogênio ocorreu provavelmente devido às diferentes performances e rotas fotossintéticas de cada espécie. Enquanto B. balansae parece utilizar nitrogênio para crescimento, Ae. distichantha aparentemente acumula nitrogênio para condições de estresse nutricional. Adicionalmente, mostramos que plantas com densidade natural de bactérias acumularam 57% mais proteínas solúveis e cresceram 13% mais do que as bromélias que tiveram a abundância de bactérias reduzidas com antibióticos. Estes resultados sugerem pela primeira vez que bactérias aceleram a ciclagem de nutrientes na filosfera e podem favorecer nutricionalmente estas plantas / Abstract: Many organisms live associated with Bromeliaceae plants and materials derived from these organisms (e.g., faeces and plant debris) may contribute to bromeliad nutrition. The spider Psecas chapoda (Salticidae) lives in Bromelia balansae, Ananas comosus and Aechmea distichantha (Bromelioideae) in a large geographic extent. Depending on the structure of the rosette and trichomes, these bromeliads may absorb more or less nitrogen derived from spiders. The acquisition of nitrogen may vary according to the seasons (e.g., dry vs. wet) and also due to the presence of microorganisms associated with bromeliad leaves. In this study we used physiological and isotopic methods to answer the following questions: (1) Do spiders contribute to the nutrition and growth of B. balansae, An. comosus and Ae. distichantha? (2) Which physiological responses (i.e., chlorophylls, carotenoids and soluble protein concentrations) the plants have by receiving nitrogen from spiders? (3) Is there seasonal variation in the absorption of nitrogen from spiders? (4) Do bacteria associated with B. balansae phyllosphere facilitate nutrient absorption by these plants? Our results showed that P. chapoda nutritionally improve their three host plants. However, our results indicate that this mutualism is seasonally restricted generating conditional outcomes. The interespecific variation in nitrogen acquisition occurred probably due to different performances and photosynthetic routes of each plant species. While B. balansae appear to use nitrogen for growth, Ae. distichantha apparently accumulate nitrogen for nutritional stress conditions. Additionally, we showed that plants with natural density of bacteria accumulated 57% more soluble proteins and grew 13% more than bromeliads that had their abundance of bacteria reduced with antibiotics. These results suggest for the first time that bacteria accelerate nutrient cycling in the phyllosphere and may nutritionally favor these plants / Mestrado / Ecologia / Mestre em Ecologia

<p>Bacterial endospores have been a topic of research interest over the last several decades given their high resistance to ultraviolet (UV) damage. Unlike vegetative bacterial cells, which form cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) as the major product upon UV irradiation, endospore bacteria form a spore photoproduct (5-(<em>R</em>-thyminyl)-5,6-dihydrothymine or SP) as the major product. Vegetative bacteria cells are subject to regular cell activities and processes such as division and deoxyribonucleic acid (DNA) replication, which are prone to damage from UV exposure. However, in endospores, which have a largely anhydrous inner environment, the DNA remains dormant when bound to spore-specific small acid-soluble proteins (SASP) and dipicolinic acid, making spores highly resistant to radiation, heat, desiccation, and chemical harm. During spore germination, SP lesions in DNA are repaired by a distinctive repair enzyme, spore photoproduct lyase (SPL). In this thesis, molecular dynamics (MD) simulations were carried out to (i) examine how the formation of the SP lesion in DNA affects the global and local structural properties of duplex DNA and (ii) study how this lesion is recognized and repaired in endospore. The first part of this work was focused on designing and developing a structurally and dynamically stable model for dinucleotide SP molecule (TpT), which was subsequently used as an SP patch incorporated into duplex DNA. Computationally, this requires modifications of the bond and nonbonded force field parameters. The stability of the patch and developed parameters was tested via solution-phase MD simulations for the SP lesion incorporated within the B-DNA dodecamer duplex (PDB 463B). The second part involved applying the new SP patch to simulate the crystallographic structure of the DNA oligomer containing SP lesions. Solution-phase MD simulations were performed for the SP-containing DNA oligomers (modeled based on PDB 4M94) and compared to the simulations of the native structure (PDB 4M95). Our analysis of the MD trajectories revealed a range of SP-induced structural and dynamical changes, including the weakened hydrogen bonds at the SP sites, increased DNA bending, and distinct conformational stability and distribution. In the third part of this thesis project, we carried out MD simulations of SP-containing DNA bound with SASPs to examine how the DNA interacts differently with SASP in the presence and absence of the SP lesion. The simulation results suggested decreased electrostatic and hydrogen bonding interactions between SASP and the damaged DNA at the SP site compared to the undamaged DNA-protein complex. In addition, decreased helicity percentage was observed in the SASPs that directly interact with the SP lesion. The last part of this this thesis work focused on the SP-dimer flipping mechanism, as the lesion is likely flipped out to its extrahelical state to be recognized and repaired by SPL. Using steered molecular dynamic (SMD) simulations and a pseudo-dihedral angle reaction coordinate, we obtained possible SP flipping pathways both in the presence and absence of SASP. Collectively, these simulation results lend new perspectives toward understanding the unique behavior of the SP lesion within the DNA duplex and the nucleoprotein complex. They also provide new insights into how the SP lesion is efficiently recognized and repaired during spore germination.</p>

La malnutrition liée au vieillissement est souvent accompagnée de nombreux dérèglements et dysfonctionnements métaboliques, notamment la perturbation de l'homéostasie énergétique (installation de l’insulinorésistance), la fragilité, la diminution de la masse musculaire et les troubles de la réponse immunitaire. Ainsi, la manipulation nutritionnelle, au cours du vieillissement, est considérée comme l'une des solutions possible pour prévenir et traiter ces troubles. Parmi les substances nutritives qui ont été largement étudiées, la composition en protéines (acides aminés), la qualité des lipides (AGPI n-3) et les micronutriments (vitamine D).L’objectif de cette thèse consiste à déterminer l’impact de la dénutrition et d’évaluer le potentiel d'une des formules de réalimentation contenant un mélange à haute teneur en protéines solubles du lait, associées à de la matière grasse laitière, enrichie en acides gras polyinsaturés de la famille omega 3 (précurseur ALA et DHA) et en vitamine D sur la composition en acides gras (AG) du plasma, des globules rouges et du cerveau ainsi que ses conséquences sur les marqueurs du statut inflammatoire, la réponse hépatique à l’insuline, l'expression de gènes impliqués dans la régulation de l'homéostasie énergétique et l’inflammation hypothalamique, chez des rats âgés préalablement soumis à une restriction alimentaire. Dans un premier temps, nous avons montré que la restriction alimentaire de trois mois, non carencée en ALA, induit une perte importante en omega3 (ALA et dérivés LCn-3) alors que le dérivé ARA de la série n-6 est peu modifié, conduisant ainsi à une élévation du statut pro-inflammatoire exprimé sous forme d’une augmentation du ratio ARA/LCn-3.Toutefois, les quatre semaines de réalimentation, notamment avec la formule contenant le mélange matière grasse laitière, colza et DHA, associé à de la caséine ou des protéines solubles du lait, restaure 1/ les valeurs de DHA du cerveau non restaurées par un régime contrôle de renutrition, 2/ augmente les valeurs des dérivés LCn-3 (EPA, DHA) à des niveaux supérieurs à ceux d’un régime contrôle non dénutris et de renutrition. Cette augmentation s’accompagne d’une réduction des valeurs d’ARA, induisant une baisse drastique dans le plasma et les globules rouges du ratio ARA/EPA. Ces formules montrent pour la première fois qu’elles peuvent induire une réduction très importante du statut inflammatoire par rapport à celui observé généralement chez les vieux rats et pourrait présenter un intérêt beaucoup plus général en prévention des pathologies associées au vieillissement, liées ou non à la dénutrition.Dans un second temps, nous avons montré que la restriction alimentaire de trois mois entraîne 1/ une augmentation de l’expression du récepteur à l'insuline dans l'hypothalamus, le foie et le tissu adipeux, accompagnée d'une augmentation du facteur pro-inflammatoire TNFα dans l’hypothalamus. Cependant, la réalimentation de quatre semaines entraîne 2/ un gain de poids similaire et maintient l’insulinosensibilité hépatique. En effet, nous avons montré, pour la première fois, qu’une réalimentation avec les régimes comportant le mélange MGLA/colza/DHA, permettrait 3/ d’augmenter la prise alimentaire et de diminuer l’inflammation hypothalamique, notamment, avec la formule complète contenant un mélange de haute teneur en protéines solubles de lait, associée à la matière grasse laitière /colza/DHA et enrichie en vitamine D. / Malnutrition related to aging is often accompanied by many metabolic disorders, including the disruption of energy homeostasis (installation of insulin resistance), fragility, decreased muscle mass and immune response deficiency. Thus, the nutritional manipulation, during aging, is considered to be a solution to prevent these disorders or to treat and limit damages. Amongst the nutrients that have been widely studied, we find the quality of proteins (or amino acids), of lipids (n-3 PUFA) and micronutrients (vitamin D).The aim of this thesis is to determine the impact of undernutrition and assess the potential of the refeeding formulas containing a high content of soluble protein of milk, associated with milk fat enriched with omega3 polyunsaturated fatty acids (ALA precursor and DHA) and vitamin D, on the fatty acid (FA) of the plasma, red blood cells and brain and its consequences on markers of inflammatory status, the hepatic response to insulin, the expression of genes involved in the regulation of energy homeostasis as well as hypothalamic inflammation, in old rats previously submitted for food restrictionAs a first step, our results showed that the dietary restriction of three months, despite being only moderately ALA deficient, induced a drastic loss omega3 (ALA and derivatives LCn-3), whereas a weak increase of ARA derived from n-6 series is observed, leading to a rise of the pro-inflammatory state expressed as an increase in the ratio ARA/LCn-3.However, we have shown that the four-week-refeeding formulas containing a blend of dairy-fat, rapeseed and DHA associated with casein or milk soluble proteins, restored 1 / DHA values of the brain not previously restored by the refeeding control diet, 2 /increases the values of LCn-3 derivatives (EPA, DHA) to levels above those obtained with the control non-malnourished and refeeding diets. This increase was accompanied by a reduction in ARA values, leading to a drastic drop in plasma and red blood cells ratio ARA / EPA. These formulas show for the first time that they can induce a very significant reduction of inflammatory status compared to that usually seen in old rats and could therefore present a more general interest in prevention of ageing diseases associated or not to undernutrition.In a second step, our results showed that dietary restriction of three months resulted 1/increased expression of the insulin receptor in the hypothalamus, liver and adipose tissue, accompanied by an increase of the proinflammatory factor TNF in the hypothalamus. However, the four-weeks-refeeding produces 2/ a similar weight gain and maintains hepatic insulin sensitivity. Indeed, we showed, for the first time, that refeeding, with diets containing the blend of dairy-fat / rapeseed / DHA, would 3/ increase food intake and decrease the hypothalamic inflammation, especially with the full formula containing a mixture of high content of soluble milk proteins, associated with dairy-fat / rapeseed / DHA fortified with vitamin D.

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