Die pflanzlichen Biopolyester Kutin und Suberin : Chemische Zusammensetzung und Biosynthese

Ziel meiner Arbeit war es, anhand ausgewählter Lipidstoffwechsel-Mutanten Rückschlüsse auf die Beteiligung der untersuchten Gene an der Polyesterbildung von Kutin und Suberin zu ziehen. Untersucht wurden die Polyesterzusammensetzungen einer CYP450-abhängigen ω-Hydroxylase Bncyp704b1 in Brassica napus, einer BAHD-Acyltransferase Sldcr in Solanum lycopersicum, einem ABC-Transporter abcg11, sowie die Transkriptionsfaktorenfamilie shn1, shn2 und shn3 in Arabidopsis thaliana. In B. napus wurde die doppelt rezessive Linie des Gens BnCYP704B1 untersucht. Die Mutante Bncyp704b1 weist im Vergleich zum Wildtyp vergrößerte Antheren mit veränderter Kutikulazusammensetzung und defekte Pollenwände, mit Folge einer Semisterilität, auf. In der Mutante wurde ein Defizit von C16- und C18-ω-Hydroxysäuren und deren Folgeprodukte im Antherenkutin nachgewiesen. In Anlehnung an den Polyesterbiosyntheseweg und anhand der Resultate konnte die putative ω-Hydroxylasefunktion für C16- und C18-Fettsäuren in der Kutinbildung der Antherenkutikula bestätigt werden. Der RNAi-Knockdown des SlDCR-Gens führte in S. lycopersicum zu enormen Veränderungen der Morphologie der Tomatenfruchtschale. Mikroskopische Untersuchungen der braungefärbten Schale zeigten, dass die Tomatenfruchtschale mehrere Zelllagen aufweist, die unter UV-Licht fluoreszieren. Durch Transportstudien mit isolierten Kutikeln konnte eine erhöhte Wasserpermeabilität nachgewiesen werden. Die Analyse der chemischen Zusammensetzung der Kutikula ergab, sowohl bei den Wachsen als auch im Kutin, drastische Veränderungen der Kompositionen, was die Beteiligung des Gens an der Kutikulabildung belegt. Die Reduktion der C16-9/10-Dihydroxyfettsäuremenge (C16-DHFS) und die Akkumulation der Vorstufen im Kutin belegen die SlDCR-Beteiligung an der C16-DHFS-Synthese. Die Sequenzhomologie zum Ortholog AtDCR und in vitro Studien der AtDCR führen zur Annahme, dass es sich bei der SlDCR ebenfalls um eine BAHD-Acyltransferase handelt die CoA-aktivierte C16 ω Hydroxysäuren mit Glycerin verknüpft, woraus folgend C16-DHFS-glycerin gebildet wird. Der ABC-Transporter ABCG11 wurde mithilfe von Cosuppressionslinien des Gens untersucht bei denen die Polyesterzusammensetzungen von Sprossachse, Blatt, Wurzel, Blüte und Schote analysiert wurden. Es konnten in allen Polyestern veränderte Zusammensetzungen beobachtet werden, welche im Kutin die Substanzklassen der ω-Hydroxysäuren, Disäuren und mittkettig oxygenierten Säuren betrafen und im Suberin die der ω-Hydroxysäuren und Disäuren. Da in allen Polyestern der Knockdownlinien nur die Disäuren eine Verringerung und in Überexpressionslinien eine Erhöhung zeigten, was schlüssig mit der ABCG11-Transportermenge ist, konnte die Substratspezifität für den ABCG11-Transporter auf gesättigte und ungesättigte C16-/C18-Disäuren eingeengt werden. Die SHINE-Transkriptionsfaktorenfamilie wurde mithilfe eines artifiziellen RNAi Knockdowns mit den drei Mitgliedern dieser Familie als Target in A. thaliana untersucht. Die shine-Linien wiesen Organfusionen in der Blüte auf. Die Analyse der Kutikulazusammensetzung ergab in der Mutante eine Verringerung des Blütenkutins und eine Erhöhung der Alkane der Blattwachse. Im Kutin konnte das Defizit an den C16-und C18-ω-Hydroxysäuren, Disäuren und mittkettig oxygenierten Säuren ausgemacht werden. Auf Grundlage des bisher bekannten Kutinsynthesewegs und durch Expressionsstudien konnten die ω-Hydroxylasegene CYP86A4 und CYP86A7 und die Mittketten-Alkan-Hydroxylase CYP96A15 als SHINE-Target ausgemacht werden

ACCELERATED CORROSION TEST FOR THE QUALITATIVE EVALUATION OF CORROSION IN CONCRETE

This paper is devoted to simulate the behavior of reinforced concrete in saline solution through the accelerated corrosion test. During this test, this method was evaluating the behavior of the steel bars that reinforce concrete for corrosion, according to the NACE 0775 standard. After the corrosion test, the steel bars were cleaned and dried according to the ASTM A380 standard and evaluated according to the NACE RP 0775, NACE SP0308, and NACE SP0187 standards. From the results obtained, the steel bars concrete showedhigh corrosion rates with structural impairment of the metal armor according to the NACE standards

SHINE Transcription Factors Act Redundantly to Pattern the Archetypal Surface of Arabidopsis Flower Organs

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions

Quantification of lateral heterogeneity in carbohydrate permeability of isolated plant leaf cuticles.

In phyllosphere microbiology, the distribution of resources available to bacterial colonizers of leaf surfaces is generally understood to be very heterogeneous. However, there is little quantitative understanding of the mechanisms that underlie this heterogeneity. Here, we tested the hypothesis that different parts of the cuticle vary in the degree to which they allow diffusion of the leaf sugar fructose to the surface. To this end, individual, isolated cuticles of poplar leaves were each analyzed for two properties: (1) the permeability for fructose, which involved measurement of diffused fructose by gas chromatography and flame ionization detection (GC-FID), and (2) the number and size of fructose-permeable sites on the cuticle, which was achieved using a green-fluorescent protein (GFP)-based bacterial bioreporter for fructose. Bulk flux measurements revealed an average permeance P of 3.39 × 10(-9) ms(-1), while the bioreporter showed that most of the leaching fructose was clustered to sites around the base of shed trichomes, which accounted for only 0.37% of the surface of the cuticles under study. Combined, the GC-FID and GFP measurements allowed us to calculate an apparent rate of fructose diffusion at these preferential leaching sites of 9.15 × 10(-7) ms(-1). To the best of our knowledge, this study represents the first successful attempt to quantify cuticle permeability at a resolution that is most relevant to bacterial colonizers of plant leaves. The estimates for P at different spatial scales will be useful for future models that aim to explain and predict temporal and spatial patterns of bacterial colonization of plant foliage based on lateral heterogeneity in sugar permeability of the leaf cuticle

Quantification of lateral heterogeneity in carbohydrate permeability of isolated plant leaf cuticles

In phyllosphere microbiology, the distribution of resources available to bacterial colonizers of leaf surfaces is generally understood to be very heterogeneous. However, there is little quantitative understanding of the mechanisms that underlie this heterogeneity. Here, we tested the hypothesis that different parts of the cuticle vary in the degree to which they allow diffusion of the leaf sugar fructose to the surface. To this end, individual, isolated cuticles of poplar leaves were each analyzed for two properties: (1) the permeability for fructose, which involved measurement of diffused fructose by gas chromatography and flame ionization detection (GC-FID), and (2) the number and size of fructose-permeable sites on the cuticle, which was achieved using a green-fluorescent protein (GFP)-based bacterial bioreporter for fructose. Bulk flux measurements revealed an average permeance P of 3.39 × 10 −9 ms −1 , while the bioreporter showed that most of the leaching fructose was clustered to sites around the base of shed trichomes, which accounted for only 0.37% of the surface of the cuticles under study. Combined, the GC-FID and GFP measurements allowed us to calculate an apparent rate of fructose diffusion at these preferential leaching sites of 9.15 × 10 −7 ms −1 . To the best of our knowledge, this study represents the first successful attempt to quantify cuticle permeability at a resolution that is most relevant to bacterial colonizers of plant leaves. The estimates for P at different spatial scales will be useful for future models that aim to explain and predict temporal and spatial patterns of bacterial colonization of plant foliage based on lateral heterogeneity in sugar permeability of the leaf cuticle

Efeito da trimegestona sobre o tecido mamário de ratas castradas Effect of trimegestone on mammary gland of castrated rats

OBJETIVO: Avaliar o efeito da trimegestona sobre a proliferação celular do tecido mamário de ratas castradas. MÉTODOS: Foram utilizadas 45 ratas adultas e virgens, da linhagem Wistar, submetidas à castração. Após o 60º dia da castração, confirmado o hipoestrogenismo, os animais foram divididos aleatoriamente em três grupos, conforme o tratamento proposto: controle (n=15) recebeu soro fisiológico 0,9%; estrogênio (n=15) recebeu 17 beta-estradiol; e combinado (n=15) recebeu 17 beta-estradiol associado à trimegestona, todos por 60 dias consecutivos. Após o término do tratamento, procedeu-se a exérese das mamas inguinais, destinadas a análise morfométrica pela coloração de hematoxilina e eosina (HE) e imuno-histoquímica pela quantificação do anticorpo anti-PCNA no tecido mamário, seguido de eutanásia. Os parâmetros morfométricos avaliados foram: proliferação celular epitelial, atividade secretora e alteração do estroma mamário. Ocorreram nove óbitos durante o experimento. As variáveis foram submetidas à análise estatística adotando-se como significante pPURPOSE: To evaluate the efect of trimegestone on the histological changes of the mammary tissue of castrated rats. METHODS: Forty-five virgin female Wistar rats were used after oophorectomy. Sixty days after surgery, with hypoestrogenisms confirmed, the experimental rats were randomly assigned to three groups of 15 animals each, when then the specific treatment for each group was started. The control group (C) and experimental groups 1 and 2 respectively received 0.9% saline solution, 17-beta-estradiol and 17-beta-estradiol in combination with trimegestone for 60 consecutive days. After the end of treatment , the inguinal mammary glands were removed, stained with hematoxylin and eosin (HE) for morphometry and examined by immunohistochemistry for the quantification of anti-PCNA antibody in the mammary tissue, followed by euthanasia. The morphometric parameters evaluated were: epithelium cell-proliferation, secretor activity and mammary stroma changes. There were nine deaths during the experiment. The variables were submitted to statistical analysis adopting the 0.05 level of significance. RESULTS:Histological changes were observed in 16/36 rats, mild epithelial hyperplasia in 13/36, moderate epithelial hyperplasia in 3/36, with no cases of severe epithelial hyperplasia. Stromal fibrosis was found in 10/36 and secretory activity in 5/36 rats. All morphometric variables were significant in the estrogen group compared to control (p=0.0361), although there were no difference between the group receiving combined treatment and the controls (p=0.405). The immunohistochemical analysis showed no difference between groups. CONCLUSIONS:The hormones administered to castrated rats, i.e., 17 beta-estradiol alone or in combination with trimegestone, increased the proliferation of breast cells, but this effect appeared to be lower in the combined treatment, the same occurring regarding fibrosis of the mammary stroma

Pain measurement as part of primary healthcare of adult patients with sickle cell disease

OBJECTIVE: The aim of this exploratory, cross-sectional study was to evaluate pain in sickle cell disease patients and aspects related to primary healthcare. METHODS: Data were obtained through home interviews. The assessment instruments (body diagram, Numerical Pain Scale, McGill Pain Questionnaire) collected information on the underlying disease and on pain. Data were analyzed using the Statistical Package for Social Sciences program for Windows. Associations between the subgroups of sickle cell disease patients (hemoglobin SS, hemoglobin SC, sickle &#946;-thalassemia and others) and pain were analyzed using contingency tables and non-parametric tests of association (classic chi-square, Fisher's and Kruskal-Wallis) with a level of 5% (p-value < 0.05) being set for the rejection of the null hypothesis. RESULTS: Forty-seven over 18-year-old patients with sickle cell disease were evaluated. Most were black (78.7%) and female (59.6%) and the mean age was 30.1 years. The average number of bouts of pain annually was 7.02; pain was predominantly reported by individuals with sickle cell anemia (hemoglobin SS). The intensity of pain (Numeric Pain Scale) was 5.5 and the quantitative index (McGill) was 35.9. This study also shows that patients presented a high frequency of moderately painful crises in their own homes. CONCLUSION: According to these facts, it is essential that pain related to sickle cell disease is properly identified, quantified, characterized and treated at the three levels of healthcare. In primary healthcare, accurate measurement of pain combined with better care may decrease acute painful episodes and consequently minimize tissue damage, thus improving the patient's overall health