A escrita da escrita em (d)enunciação no espaço sócio-escolar: um espaço (em vão) para a (cri)atividade escrita

This thesis is a writing of (d)enunciation because it reveals the language experiencies of those individuals who take, in social school space, the social role of the teacher and of the student, for some reason, they feel restrained by a demand which prevents them of teaching and learning how to write – and, even, their own writing. On this basis, we consider some questions concerning Benveniste‘s theory of enunciation and the teaching and learning process in Portuguese from the triad among teacher – teaching of writing/ knowledge – student, making this theoretical area closer to the pedagogical one. We support in the working hypothesis that is based on the position of the loosening of this triad has its effects in the process of intervention-interference to the point where it does not leave a scar which can remain working on productive effects on the student‘s writing. As a consequence, we show two findings which allow us to think the teaching of writing in Portuguese is a space for writing creativity in such a way it reveals itself as a sine qua non condition for the institutionalized and subjective writing. The first finding is that taking genre as a model has some implications in the teaching and learning process of writing according to the instructions of the official documents. The second finding, as a result of the first one, is the assumption that genre is at the same time a model and a transgression and taking it as a (ex)sample opens space for writing creativity which ―is a transposition of the interior language‖ (BENVENISTE, 2014 [1968-1969], p. 132) in a way this creativity makes it intelligible and highlights what is a model (interactiveness) and a transgression (inventiveness). From such findings, we organize our heterogeneous corpus according to these procedures: recording the classes in audio; recording the interviews we made with teachers and students in audio; and constituting an archive with the students‘ writing production. From this corpus, we analyzed the (d)enunciative mo(ve)ments in the teacher – teaching of writing/ knowledge – student triad and the history of (d)enunciations, of language experiences. Besides, we analyzed the teacher‘s discourse in relation to the way he plans his classes based on the knowledge that must be taught and his own way of managing this knowledge in order to understand his interference in his students‘ writings. Our analyses (re)affirm that when the loosening of the triad happens, the students become lost, what makes them unable of working upon their own writing epilinguisticaly. Because of it, schools have lost their specificity as a place for teaching students how to read and write as they open space for training, only. On this basis, we consider that the effect of such a loosening generates complaints and denunciations. Therefore, based on such (d)enunciations, we present the Primary sketches of a proposition for intervention-interference in teaching how to write and we aim at introducing much knowledge to teachers and students in order to allow them to think about possible ways out.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead β-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples

BACKGROUND: There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints. MATERIALS AND METHODS: A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed. RESULTS: The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR. CONCLUSION: The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy.

A quantitative phenytoin GC-MS method and it's validation for samples from human ex situ brain microdialysis, blood and saliva using solid-phase extraction

This study describes the development and validation of a gas chromatography-mass spectrometry (GC-MS) method to identify and quantitate phenytoin in brain microdialysate, saliva and blood from human samples. A solid-phase extraction (SPE) was performed with a nonpolar C8-SCX column. The eluate was evaporated with nitrogen (50°C) and derivatized with trimethylsulfonium hydroxide before GC-MS analysis. As the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin was used. The MS was run in scan mode and the identification was made with three ion fragment masses. All peaks were identified with MassLib. Spiked phenytoin samples showed recovery after SPE of ≥94%. The calibration curve (phenytoin 50 to 1,200 ng/mL, n = 6, at six concentration levels) showed good linearity and correlation (r² > 0.998). The limit of detection was 15 ng/mL; the limit of quantification was 50 ng/mL. Dried extracted samples were stable within a 15% deviation range for ≥4 weeks at room temperature. The method met International Organization for Standardization standards and was able to detect and quantify phenytoin in different biological matrices and patient samples. The GC-MS method with SPE is specific, sensitive, robust and well reproducible, and is therefore an appropriate candidate for the pharmacokinetic assessment of phenytoin concentrations in different human biological samples.

Ex situ respiration rates measured in water column samples during the METEOR cruise M87/1 in April 2012

Oxygen concentration and rate of change of oxygen were measured using the Unisense Oxygen Microsensor System. Water from different depth was taken from CTD attached niskin bottle. Measurements were conducted in 2 ml vials provided by Unisense and lasted for a minimum of two minutes after a stable rate was achieved. The sampling interval was 6 seconds. Transport containers, tubes and vials for measurements were covered with light proof black foil for dark-measurements. Measurements labeled "unfiltered" were passed through a 200 µm sieve in order to remove potential biases stemming from individual meso-zooplankton. Measurements labeled "filtered" were passed through a 0.8 µm polycarbonate filter placed on top of a wetted GF/F filter.

Ex situ respiration rates measured in water column samples during the James Cook cruise JC087 in June 2013

Oxygen concentration and rate of change of oxygen were measured using the Unisense Oxygen Microsensor System. Water from different depth was taken from CTD attached niskin bottle. Measurements were conducted in 2 ml vials provided by Unisense and lasted for a minimum of two minutes after a stable rate was achieved. The sampling interval was 6 seconds. Transport containers, tubes and vials for measurements were covered with light proof black foil for dark-measurements.

Quantitation of gammaH2AX foci in tissue samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.

Macro- and micronutrient disposition in an ex vivo model of extracorporeal membrane oxygenation

Background Extracorporeal membrane oxygenation (ECMO) circuits have been shown to sequester circulating blood compounds such as drugs based on their physicochemical properties. This study aimed to describe the disposition of macro- and micronutrients in simulated ECMO circuits. Methods Following baseline sampling, known quantities of macro- and micronutrients were injected post oxygenator into ex vivo ECMO circuits primed with the fresh human whole blood and maintained under standard physiologic conditions. Serial blood samples were then obtained at 1, 30 and 60 min and at 6, 12 and 24 h after the addition of nutrients, to measure the concentrations of study compounds using validated assays. Results Twenty-one samples were tested for thirty-one nutrient compounds. There were significant reductions (p < 0.05) in circuit concentrations of some amino acids [alanine (10%), arginine (95%), cysteine (14%), glutamine (25%) and isoleucine (7%)], vitamins [A (42%) and E (6%)] and glucose (42%) over 24 h. Significant increases in circuit concentrations (p < 0.05) were observed over time for many amino acids, zinc and vitamin C. There were no significant reductions in total proteins, triglycerides, total cholesterol, selenium, copper, manganese and vitamin D concentrations within the ECMO circuit over a 24-h period. No clear correlation could be established between physicochemical properties and circuit behaviour of tested nutrients. Conclusions Significant alterations in macro- and micronutrient concentrations were observed in this single-dose ex vivo circuit study. Most significantly, there is potential for circuit loss of essential amino acid isoleucine and lipid soluble vitamins (A and E) in the ECMO circuit, and the mechanisms for this need further exploration. While the reductions in glucose concentrations and an increase in other macro- and micronutrient concentrations probably reflect cellular metabolism and breakdown, the decrement in arginine and glutamine concentrations may be attributed to their enzymatic conversion to ornithine and glutamate, respectively. While the results are generally reassuring from a macronutrient perspective, prospective studies in clinical subjects are indicated to further evaluate the influence of ECMO circuit on micronutrient concentrations and clinical outcomes.

Dinâmica de crescimento de Centrolobium robustum (Vell.) Mart. ex Benth. (Leguminosae-Papilionoideae) na Floresta Atlântica, Rio de Janeiro, Brasil

O conhecimento sobre o ritmo de crescimento radial e a idade das árvores é um aspecto básico para compreender a dinâmica das populações, bem como o desenvolvimento e a sobrevivência das espécies. Nos trópicos, entretanto, estudos populacionais com este enfoque ainda são escassos, a despeito da urgente necessidade de preservação e manejo de suas florestas. Este trabalho tem por objetivo: i) Descrever a atividade cambial e o comportamento fenológico de Centrolobium robustum (Vell.) Mart. ex Benth., correlacionando estes parâmetros entre si; ii) Avaliar a influência da sazonalidade climática e do fotoperíodo sobre a atividade cambial e o comportamento fenológico e iii) Caracterizar o padrão estrutural dos anéis de crescimento e determinar, a partir destes, a idade e as taxas de crescimento radial da espécie na Reserva Biológica do Tinguá, RJ. Para a análise da atividade cambial, amostras de câmbio foram coletadas trimestralmente, processadas segundo técnicas usuais de anatomia vegetal e observadas sob microscopia ótica de campo claro, de fluorescência, de polarização e microscopia eletrônica de transmissão. O acompanhamento fenológico foi realizado mensalmente e os índices de atividade e de intensidade foram utilizados para analisar as fenofases reprodutivas e vegetativas, respectivamente. Para a investigação dendrocronológica, foram coletadas amostras com auxílio de sonda de Pressler, as quais foram polidas e observadas sob microscópio estereoscópico. Os resultados evidenciaram um ciclo anual de atividade e dormência cambial, caracterizados, respectivamente, pela presença de células em processo de divisão e diferenciação junto ao câmbio e de células completamente diferenciadas e deposição de calose em elementos de tubo crivado adjacentes à zona cambial. A dormência cambial coincidiu com a senescência e queda foliar, enquanto a atividade foi mais evidente na presença de folhas adultas na copa. A sazonalidade da atividade cambial apresentou correlação significativa com os dados de temperatura, precipitação e fotoperíodo do mês de realização das coletas. Foi constatado o regime sazonal da atividade cambial em associação ao clima e ao comportamento fenológico da espécie, conferindo caráter anual aos anéis de crescimento. Os resultados permitiram estabelecer o padrão dendroecológico de C. robustum e as idades e taxas de crescimento da população estudada.

Dinâmica de crescimento em Tabebuia rosea (Bertol.) Bertero ex A.DC. (Bignoniaceae): fenologia apical e cambial caulinar

O estudo da dinâmica de crescimento em espécies arbóreas permite melhor compreender a adaptação de uma espécie ao ambiente em que se desenvolve. Dados dessa natureza ainda são escassos no Brasil considerando-se a diversidade de espécies e fitofisionomias existentes. Esse tipo de estudo tem sido apontado como de grande relevância principalmente diante das situações atuais de desmatamento e mudanças climáticas, que exigem recuperação de áreas degradadas e prospectar como as espécies nativas sobreviveram diante deste ambiente de aceleradas mudanças. Nesta perspectiva, o objetivo deste trabalho foi investigar a dinâmica de crescimento de Tabebuia rosea, por meio do monitoramento mensal da fenologia apical e cambial caulinar e dos teores dos pigmentos fotossintetizantes: clorofilas a, b, totais e carotenoides. Os resultados obtidos foram correlacionados entre si e também com a sazonalidade das variáveis ambientais: temperatura, precipitação e fotoperíodo. No que se refere aos teores dos pigmentos fotossintetizantes, foi também avaliada as oscilações observadas nas folhas obtidas nas orientações geográficas: norte, sul, leste e oeste. O trabalho foi desenvolvido no Campus da Universidade do Estado do Rio de Janeiro, RJ, Brasil. A fenologia dos ápices caulinares foi acompanhada mensalmente a partir de observações qualitativas e quantitativas em 15 indivíduos. As amostras contendo a zona cambial foram obtidas por método não destrutivo e processadas segundo as técnicas usuais em histologia vegetal. A extração dos pigmentos fotossintéticos foi realizada em acetona 80%, com posterior centrifugação e análise ao espectrofotômetro em diferentes comprimentos de onda. Tabebuia rosea apresentou aumento na largura da zona cambial em número de camadas celulares e em micrômetros, diminuição do diâmetro radial da célula inicial fusiforme e aumento em micrômetros da camada de células em processo de alongamento e diferenciação do xilema secundário no mesmo período em que a copa das árvores estava ocupada por folhas adultas e com os teores mais elevados de clorofilas nas folhas, coincidindo com o período em que as variáveis ambientais apresentaram-se elevadas, corroborando os resultados já obtidos para outras espécies nativas da Mata Atlântica se desenvolvendo no estado do Rio de Janeiro. A maior concentração de carotenoides foi observada no período em que as variáveis ambientais apresentavam índices elevados e as plantas estavam mais expostas à radiação luminosa, possivelmente em função de um maior investimento em fotoproteção. Cabe destacar que o lado oeste da copa das árvores mostrou uma tendência em apresentar maiores concentrações dos pigmentos fotossintéticos estudados, resultado ainda não observado na literatura até o momento, o que pode ser uma estratégia da espécie em compensar níveis baixos de radiação solar com acúmulo de clorofilas.

Capacidade de invasão, persistência intracelular e atividade citotóxica de cepas de Aeromonas spp. em modelo celular in vitro e ex vivo

As Aeromonas são consideradas patógenos em potenciais para o homem e animais e estão amplamente distribuídas no ambiente sendo a água e os alimentos importantes veículos de transmissão. Muitos estudos têm demonstrado que a patologia causada pela infecção por Aeromonas é complexa e envolvem inúmeros fatores de virulência, dentre eles a aderência, invasão, enterotoxinas, hemolisinas, exoenzimas, sideróforos, flagelos, formação de biofilme e mecanismos de secreção. No presente estudo, analisamos os mecanismos de patogênese mediados por A. caviae e A. hydrophila, avaliando a participação desses microrganismos nos processos de adesão, invasão, persistência intracelular e citotoxidade celular. Foram utilizados ensaios quantitativos in vitro para testar associação, invasão e persistência intracelular em linhagens celulares HEp-2 e/ou T84. A interação de tecidos intestinais de coelho cultivados in vitro (IVOC) com três cepas de A. caviae originárias de fezes diarréicas também foi avaliada. Observamos que 10 (62,5%) das 16 cepas de Aeromonas spp. de diferentes origens, submetidas aos testes de invasão quantitativos foram capazes de invadir células HEp-2 e T84 em 6 horas de incubação. As cepas positivas nos testes de invasão foram submetidas ao teste quantitativo de persistência em células HEp-2 e sobreviveram no ambiente intracelular por 48 e/ou 72 horas sem multiplicação. A interação de três cepas de A. caviae com a mucosa intestinal de coelho ex vivo resultou em aderência, produção de muco e alterações como, intensa vacuolização e drástica desorganização estrutural que levaram a destruição das microvilosidades intestinais. Este estudo demonstrou que subconjuntos de cepas de A. caviae e A. hydrophila de diversas origens, foram capazes de invadir, persistir ou destruir linhagens celulares in vitro. Nosso estudo também evidenciou que cepas de A. caviae causaram expressivas alterações morfológicas que resultaram na destruição de epitélios intestinais de coelho ex vivo. Finalmente, nossos resultados contribuíram para reforçar o potencial patogênico de cepas de Aeromonas, em especial, as de origem vegetal e clínica.

中国蔷薇属芹叶组（Rosa Sect. Pimpinellifoliae DC. ex Ser.）的分类学研究

本研究通过我国CDBI、 KUN、PE、SZ等主要标本馆约3, 500份馆藏标本的研究和野外考察相结合，对我国蔷薇属（Rosa L.）芹叶组(Sect. pimpinellifoliae DC. ex Ser.）植物以及相关组的一些种进行了性状特征、形态和微形态的研究，对该组的一些种的形态特征描述进行了补充，同时给出详细的地理和海拔范围分布图。综合花粉以及种子（瘦果）形态的研究结果重新制订了分种检索表，同时，对该组一些形态相近容易混淆的种进行了对比研究，特别对一直存在争议的绢毛复合体（绢毛蔷薇R. sericea Lindl.和峨眉蔷薇R. omeiensis Rolfe）进行了大量宏观形态特征的研究，并用光学显微镜（LM）和扫描电镜(SEM)对二者的花粉及种子形态、微形态进行对比研究和分析，主要研究内容包括： 1. 芹叶组孢粉研究 对芹叶组的10个种及相关的4个组共17个种（18个样品）的植物花粉进行了光镜和扫描电镜观察和比较研究。研究结果表明：蔷薇属植物花粉粒大小为中等偏小，极轴长23.98[21.82(R. graciliflora Rehd. et Wils.)～29.18(R. tsinglingensis Pax. et Hoffm.)] μm，赤道轴长28.65[24.15（R. graciliflora）～34.70(R. davidii Crép.)] μm；花粉属辐射对称等极单花粉，花粉形态赤道面观呈球形到超长球形；极面观为三裂圆形或近圆形,三孔沟，孔缘加厚,具中部突起的桥状盖。花粉外壁纹饰为条纹状，光镜下形态特征相差不大；在电镜下外壁条纹和脊沟内穿孔的形状、大小和频度等特征，常具组至种水平上的可见变异，可作为组至种水平划分的依据。 根据花粉外壁条纹特征及穿孔形状和数目等特征，本研究将这些植物的花粉归为5个类型，并编制了分组检索表。同时，根据条纹状的清晰度，排列方式、条纹形状、穿孔大小及其频度等方面的差异，各有特点，对该组的10个种编制了分种检索表。 2. 芹叶组种子形态研究 应用光学显微镜和扫描电镜对我国蔷薇属芹叶组14个种及相关组5个组共36种植物的种子宏观形态及种皮微形态特征进行了观察研究。结果显示，蔷薇属种子形态多样,形状分别为肾形、卵形或锥形等；种子颜色以淡棕色、褐色以及土黄色为主；种子大小种间相差悬殊，相对体积为（长×宽×厚）36.66（4.79～114.47) mm3。光镜下，种子宏观形态特征具组内一致性，在扫描电镜下种子表面结构特征因种而异，其纹饰以网纹为主，可分为3种类型，即近平滑型、负网纹型和网纹型。研究结果表明，蔷薇属种子表面纹饰与地理分布关系不大,具有组及种内稳定性。其种子形态、大小、表而纹饰类型等特征可作为蔷薇属组及种水平上的分类依据。 结合蔷薇属花粉形态研究结果，得出蔷薇属种皮微形态特征与花粉外壁纹饰特征相吻合，在代表组及种的特征上具相关性的结论。同时根据种子形态、微形态结构特征的组间区别和种间差异编制了分组及芹叶组14个种的分种检索表。 3. 绢毛蔷薇复合体的研究 通过对大量标本的研究、野外观察以及扫描电镜对绢毛蔷薇复合体的花粉形态和种皮表面结构进行研究，通过对小叶、花粉及种子的形态定量分析结果支持Rowley (1959)的观点，将峨眉蔷薇处理为绢毛蔷薇的一个变种。 综上研究结果得出，蔷薇属植物的小叶片数目、花被基数以及花粉及种子形态等性状是较为稳定的，这些特征可很好的作为分类学依据。 The morphology, pollen exine sculpture and seed coat structure of the species of Rosa sect. Pimpinellifoliae and related sections were studied.About 3,500 herbarium specimens at CDBI, KUN, PE, and SZ were examined. Field work in Sichuan and Yunnan were conducted. Revisions of some species were carried out and a new key to species of sect. Pimpinellifoliae was proposed based on morphology, pollen exine sculpture and seed coat structure, Detailed morphological descriptions, geographical distributions and the altitudinal ranges of some taxa are given. The systematics of the species complex, the Rosa sericea complex (R. sericea Lindl. & R. omeiensis Rolfe), was emphasized. This thesis focused on the following three aspects: 1. Pollen morphology of Rosa sect. Pimpinellifoliae The pollen morphology of 18 samples representing 10 species of the Eurasian Rosa sect. Pimpinellifoliae and 7 additional species of related sections was investigated under LM and SEM. The pollen grains are monadic, actinomorphic, equipolar, medium-sized, spheroidal to perprolate in equatorial view, 3-lobed circular or semi-circular in polar view, crassimarginate, pontoperculate, and with striate exine sculpture. The striate sculpture varies among sections and species. The equatorial axis ranges from 17.97 μm (R. sikangensis) to 29.18 μm (R. tsinglingensis) with an average of 23.98 μm in length, while polar axis varies from 24.15 μm (R. gracilifolra) to 34.70 μm (R. davidii) with an average of 28.65 μm in length. The pollens can be divided into five types based on striate sculpture and a key to the sections sampled was proposed accordingly. The pollen morphology of species of sect. Pimpinellifoliae is more homogeneous and different from other sections sampled and did not support the two-series subdivisions in sect. Pimpinellifoliae. A key is also provided based on characers of pollen morphology among species in sect. Pimpinellifoliae. 2. Seed coat structure of Rosa sect. Pimpinellifoliae The seed coat structure of 39 samples representing 14 species of Rosa sect. Pimpinellifoliae and 12 additional species of related sections was investigated under LE and SEM. The seed relative volume (Length × width × thickness) ranges from 4.79 to 114.47 mm3 with an average of 36.66. mm3. The seeds are reniform, ovate or oblong in shape, with orange-brown, light brown or deep brown color. Seed coat sculpture was reticulate or striate-like reticulate. There was no difference in sculpture character of various speices under LM, while three types of seed coat sculpture were identified under SEM and a key to species based on the seed coat sculpture was provided. The three types of seed coat sculpture were nearly smooth, areolate and reticulate. The study of the seed coat sculpture of same species sampled from different populations showed that characters on the seed coat are stable, and thus the size, shape and seed coat sculpture can be used in species level identification. Interestingly, characters in the seed coat sculpture and the pollen morphology in sect. Pimpinellifoliae are consistent at in specific or sectional levels. A key to the 14 species sampled was given based on seed coat sculpture. 3. The study on Rosa sericea complex The Rosa sericea complex contains R. omeiensis and R. sericea. They are morphologically similar to one another and the systematic status of R. omeiensis has been controversial. In this study we examined large numbers of herbarium specimens of R. omeiensis and R. sericea and conducted field observations in the Hengduan Mts.. We also performed SEM study of pollen morphology and seed coat structure of R. omeiensis and R. sericea. We further carried out intensive morphometric study on the leaflet, pollen, and seed morphology. Our results showed that R. omeiensis should be sunk to be a variety of R. sericea, just as Rowley’s treatment in 1959. In conclusion, the features in the number of leaflet and petal, and the morphological character on pollen and seed are relatively stable. Therefore these characters are very useful in taxon delimition.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P

Exposição profissional a chumbo : utilização da protoporfirina-zinco (PPZ) na vigilância de saúde de trabalhadores ex

Resumo Uma estratégia de avaliação e prevenção de riscos na exposição a agentes químicos deve ter sempre em conta que a vigilância do ambiente de trabalho e a da saúde dos trabalhadores são aspectos complementares de uma mesma realidade – os riscos resultantes da interacção entre um agente químico e os trabalhadores a ele expostos. Se à Vigilância Ambiental compete apreciar o risco, pela caracterização do agente no ambiente de trabalho, a Vigilância Biológica pronuncia-se sobre a interacção entre o tóxico e o organismo, avaliando a resposta à agressão química e a evolução das reacções de adaptação ou de desajuste face à absorção do tóxico. Os Indicadores Biológicos, deste modo, assumem um estatuto de instrumento privilegiado na vigilância da saúde dos trabalhadores expostos, na medida em que medem a quantidade de tóxico que efectivamente penetrou e foi absorvido, ou o resultado (efeito) determinado por essa mesma dose. O presente estudo procura contribuir para a definição de um quadro metodológico de utilização dos Indicadores Biológicos na avaliação/gestão da exposição profissional ao chumbo, designadamente apreciando a variação da protoporfirina-zinco (PPZ), indicador até ao presente ainda não utilizado em Portugal. O chumbo é um metal de ocorrência natural, cujos níveis nos diversos ecossistemas resultam, principalmente, das actividades antropogénicas de natureza doméstica e industrial. A sua capacidade poluente é assinalável, representando uma fonte de exposição permanente para o homem, demonstrável pela sua constante presença no organismo apesar de não desempenhar qualquer tipo de função fisiológica. São actualmente inúmeras as suas aplicações, tornando a exposição profissional ao chumbo uma realidade vasta: indústrias de acumuladores eléctricos, de vidros, de plásticos e de munições, construção civil, manutenção e reparação automóvel e de navios, fabrico de tintas, indústrias electrónicas, fundições e actividades de soldadura são, entre outras, situações onde é uma realidade a ter em conta. A penetração dos compostos inorgânicos de chumbo no organismo efectua-se principalmente por via respiratória, não sendo, no entanto, desprezável, a sua penetração por via digestiva. As partículas absorvidas são transportadas pelo sangue principalmente ligadas aos eritrocitos (95%), distribuem-se pelos tecidos moles e depositam-se essencialmente no tecido ósseo, onde representam mais de 90% da carga corporal do total absorvido e tendo aí um elevado tempo de semi-vida (mais de 20 anos). Não é metabolizado no organismo e a sua eliminação efectua-se essencialmente por via renal,sendo igualmente excretado, em menor escala, através das fezes, do suor, da saliva, das faneras e do leite materno. O conhecimento científico evidencia que concentrações sanguíneas de chumbo entre 20 e 50 mg/dL são susceptíveis de determinar efeitos adversos no homem, podendo ser afectados o sistema hematopoiético, o sistema nervoso, o sistema cardiovascular, o sistema reprodutor e o sistema imunitário. Contudo, ainda muito há a clarificar no âmbito da toxicidade do chumbo. Os níveis de exposição a que correspondem as alterações nos diversos órgãos e sistemas continuam a ser motivo de alguma controvérsia. As características carcinogénicas e mutagénicas do chumbo são, ainda, um campo de vasta exigência de investigação. A intoxicação por chumbo e seus sais (Saturnismo) de origem ocupacional é reconhecida em Portugal como doença profissional (grupo 1 - Doenças Provocadas por Agentes Químicos, da Lista das Doenças Profissionais). É uma intoxicação do tipo crónico, fruto da absorção contínua de doses relativamente pequenas durante longo período, evidenciando-se no seu início por sinais e sintomas vagos e difusos de grande inespecificidade, que podem incluir, nomeadamente, perda de apetite, sabor metálico na boca, palidez, mal-estar e fadiga, cefaleias, mialgias e artralgias, irritabilidade, tremores finos, obstipação, cólicas abdominais, insónias, déficit da memória de curto prazo e da capacidade de concentração. Um importante conjunto de indicadores biológicos pode ser utilizado na vigilância periódica da saúde de trabalhadores nestas condições de exposição. Tais indicadores (de dose ou de efeito), encerram diferentes significados e comportam distintas exigências, competindo ao Médico do Trabalho, no âmbito dos programas de prevenção dos efeitos adversos relacionados com a exposição profissional a chumbo, seleccionar a sua utilização e interpretar a sua informação, de modo a avaliar a interacção do tóxico com o organismo numa fase de reversibilidade. O presente estudo envolveu 180 trabalhadores dos quais 110 apresentavam plumbémias (Pb-S) iguais ou superiores a 40 mg/dL. Além da Pb-S, a todos foi doseada a protoporfirina-zinco (PPZ) e efectuado o Hemograma e a cerca de 25% foi determinada a concentração do ácido d-aminolevulínico urinário (ALA-U). Os doseamentos da PPZ efectuados em amostra de sangue capilar através de um hematofluorímetro portátil revelaram-se de total fiabilidade, dando significado a uma técnica de fácil execução e baixo custo. A avaliação do tipo de colheita urinária para doseamento do ALA-U concluiu pela necessidade de recurso a urinas de 24 horas.Os resultados do estudo evidenciaram uma elevada associação entre a PPZ e a Pb-S, com uma maior magnitude e de início mais precoce do que o que registado na associação da Pb-S com o ALA-U. Revelaram, ainda, fracos níveis de associação da hemoglobina (e outros parâmetros hematológicos) com a Pb-S. E demonstraram para um cut-off de 100 mg/ dL de PPZ, taxa de falsos negativos e falsos positivos, para plumbémias a partir de 70 mg/dL, inferiores a 20%. Assim, concluiu-se que, nos protocolos de vigilância de saúde de trabalhadores expostos a chumbo, o doseamento da PPZ por hematofluorímetro, em sangue de colheita capilar, é adequado, fiável e de realização preferencial em relação ao do ALA-U. Concluiu-se, também, que a realização do hemograma apenas se justifica em situações individuais que clinicamente o tornem aconselhável. E que estes protocolos devem incluir a realização da Pb-S e da PPZ, podendo, em situações de controlo rigoroso (ambiental, biológico e clínico), basear-se apenas na determinação da PPZ reservando os outros indicadores para aprofundar a investigação médica nos casos de taxas elevadas desta ou de situações limitantes. ■ Résumée Une stratégie d’évaluation et de prévention des risques d’exposition aux agents chimiques doit toujours tenir en considération que la vigilance du lieu de travail et de la santé des travailleurs sont des aspects complémentaires d’une même réalité – les risques résultant d’une interaction entre l’agent chimique et les travailleurs exposés. Si c’est à la Vigilance Ambiantale de juger le risque, par la caractérisation de l’agent dans le lieu de travail, la Vigilance Biologique, elle, se prononce sur l’interaction entre le toxique et l’organisme, évaluant la réponse à l’agression chimique et l’évolution des réactions d’adaptation ou de rupture face à l’absorption du toxique. Les Indicateurs Biologiques assument ainsi un statut d’instrument privilégié de vigilance de la santé des travailleurs exposés, dans la mesure où ils déterminent la quantité de toxique qui a effectivement été pénétré et absorbé, ou le résultat (effet) déterminé par cette dose. Cette étude-ci cherche à contribuer à la définition d’un cadre méthodologique d’utilisation des Indicateurs Biologiques dans l’évaluation/ gestion de l’exposition professionnelle au plomb inorganique, évaluant spécialement le comportement de la protoporphirine-zinc (PPZ), indicateur pas encore utilisé au Portugal.Le plomb est un métal d’occurrence naturelle dont les niveaux dans les différents écosystèmes en résultent, principalement, des activités anthropogéniques de nature domestique et industrielle. Sa capacité polluante peut être signalée, représentant une source d’exposition permanente pour l’homme, celle-ci démontrable par sa présence continue dans l’organisme, même si elle n’y accomplit aucune fonction physiologique. Actuellement ses applications sont innombrables, faisant de l’exposition professionnelle au plomb une réalité de grande ampleur : industries d’accumulateurs électriques, de verre, de plastique et de munitions, bâtiments, manutention et réparation automobile et navale, fabrication d’encres, industries électroniques, fontes et activités de soudure sont, entre autres, des situations réelles a en tenir compte. La pénétration du plomb inorganique dans l’organisme se fait principalement par voie respiratoire, pouvant se faire également par voie digestive. Les particules absorbées sont transportées par le sang, surtout liées aux érythrocytes (95%), se repartent à travers les tissus mous et se déposent essentiellement dans le tissu osseux, où elles représentent plus de 90% de la charge corporelle de ce qui a été absorbé et ont un temps de demi-vie élevé (plus de 20 ans). Le plomb n’est pas métabolisé dans l’organisme et son élimination se fait essentiellement par voie rénale, pouvant tout de même, à une moindre échelle, être excrété dans les fèces, de la sueur, de la salive, des ongles, des cheveux et du lait maternel. La connaissance scientifique met en évidence que des concentrations sanguines de plomb entre 20 et 50 mg/dL sont susceptibles de déterminer des effets adverses dans l’homme, pouvant les systèmes hématopoïétique, nerveux, cardiovasculaire, reproducteur et immunitaire en être affectés. Cependant, il en reste beaucoup à éclaircir dans le domaine de la toxicité du plomb. Les niveaux d’exposition auxquels correspondent les modifications des divers organes et systèmes, demeurent toujours sujet de quelque controverse. Les caractéristiques carcinogèniques et mutagèniques du plomb restent toujours un champ d’investigation d’une grande exigence. L’intoxication par le plomb et ses sels (Saturnisme) d’origine occupationnelle est reconnue, au Portugal, comme une maladie professionnelle (groupe 1- Maladies Provoquées par des Agents Chimiques, de la Liste des Maladies Professionnelles). C’est une intoxication du tipe chronique, due à l’absorption continue de doses relativement petites pendant une longue période, mise en évidence à travers des signes et des symptômes vagues et diffus sans grande spécificité, lesquels peuvent inclure, particulièrement, le manque d’appétit, goût métallique dans la bouche, pâleur, malaise et fatigue, céphalées, myalgies et arthralgies, irritabilité, tremblements fins, constipation, coliques abdominales, insomnies, déficit de la mémoire à court terme et de la capacité de concentration.Un ensemble important d’indicateurs biologiques peut être employé dans la vigilance périodique de la santé des travailleurs dans ces conditions d’exposition. Ces indicateurs (de dose ou d’effet) renferment différentes significations et comportent diverses exigences, devant le Médecin de Travail, dans le domaine des programmes de prévention des effets adverses qui sont en relation avec l’exposition professionnelle au plomb, sélectionner son utilisation et interpréter son information de façon à évaluer l’interaction de l’élément toxique avec l’organisme à un stade de réversibilité. L’étude ci-présent engloba 180 travailleurs desquels 110 présentaient des plombémies (Pb-S) égales ou supérieures à 40 mg/dL. À part la Pb-S, la protoporphyrine-zinc (PPZ) leur a été prise en dosage et un Hémogramme fut effectué et fut déterminé l’acide d- aminolévulinique urinaire (ALA-U) sur environ 25% des travailleurs. Le dosage de la PPZ efectué en échantillon de sang capillaire par un fluorimètre portable, s’est accomplit d’une fiabilité total, donnant du sgnificat à une téchnique de facile execution et bas prix. L’évaluation de la prise urinaire par dosage du ALA-U conclut au besoin d’un recours aux urines de 24 heures Les résultats de l’étude ont mis en évidence une association élevée entre la PPZ et la Pb- S, avec une intensité majeure et de début plus précoce par rapport à celui qui fut registré lors de l’association de la Pb-S avec la ALA-U. Ces résultats ont également montré de faibles niveaux d’association entre l’hémoglobine (et autres paramètres hématologiques) et la Pb-S. Ils ont démontré aussi, une valeur de cut-off de 100 mg/dL de PPZ, des taux de faux négatifs et faux positifs, pour des plombémies de 70 mg/dL, inférieurs à 20%. On peut donc conclure que dans les protocoles de vigilance de la santé des travailleurs exposés au plomb, le dosage de la PPZ par fluorimetrie dans le sang capillaire est adéquat, fiable et de réalisation préférentielle par rapport à celui du ALA-U. On peut également conclure que la réalisation de l’hémogramme ne se justifie que dans les cas individuels où, cliniquement, celui-ci est conseillé. De plus, ces protocoles doivent inclure la réalisation de la Pb-S et de la PPZ, pouvant, en cas de contrôle rigoureux (ambiantal, biologique et clinique), s’appuyer que dans la détermination de la PPZ réservant les autres indicateurs pour approfondir l’investigation médicale dans les cas où les taux de celle-ci sont élevés ou dans les cas de situations limitantes. ■ Summary Any strategy to evaluate and prevent the risks of chemical agents exposure must always regard the work environment and workers health as complementary aspects of one reality - the resulting risks from the interaction between the chemical agent and the exposed workers. It is the responsibility of Environmental Monitoring to evaluate the risks of exposure by the characterization of the chemical agent in the work environment. Biological Monitoring, on the other hand, pronounces itself over the toxin and body interaction, evaluating human response to the chemical aggression and the body adaptations to the toxic absorption. Biological Exposure Indices (BEI) assume, therefore, a privileged status among exposed workers' health monitoring instruments, as they measure the actual penetrated and absorbed toxic quantity and the effect it produces. This research study aims to contribute to the definition of a methodological strategy on the utilization of BEI’s in evaluating inorganic lead's occupational exposure, more specifically appreciating the zinc protoporphyrin (ZPP) variation, an index that has never been taken under consideration in Portugal until now. Lead is a natural metal whose ecosystem’s levels are mainly due to domestic and industrial anthropogenic activities. Its pollutant capacity is notable, representing a permanent exposure risk shown by its constant presence in the human body, although it has no physiologic function. Nowadays, lead's applications are countless, turning its professional exposure a huge reality: storage batteries industries, glass industries, plasterers and munitions industries, building construction, ships and motor car maintenance and repairing, ink manufacture, electronics industries, foundries and other soldering activities are, among so many other, realities to attend to. Respiration is the main cause of human body's inorganic lead absorption, although digestive pathway must not to be ignored. The absorbed particles are transported by blood, essentially bounded to erythrocytes (95%). It is distributed by soft tissues and settled mainly on bone tissues, where it represents approximately 90% of the total body charge and has a high half-life time (more than 20 years). It is not metabolized by the organism, its elimination being effectuated by renal activity and, in smaller scale, through lees, sweat, saliva, nails, hair and maternal milk.Scientific knowledge shows that concentrations of lead in blood between 20 e 50 mg/dL are susceptible to determine adverse effects in man and able to affect the hematopoietic system, the nervous system, the cardiovascular system, the reproductive system and the immunological system. Nevertheless, there's still much to be learned and clarified about lead's toxicity. The correlation between exposure levels and human's systems and organs alteration levels continues to be a centre of controversies. Still, lead's carcinogenic and mutagenic characteristics continue to be a high demanding research field. Intoxication by lead and its compounds (saturnism), from occupational origin, is recognized in Portugal as an occupational disease, included in Group 1 - Chemical Agents Caused Diseases, on the Occupational Diseases List. It is a chronic intoxication caused by a continuous absorption of small doses, throughout a long period of time. Its signs and symptoms are diffuse and imprecise, of great unspecificity, such as loss of appetite, metallic flavor in the mouth, paleness, ailment and fatigue, headaches, myalgia and arthralgia, irritability, thin tremors, constipation, abdominal pain, insomnias, short memory loses and inability to concentrate. A considered number of BEI’s can be used in Periodic Health Monitoring of workers in such exposure conditions. Such BEI (dose indices or effect indices) provide different meanings and imply different procedures, being Occupational Doctors responsibility, in the context of lead related adverse effects preventive programmes, to select and interpret its information, in order to evaluate the interaction between toxic and organism in a reversible phase of the toxic action. The present research study involved 180 workers, 110 of which presented blood lead levels (PbB) above or equal to 40 mg/dL. Besides PbB, all workers has been evaluated for zinc protoporphyrin levels (PPZ) and submitted to a haemogram. About 25% of the workers were selected for d-aminolevulinic urinary acid (ALA-U) determination. The evaluation of PPZ, by a portable hematofluorometer using capillary blood samples, turned out to be an easy procedure with low costs and total warrantability. As in regard for ALA- U procedure, it was concluded the necessity of 24 hours urine samples. This research results underlined a strong connection between ZPP and PbB, which was found to to be stringer and to begin earlier than it was registered for PbB and ALA-U association. The same study also revealed a low association level between PbB and hemoglobin or other hematological indices. It was also verified less than 20% of false negatives and false positives cases when admitted a ZPP 100 mg/dL cut off value for PbB³ 70 mg/dL. As in result it was concluded that in Health Monit

Peptides generated ex vivo from serum proteins by tumor-specific exopeptidases are not useful biomarkers in ovarian cancer

BACKGROUND: The serum peptidome may be a valuable source of diagnostic cancer biomarkers. Previous mass spectrometry (MS) studies have suggested that groups of related peptides discriminatory for different cancer types are generated ex vivo from abundant serum proteins by tumor-specific exopeptidases. We tested 2 complementary serum profiling strategies to see if similar peptides could be found that discriminate ovarian cancer from benign cases and healthy controls. METHODS: We subjected identically collected and processed serum samples from healthy volunteers and patients to automated polypeptide extraction on octadecylsilane-coated magnetic beads and separately on ZipTips before MALDI-TOF MS profiling at 2 centers. The 2 platforms were compared and case control profiling data analyzed to find altered MS peak intensities. We tested models built from training datasets for both methods for their ability to classify a blinded test set. RESULTS: Both profiling platforms had CVs of approximately 15% and could be applied for high-throughput analysis of clinical samples. The 2 methods generated overlapping peptide profiles, with some differences in peak intensity in different mass regions. In cross-validation, models from training data gave diagnostic accuracies up to 87% for discriminating malignant ovarian cancer from healthy controls and up to 81% for discriminating malignant from benign samples. Diagnostic accuracies up to 71% (malignant vs healthy) and up to 65% (malignant vs benign) were obtained when the models were validated on the blinded test set. CONCLUSIONS: For ovarian cancer, altered MALDI-TOF MS peptide profiles alone cannot be used for accurate diagnoses.