Bothrops aff. Leucurus (Wagler, 1824) populazio irlatar baten deskribapen morfologikoa: suge populazio irlatar baten morfologia

[EUS] Atrox konplexuko espezieen barruan, Bothrops leucurus (Wagler, 1824) Bahia estatuko oihan atlantikoko sugegorri espezie arruntena da. Polimorfikoa da eta ezaugarri geografikoekin, ontogenikoekin eta sexuarekin erlazionatutako kolore aldakortasuna aurkezten du. Jadanik hainbat ikerketa burutu dira kontinenteko populazioei buruz, baina, oraindik ez da lanik egin BTS badiako populazio uhartetarrekin. Lan honen xedea Bothrops aff. leucurus populazio uhartetarraren deskribapena burutzea izan da, orbanen ereduetan, folidosian eta gorputz tamainan oinarriturik. Lagin tamaina erlatiboki txikia izanik (n=20), Student t testeko emaitzak type II erroreen menpe daude eta kontinenteko eta uharteko populazioen arteko ezberdintasunak estatistikoki ikertu izan ez diren arren, gure datuak garrantzia dute. Izan ere, ikerketa sakonagoetan oinarri bezala erabili ahal izango dira. Biometriari dagokionez, gure populazio uhartetarrean batazbesteko MKL txikiagoa ikusi dugu eta balioen tarteak murritzagoak dira. Ez da sexuen arteko ezberdintasun esangarririk topatu gorputzeko neurrientzat. Folidosian (ezkata zenbateka) lortutako datuak, beste autoreek Bothrops leucurus-entzat deskribatutako parametroekin bat datozen arren, gure laginak ezkata tarte mugatuagoak ageri ditu. Orban ereduak ale guztietan behatu eta irudikatu ostean, oro har bi eredu ezberdindu eta deskribatzea posible izan da. Behatuak izan diren ezberdintasunak, uhartetako baliagai-hornidura mugatuen ondorio gisa uler daitezke, izan ere, halako baldintzetan ale txikiagoak positiboki hautatuak izango liratekeelako.

Formação de biofilmes por Enterococcus faecium sensível e multirresistente aos antimicrobianos: características à proteômica

Enterococcus faecium tem se destacado no cenário das infecções hospitalares, particularmente, amostras portadoras de características de multirresistência. Apesar de serem responsabilizados como agentes etiológicos em diferentes quadros clínicos, fatores associados a patogênese das infecções ainda não estão esclarecidos. Entretanto, sabe-se que a capacidade de formação de biofilmes pode ser responsabilizada como um dos atributos capazes de promover o papel patogênico desses microrganismos. Assim sendo, este estudo se propôs a investigar a capacidade de formação de biofilmes por duas amostras de E. faecium: SS-1274 (derivada da amostra tipo da espécie) e CL-6729 (multirresistente e pertencente a um complexo clonal globalmente disperso). As amostras foram caracterizadas fenotipica e genotipicamente para confirmação da identificação, determinação da susceptibilidade a 17 antimicrobianos, caracterização da concentração mínima inibitória para ampicilina, gentamicina e vancomicina, determinação do genótipo vanA e detecção de genes associados a expressão dos genes asa1, cylA, esp, gelE e hyl. A análise quantitativa da formação por 24h e 72h dos biofilmes foi realizada por metodologia do cristal violeta, em placas de microtitulação de poliestireno. Foram construídas curvas de formação pela avaliação da DO570nm das preparações coradas por cristal violeta em períodos de tempo de 2h a 74h. A influência de subCMIs de ampicilina, gentamicina e vancomicina na formação de biofilmes de E. faecium foi também caracterizada em ensaios de quantificação da biomassa. A presença de DNA e proteínas foi avaliada em ensaios de destacamento e por fluorimetria. A arquitetura, distribuição espacial e reação aos fluorocromos Syto9 e SYPRO Ruby Protein foram evidenciadas por microscopia confocal de varredura a laser. Análises proteômicas através da avaliação por SDS-PAGE e por ESI-Q-TOF também foram empregadas para avaliação de biofilmes versus crescimento planctônico. Nossos resultados demonstraram que a amostra CL-6729 apresentou uma maior biomassa nos tempos analisados. Apesar da menor quantidade de biomassa da amostra SS-1274, o perfil da curva de formação foi semelhante a CL-6729. As análises da matriz por ensaios quantitativos e microscopia confocal revelaram que proteína parece ser um importante constituinte para ambas as amostras, entretanto biofilmes formados na presença de da CMI e durante 24h, parecem sofrer alterações na constituição. Os resultados relativos às espectrometrias de massa sugerem que células de biofilmes de E. faecium podem também estar metabolicamente ativas, devido a identificação de um considerável número de proteínas relacionadas ao metabolismo e divisão celular, similarmente às células planctônicas. No entanto, investigações complementares são necessárias para quantificar as diferenças relacionadas a sua expressão. Foi observado que ambas as amostras independente das variações fenotípicas e genotípicas são capazes de formar biofilmes maduros exibindo constituição e arquitetura similares. Entretanto, nossos resultados sugeriram que cada amostra responde as diferentes situações de acordo com seus determinantes de virulência e resistência.

联合固氮菌Klebsiella oxytoca SA2和水稻幼苗的相互作用

从内蒙流沙地先锋植物沙竹（Psammochloa mongokca）内分离到一株内生细菌，经鉴定定名为Klebsiella oxytoca SA-2 K.oxytoca SA-2兼性厌氧固氮，NH4+抑制其固氮酶合成。部分抑制固氮酶活性;N03 -抑制其固氮酶的合成和活性。 60℃灭活K.oxytoca SA-2整体菌免疫兔子得到抗血清。免疫印迹表明此抗血清具K.oxytoca SA-2种特异性。石蜡切片免疫金银染色结合显微观察发现K.oxytoca SA-2定殖于沙竹叶鞘薄壁细胞和叶片的薄壁细胞内。 K.oxytoca SA-2在半固体培养基中接种水稻幼苗，限菌培养21天，根内重新分离的数量达l06 cfu/g．FWroot，但K.oxytoca SA-2在富养的土壤中生长良好，表现为兼性内生菌。 限菌培养水稻（Oryza sativa）幼苗，石蜡切片免疫金银染色结合显微观察研究了的K.oxytoca SA-2的侵染特性．K.oxytoca SA-2可以通过侧根发生处和表皮细胞胞间层进入根内，在皮层薄壁细胞间隙大量定殖，在解体和看似完整的薄壁细胞内也有定殖，在根和茎中柱内K.oxytoca SA-2进入了木质部导管。在根基，K.oxytoca SA-2大量侵入了已解体的内皮层和中柱鞘细胞，植物细胞在K.oxytoca SA-2侵入后解体，可能表现为严格的局部超敏反应。 接种K.oxytoca SA-2 21天，水稻地上苗部分没有发现肉眼和显微可见的病症。与对照相比，接种K.oxytoca SA-2显著促进限氮培养水稻幼苗的生长。由于K.oxytoca SA-2在限碳限氮培养基和水稻幼苗共培养时能分泌NH4+和植物激素，它可能通过向水稻幼苗提供氮素和分泌植物激素促进植物生长。而且用固氮酶铁蛋白抗血清进行免疫金银染色发现定殖在根基皮层薄壁细胞胞间层和细胞间隙，木质部导管和茎基木质部导管的K.oxytoca SA-2可以表达固氮酶，固氮参与了K.oxytoca SA-2在水稻幼苗中的内生。 培养基内碳源（苹果酸）和培养温度对K.oxytoca SA-2和水稻幼苗相互作用的影响也进行了研究。 研究表明，K.oxytoca SA-2作为兼性内生固氮菌，能够和植物紧密联合，并在植物体内开拓一个有利的生态位固氮，而且K.oxytoca SA-2可以分泌NH4+和植物激素，在和植物相互作用中使植物受益。

Seismic behaviour of anchored quay walls with dry backfill

The seismic behaviour of anchored sheet pile walls is a complex soil-structure interaction problem. Damaged sheet pile walls are very expensive to repair and their seismic behaviour needs to be investigated in order to understand their possible mechanisms of failure. The research described in this paper involves both centrifuge testing and Finite Element (FE) analyses aimed at investigating the seismic behaviour of an anchored sheet pile wall in dry sand. The model wall is tied to the backfill with two tie rods connected to an anchor beam. The accelerations of the sheet pile wall, the anchor beam and the soil around the wall were measured using miniature piezoelectric accelerometers. The displacement at the tip of the wall was also measured. Stain gauges at five different locations on the wall were used to measure the bending moments induced in the the wall. The anchor forces in the tie rods were also measured using load cells. The results from the centrifuge tests were compared with 2-D, plane strain FE analyses conducted using DIANA-SWANDYNE II and the observed seismic behaviour was explained in the light of these findings. © 2011 Taylor & Francis.

A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.

Mouse Toxicity of Anabaena flos-aquae from Lake Dianchi, China

Some species of the genera Anabaena can produce various kinds of cyanotoxins, which may pose risks to environment and human health. Anabaena has frequently been observed in eutrophic freshwater of China in recent years, but its toxicity has been reported only in a few studies. In the present study, the toxicity of an Anabaena flos-aquae strain isolated from Lake Dianchi was investigated. Acute toxicity testing was performed by mouse bioassay using crude extracts from the lyophilized cultures. The mice exposed to crude extracts showed visible symptoms of toxicity and died within 10-24 h of the injection. Serum biochemical parameters were evaluated by the use of commercial diagnostic kits. Significant alterations were found in the serum biochemical parameters: alkaline phosphatase (AKP), gamma-glutamyl transpepticlase (gamma-GT), aspartate amino transferase (AST), alanine amino transferase (ALT), AST/ALT ratio, total protein content, albumin content, albumin/globulin (A/G) ratio, blood urea nitrogen (BUN), serum creatinine (Ssr), and total antioxidative capacity (T-AOC). Histopathological observations were carried out with hematoxylin and eosin (HE) stain under light microscope. Severe lesions were seen in the livers, kidneys, and lungs of the mice injected with crude extracts. The alterations of biochemical parameters were in a dose-dependent manner, and the severities of histological lesions were in the same manner. Based on biochemical and histological studies, this research firstly shows the presence of toxin-producing Anabaena species in Lake Dianchi and the toxic effects of its crude extracts on mammals. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 10-18, 2009.

Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate

The RNA helicase Vasa is a germ cell marker in animals, and its homolog in vertebrates to date has been limited to bisexual reproduction. We cloned and characterized CagVasa, a Vasa homolog from the gibel carp, a fish that reproduces bisexually or gynogenetically. CagVasa possesses 14 RGG repeats and eight conserved motifs of Vasa proteins. In bisexually reproducing gibel carp, vasa is maternally supplied and its zygotic expression is restricted to gonads. By in situ hybridization on testicular sections, vasa is low in spermatogonia, high in primary spermatocytes, reduced in secondary spermatocytes, but disappears in spermatids and sperm. In contrast, vasa persists throughout oogenesis, displaying low-high-low levels from oogonia over vitellogenic oocytes to maturing oocytes. A rabbit anti-Vasa antibody (alpha Vasa) was raised against the N-terminal CagVasa for fluorescent immunohistochemistry. On testicular sections, Vasa is the highest in spermatogonia, reduced in spermatocytes, low in spermatids, and absent in sperm. In the ovary, Vasa is the highest in oogonia but persists throughout oogenesis. Subcellular localization of vasa and its protein changes dynamically during oogenesis. The aVasa stains putative primordial germ cells in gibel carp fry. It detects gonadal germ cells also in several other teleosts. Therefore, Cagvasa encodes a Vasa ortholog that is differentially expressed in the testis and ovary. Interestingly, the alpha Vasa in combination with a nuclear dye can differentiate critical stages of spermatogenesis and oogenesis in fish. The cross-reactivity and the ability to stain stage-specific germ cells make this antibody a useful tool to identify fish germ cell development and differentiation. (c) 2005 Wiley-Liss, Inc.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

Evaluation of both composition and strain distributions in InGaN epitaxial film using x-ray diffraction techniques

The composition and stain distributions in the InGaN epitaxial films are jointly measured by employing various x-ray diffraction (XRD) techniques, including out-of-plane XRD at special planes, in-plane grazing incidence XRD, and reciprocal space mapping (RSM). It is confirmed that the measurement of (204) reflection allows a rapid access to estimate the composition without considering the influence of biaxial strain. The two-dimensional RSM checks composition and degree of strain relaxation jointly, revealing an inhomogeneous strain distribution profile along the growth direction. As the film thickness increases from 100 nm to 450 nm, the strain status of InGaN films gradually transfers from almost fully strained to fully relaxed state and then more in atoms incorporate into the film, while the near-interface region of InGaN films remains pseudomorphic to GaN.

Nano-size Effect of Interface Energy and Its Effect on Interface Fracture

An analytical model about size-dependent interface energy of metal/ceramic interfaces in nanoscale is developed by introducing both the chemical energy and the structure stain energy contributions. The dependence of interface energy on the interface thickness is determined by the melting enthalpy, the molar volume, and the shear modulus of two materials composing the interfaces, etc. The analytic prediction of the interface energy and the atomic scale simulation of the interface fracture strength are compared with each other for Ag/MgO and Ni/Al2O3 interfaces, the fracture strength of the interface with the lower chemical interface energy is found to be larger. The potential of Ag/MgO interface related to the interface energy is calculated, and the interface stress and the interface fracture strength are estimated further. The effect of the interface energy on the interface strength and the behind mechanism are discussed.

PVA降解菌的产酶条件及特性研究

目前对PVA生物降解研究重点逐渐转移到对PVA降解菌和PVA降解酶的研究开发上，随着对PVA降解高效新菌株的不断发现和PVA降解酶作用机理和分泌机制的深入了解，利用高效微生物或酶法治理PVA这类高聚物的污染将具有较大的应用潜力。本论文研究工作正是基于这种客观条件下进行的，对本实验室前期分离的PVA降解菌株P1、共生菌B1+B2、Pa、Pb为研究对象，重点研究了菌株P1和共生菌B1+B2的产酶条件和产酶特性，验证找出了影响菌株P1产酶的生长因子，论证了菌株B1+B2的产酶特性，优化得出了菌株B1+B2的最佳产酶条件；然后对共生菌B1+B2的PVA降解酶的稳定性进行了研究；最后研究了最佳组合菌的产酶特性和最佳产酶条件。主要研究结果如下： 1 通过对菌株P1产酶因子的研究，找出了核黄素是菌株P1产酶的必须因子，在以淀粉为碳源时，核黄素只是产酶的必须因子，而不是菌体生长的必须因子；在以PVA为碳源时，核黄素既是生长的必须因子，也是产酶的必须因子，是菌株P1的生长因子。 2 对共生菌B1+B2的产酶条件和产酶特性进行了研究，并通过正交实验找出了影响菌株产酶的主要条件和菌株产酶的最佳条件。 3 对共生菌PVA降解酶的稳定性进行了研究，确定了影响酶稳定性的主要理化条件。 4 通过对菌株降解性能的比较，确定菌株Pa、Pb、共生菌、P1的作为组合菌的组成菌，然后通过复配实验确定出菌株的最佳组合为菌株Pa、P1、共生菌，最后通过正交实验确定最佳组合菌的最佳配比。 5对影响组合菌产酶的因素进行了研究，通过正交实验确定了影响组合菌产酶的主要因素和最佳产酶条件。 本文通过对PVA降解菌株产酶条件和特性的研究，旨在为PVA降解菌生产酶制剂及进一步优化PVA降解菌在PVA废水治理中的应用提供理论和应用依据。 Now the PVA-degrading bacteria and polyvinyl alcohol-degrading enzyme are the key studies on the PVA biological degradation. It has great application potential using special bacteria and enzyme to treat pollution of PVA, with some high efficient Strain and enzyme were found. The study of this paper was based on that objective condition. The stain P1, symbiotic bacteria B1+B2, stain Pa and strain Pb were studied .The conditions of enzyme production and enzyme production characteristic of stain P1, symbiotic bacteria B1+B2 were our key study, we tested and verified the growth factor which effected enzyme production of strain P1, demonstrated enzyme production characteristic of symbiotic bacteria B1+B2, optimized and obtained the optimum conditions of enzyme production; then we studied the stability of polyvinyl alcohol-degrading enzyme of strain B1+B2; last the enzyme characteristic and the optimum conditions of alcohol-degrading enzyme production of optimum combination stains were studied. The main study results are below: 1. Through the study of enzyme production factor we found that lactoflavin is the necessary factor in strain P1 enzyme production. When we used starch to be carbon energy, lactoflavin is only the necessary factor of enzyme production, but not growth factor. When we used PVA to be carbon energy, lactoflavin was not only the necessary growth factor ,but also the necessary enzyme production factor.So it was the growth factor of strain P1 2. The enzyme production conditions and enzyme production characteristic of symbiotic bacteria B1+B2 were studied. Through the orthogonal experimental design, the main conditions which effected the enzyme production and the optimum conditions of enzyme production were obtained 3. Through the study of the stability of polyvinyl alcohol-degrading enzyme, the main physical and chemical conditions which effected the enzyme stability were 4. The stain P1,symbiotic bacteria B1+B2, stain Pa and strain Pb were selected to form combination bacteria. The stain P1,symbiotic bacteria B1+B2,stain Pa were the optimum combination through duplication experiment. Then the optimum ratio was obtained through orthogonal experiment. 5. Studied the factors which effected the polyvinyl alcohol-degrading enzyme activity, then through orthogonal experiment, the main factors and condition of enzyme production which effected the combination bacteria were achieved. The result of the study was valuable for the ferment of the PVA-degrading enzyme and the optimization of the PVA-degrading performance in the PVA wastewater.

Simultaneous enumeration of diatom, protozoa and meiobenthos from marine sediments using Ludox-QPS method

The Ludox-QPS method is a newly developed technique, which combines the Ludox HS 40 density centrifugation and quantitative protargol stain, to enumerate marine ciliates with good taxonomic resolution. We tested the method for simultaneous enumeration of diatoms, protozoa and meiobenthos and compared its extraction efficiency for meiobenthos with that of the routine Ludox-TM centrifugation and a modified protocol using Ludox HS 40. We conducted the evaluation with a sample size of 8.3 ml each from sandy, muddy-sand and muddy sediments collected from the intertidal area of the Yellow Sea in summer 2006 and spring 2007. The Ludox-QPS method not only produced high extraction efficiencies of 97 +/- 1.3% for diatoms and 97.6 +/- 0.8% for ciliates, indicating a reliable enumeration for eukaryotic microbenthos, but also produced excellent extraction efficiencies of on average 97.3% for total meiobenthos, 97.9% for nematodes and 97.8% for copepods from sands, muddy sands and mud. By contrast, the routine Ludox-TM centrifugation obtained only about 74% of total meiobenthos abundance with one extraction cycle, and the modified Ludox HS 40 centrifugation yielded on average 93% of total meiobenthos: 89.4 +/- 2.0% from sands, 93 +/- 4.1% from muddy sands and 97.1 +/- 3.0% from mud. Apart from the sediment type, sample volume was another important factor affecting the extraction efficiency for meiobenthos. The extraction rate was increased to about 96.4% when using the same modified Ludox centrifugation for a 4 ml sediment sample. Besides the excellent extraction efficiency, the Ludox-QPS method obtained higher abundances of meiobenthos, in particular nematodes, than the routine Ludox centrifugation, which frequently resulted in an uncertain loss of small meiobenthos during the sieving process. Statistical analyses demonstrated that there were no significant differences between the meiobenthos communities revealed by the Ludox-QPS method and the modified Ludox HS 40 centrifugation, showing the high efficiency of the Ludox-QPS method for simultaneous enumeration of diatom, protozoa and meiobenthos. Moreover, the comparatively high taxonomic resolution of the method, especially for diatoms and ciliates, makes it feasible to investigate microbial ecology at community level.

华南地壳岩石弹性和变形性质的高温高压实验研究

The South continent of China lies to southeast of Eurasia block. It is an active area from the view of crust growth and continent spread and is a transition zone between continental crust and oceanic crust. The compressional wave velocities and anisotropies of typical crustal metamorphic rocks were determined at high temperature (up to 1000 ℃) and high pressure(up to 800MPa). The experimental results show that the velocities generally increase with pressure, and is unaffected by temperature up to around 550 ℃. But the velocities of all experimental samples start to drop above a temperature point. For an example, this greatly reduce the speed of wave propagation in amphibolite and serpentinite above 760 ℃ and above 550 ℃ respectively, which may be due to dehydrate of amphibole and serpentine. P-wave anisotropy coefficients of those rocks range from 2% to 10% at 800MPa and 500 ℃. The anisotropies decrease with increasing pressure at room temperature, but hardly change as function of temperature at constant 800MPa or 600MPa pressure. The average velocity of the six crustal rocks is 6.28km/s under the condition of 800MPa and 550 ℃, which is consistent with the result of deep seismic sounding data. Based on this experimental result, we deduce there may exist a lot of felsic granulites and amphibolites at the depth of 15-25km underground. With increasing temperature and pressure, the deformation behavior of the rocks undergoes from localized brittle fracture, semi-brittle deformation (cataclastic flow or semi-brittle faulting, semi-brittle flow) to homogeneous crystal-plastic flow. This transition is associated with mechanical behavior and micro-mechanism. It is very important to understanding earthquake source mechanics, the strength of the lithosphere and the style of deformation. The experiments were conducted at temperature of 600-1000 ℃, confining pressure of 500MPa, and stain rates of 10~(-4)-10~(-6) S~(-1). For fine-grained natural amphibolite, the results of experiments show that brittle faulting is major failure mode at temperature <600 ℃, but crystal-plastic deformation is dominate at temperature >800 ℃, and there is a transition with increasing temperature from sembrittle faulting to cataclastic flow and sembrittle flow at temperature of 670-750 ℃. For medium-grained natural Felsic granulite, the results of experiments show that brittle faulting is major failure mode at temperature <500 ℃, but crystal-plastic deformation is dominate at temperature >700 ℃, and there is a transition with increasing temperature from semibrittle faulting to cataclastic flow and sembrittle flow at temperature of 500-600 ℃.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Caratterizzazione proteomica di fluidi e tessuti in diverse condizioni fisio-patologiche

This thesis has been focused on the proteomic characterization of human saliva from donors of different ages, starting from birth up to adult age, and pediatric brain tumor tissues. The first study has been performed in order to compare the acid-insoluble fraction of saliva from preterm with at-term newborns and adults and establish if differences exist. In the second study medulloblastoma and pilocytic astrocytoma pediatric brain tumor extracts have been compared. In both studies 2- DE analysis was coupled with high resolution tandem mass spectrometry (MS/MS). The proteomic characterization of the acid-insoluble fractions of saliva from preterm newborns allowed to integrate data previously obtained on the acid-soluble fraction by HPLC-electrospray ionization (ESI)-mass spectrometry (MS), and to evidence several differences between preterm newborns, at-term newborns and adults. Spots differentially expressed between the three groups, according to image analysis of the gels, were submitted to in-gel tryptic digestion and the peptide mixture analyzed by high performance HPLC-ESI-MS/MS for their characterization. By this strategy, we identified three over-expressed proteins in atterm newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, two proteoforms of annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100A6, S100A7, two proteoforms of S100A9, several proteoforms of prolactin-inducible protein, Ig kappa chain, two proteoforms of cystatin SN, one proteoform of cystatin S and several proteoforms of α-amylase 1). Moreover, for the first time, it was possible to assign by MS/MS four spots of human saliva 2-DE, already detected by other authors, to different proteoforms of S100A9. The strategy applied used a sequential staining protocol to the 2-DE gels, first with Pro-Q Diamond, that allows specific detection of phosphoproteins, and successively with total protein SYPRO Ruby stain. In the second study, proteomic analysis of two pediatric brain tumor tissues pointed out differences between medulloblastoma, the prevalent malignant tumor in childhood, and pilocytic astrocytoma, the most common, that only rarely shows a malignant progression. Due to the limited availability of bioptic tissue, the study was performed on pooled tumor tissues, and was focused on acid-insoluble fraction to integrate the characterization performed by a group of colleagues in Rome on the acid-soluble fraction by high performance HPLC-ESI-MS/MS. The results indicated that the two tumors exhibit different proteomic profiles and evidenced interesting differential expression of several proteins. Among them, peroxiredoxin- 1, peptidyl-prolyl cis–trans isomerase A, heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial isoform of malate dehydrogenase, nucleoside diphosphate kinase A, glutathione S-transferase P and fructose bisphosphate aldolase A resulted significantly over-expressed in medulloblastoma while glial fibrillary acidic protein, serotransferrin, α crystallin B chain, ferritin light chain, annexin A5, fatty acid-binding protein (brain), sorcin and apolipoprotein A-I resulted significantly over-expressed in pilocytic astrocytoma. In conclusion, the work done allowed to evidence the usefulness of using an integrated bottom-up/top-down approach, based on 2-DE-MS analysis and high performance MS in order to obtain a complete characterization of the proteome under investigation, revealing and identifying, not only peptides and small proteins, but also proteins with higher MW, that often it is not possible to identify by using exclusively a top-down ESI-MS approach.