Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Ultrasonic measurement technique of burning metals in normal and reduced gravity

The in situ real time measurement of the regression rate of a melting interface (RRMI) is performed by the ultrasonic measurement system reported here. The RRMI is the rate at which a solid/liquid interface (SLI) moves along a metallic rod while burning in an oxygen-enriched atmosphere and is an important flatnmability indicator. The ultrasonic transducer and associated equipment used to drive the transducer and record the echo signal is described, along with the process that transforms the acquired signals into a RRMI value. Test rods of various metals and geometric shapes were burned at several test conditions in different test facilities. The RRMI results with quantified errors are presented and reviewed. The effect of reduced gravity on burning metals is important to space-applications and RRMI results obtained in a reduced gravity environment are also presented.

In-situ measurement and the reconstruction in 3D of femtosecond inscription induced complex permittivity modification in glass

We demonstrate a new approach to in-situ measurement of femtosecond laser pulse induced changes in glass enabling the reconstruction in 3D of the induced complex permittivity modification. The technique can be used to provide single shot and time resolved quantitative measurements with a micron scale spatial resolution.

A method for the in-situ determination of the hydraulic conductivity of gravels as used in constructed wetlands for wastewater treatment

A new instrument and method are described that allow the hydraulic conductivities of highly permeable porous materials, such as gravels in constructed wetlands, to be determined in the field. The instrument consists of a Mariotte siphon and a submersible permeameter cell with manometer take-off tubes, to recreate in-situ the constant head permeameter test typically used with excavated samples. It allows permeability to be measured at different depths and positions over the wetland. Repeatability obtained at fixed positions was good (normalised standard deviation of 1–4%), and results obtained for highly homogenous silica sand compared well when the sand was retested in a lab permeameter (0.32 mm.s–1 and 0.31 mm.s–1 respectively). Practical results have a ±30% associated degree of uncertainty because of the mixed effect of natural variation in gravel core profiles, and interstitial clogging disruption during insertion of the tube into the gravel. This error is small, however, compared to the orders of magnitude spatial variations detected. The technique was used to survey the hydraulic conductivity profile of two constructed wetlands in the UK, aged 1 and 15 years respectively. Measured values were high (up to 900 mm.s –1) and varied by three orders of magnitude, reflecting the immaturity of the wetland. Detailed profiling of the younger system suggested the existence of preferential flow paths at a depth of 200 mm, corresponding to the transition between more coarse and less coarse gravel layers (6–12 mm and 3–6 mm respectively), and transverse drift towards the outlet.

A comparison of in situ constant and falling head permeameter tests to assess the distribution of clogging within horizontal subsurface flow constructed wetlands

Clogging is the main operational problem associated with horizontal subsurface flow constructed wetlands (HSSF CWs). The measurement of saturated hydraulic conductivity has proven to be a suitable technique to assess clogging within HSSF CWs. The vertical and horizontal distribution of hydraulic conductivity was assessed in two full-scale HSSF CWs by using two different in situ permeameter methods (falling head (FH) and constant head (CH) methods). Horizontal hydraulic conductivity profiles showed that both methods are correlated by a power function (FH= CH 0.7821, r 2=0.76) within the recorded range of hydraulic conductivities (0-70 m/day). However, the FH method provided lower values of hydraulic conductivity than the CH method (one to three times lower). Despite discrepancies between the magnitudes of reported readings, the relative distribution of clogging obtained via both methods was similar. Therefore, both methods are useful when exploring the general distribution of clogging and, specially, the assessment of clogged areas originated from preferential flow paths within full-scale HSSF CWs. Discrepancy between methods (either in magnitude and pattern) aroused from the vertical hydraulic conductivity profiles under highly clogged conditions. It is believed this can be attributed to procedural differences between the methods, such as the method of permeameter insertion (twisting versus hammering). Results from both methods suggest that clogging develops along the shortest distance between water input and output. Results also evidence that the design and maintenance of inlet distributors and outlet collectors appear to have a great influence on the pattern of clogging, and hence the asset lifetime of HSSF CWs. © Springer Science+Business Media B.V. 2011.

Redução in situ de NIO durante a sinterização de misturas NI/NIO produzidas por metalurgia do pó

The nickel alloys are widely used in the production of various materials, especially those that require mechanical strength characteristics associated with resistance to corrosion, for example, the stainless steel. Another use is the production of nickel alloy sintered from powder of metallic nickel. A promising alternative for the production of sintered components of nickel with an important reduction in costs of starting material is the use of mixtures of powders of Ni-NiO. This work aimed to study in situ reduction of NiO during sintering mixtures of Ni / NiO produced by powder metallurgy. The nickel mixtures have been processed by the technique of powder metallurgy and were pre-sintered in an oven under plasma reducing atmosphere of hydrogen. Mixtures Ni +15%NiO, Ni +25%NiO and Ni +35%NiO were studied and compared with samples consisting only of metallic Ni. Dilatometric tests were performed to study the sintering conditions of the mixtures. The consolidated material was analyzed for their microstructure and microhardness. Dilatometry graphs showed that the addition of nickel oxide in all compositions the active sintering the mixtures studied. In tests of microhardness indentations were made at different points of the sample surface. All compositions showed microhardness values close to the consolidated material from metallic nickel. However, sample containing Ni+35% NiO, showed a large dispersion of the values of microhardness tests performed at different points of the sample surface. Microstructural analysis of the material showed a higher concentration of voids and the presence of oxides in the waste composition of the mixtures Ni 35% NiO. The samples containing Ni+15%NiO showed microstructural characteristics and mechanical properties similar to metallic nickel consolidated under the same conditions of the compositions studied in this work and therefore had great potential for production of sintered nickel alloys

Análise comparativa de diferentes configurações de poços no processo de combustão in-situ

With an increasing number of mature fields, heavy oil recovery has performed one of the great challenges of the oil industry. The Brazilian Northeast, for example, has numerous heavy oil reservoirs are explored with the use of thermal methods. Among the types of methods used for heavy oil, there is the method of in-situ combustion, a technique in which heat is produced within the container, unlike the injection of heated fluid when the heat is generated at the surface and transported to the reservoir. In this type of process, it is common to use vertical wells as injectors and producers. However, methods which use horizontal wells like oil producers are increasingly studied because of greater contact area between the formation and combustion front. Thus, the main objective of this work was to study the different configurations of wells (CIS THAITM and CAGD) in the process of in-situ combustion in oil recovery using a semi-synthetic tank with Brazilian Northeast features. The method "toe-to-heel air injection" (THAITM) is a process of enhanced oil recovery, which is the integration of in-situ combustion with technological advances in drilling horizontal wells. This method uses horizontal wells such as oil producers, keeping vertical injection wells for injecting air. The oil drain process by differential gravitational assisted with combustion (CAGD) is an integrated, in this configuration the horizontal injector well is drilled at the top formation with a horizontal production well in the lower section. The simulations were performed in a commercial program of thermal processes, called "STARS" (Steam, Thermal, and Advanced Processes Reservoir Simulator), the company CMG (Computer Modelling Group). An analysis of the air flow injection was performed and it was found that each method had a maximum injection to the base model, a show that through this air injection limit was reduced cumulative production of oil. Analyses of operating parameters were used: injection flow, configuration and completion of wells. In the sensitivity analysis we found that the air injection flow showed greater influence on THAI method, since the CIS method the completion of the wells was the most influential parameter and CAGD configuration wells showed the greatest influence in the recovered fraction. The economic results have shown that the best case obtained in CAGD method because, despite having higher initial cost showed the best financial return compared to the best cases the CIS and THAI.

Análise comparativa de diferentes configurações de poços no processo de combustão in-situ

With an increasing number of mature fields, heavy oil recovery has performed one of the great challenges of the oil industry. The Brazilian Northeast, for example, has numerous heavy oil reservoirs are explored with the use of thermal methods. Among the types of methods used for heavy oil, there is the method of in-situ combustion, a technique in which heat is produced within the container, unlike the injection of heated fluid when the heat is generated at the surface and transported to the reservoir. In this type of process, it is common to use vertical wells as injectors and producers. However, methods which use horizontal wells like oil producers are increasingly studied because of greater contact area between the formation and combustion front. Thus, the main objective of this work was to study the different configurations of wells (CIS THAITM and CAGD) in the process of in-situ combustion in oil recovery using a semi-synthetic tank with Brazilian Northeast features. The method "toe-to-heel air injection" (THAITM) is a process of enhanced oil recovery, which is the integration of in-situ combustion with technological advances in drilling horizontal wells. This method uses horizontal wells such as oil producers, keeping vertical injection wells for injecting air. The oil drain process by differential gravitational assisted with combustion (CAGD) is an integrated, in this configuration the horizontal injector well is drilled at the top formation with a horizontal production well in the lower section. The simulations were performed in a commercial program of thermal processes, called "STARS" (Steam, Thermal, and Advanced Processes Reservoir Simulator), the company CMG (Computer Modelling Group). An analysis of the air flow injection was performed and it was found that each method had a maximum injection to the base model, a show that through this air injection limit was reduced cumulative production of oil. Analyses of operating parameters were used: injection flow, configuration and completion of wells. In the sensitivity analysis we found that the air injection flow showed greater influence on THAI method, since the CIS method the completion of the wells was the most influential parameter and CAGD configuration wells showed the greatest influence in the recovered fraction. The economic results have shown that the best case obtained in CAGD method because, despite having higher initial cost showed the best financial return compared to the best cases the CIS and THAI.

Controlling Nanostructures for in-situ TEM Characterization

Low dimensional nanostructures, such as nanotubes and 2D sheets, have unique and promising material properties both from a fundamental science and an application standpoint. Theoretical modelling and calculations predict previously unobserved phenomena that experimental scientists often struggle to reproduce because of the difficulty in controlling and characterizing the small structures under real-world constraints. The goal of this dissertation is to controlling these structures so that nanostructures can be characterized in-situ in transmission electron microscopes (TEM) allowing for direct observation of the actual physical responses of the materials to different stimuli. Of most interest to this work are the thermal and electrical properties of carbon nanotubes, boron nitride nanotubes, and graphene. The first topic of the dissertation is using surfactants for aqueous processing to fabricate, store, and deposit the nanostructures. More specifically, thorough characterization of a new surfactant, ammonium laurate (AL), is provided and shows that this new surfactant outperforms the standard surfactant for these materials, sodium dodecyl sulfate (SDS), in almost all tested metrics. New experimental set-ups have been developed by combining specialized in-situ TEM holders with innovative device fabrication. For example, electrical characterization of graphene was performed by using an STM-TEM holder and depositing graphene from aqueous solutions onto lithographically patterned, electron transparent silicon nitride membranes. These experiments produce exciting information about the interaction between graphene and metal probes and the substrate that it rests on. Then, by adding indium to the backside of the membrane and employing the electron thermal microscopy (EThM) technique, the same type of graphene samples could be characterized for thermal transport with high spatial resolution. It is found that reduced graphene oxide sheets deposited onto a silicon nitride membrane and displaying high levels of wrinkling have higher than expected electrical and thermal conduction properties. We are clearly able to visualize the ability of graphene to spread heat away from an electronic hot spot and into the substrate.

In situ characterization of sediment oxygen demand across the spectrum of non-resuspension to full resuspension with varying bed shear stress

Sediment oxygen demand (SOD) can be a significant oxygen sink in various types of water bodies, particularly slow-moving waters with substantial organic sediment accumulation. In most settings where SOD is a concern, the prevailing hydraulic conditions are such that the impact of sediment resuspension on SOD is not considered. However, in the case of Bubbly Creek in Chicago, Illinois, the prevailing slack water conditions are interrupted by infrequent intervals of very high flow rates associated with pumped combined sewer overflow (CSO) during intense hydrologic events. These events can cause resuspension of the highly organic, nutrient-rich bottom sediments, resulting in precipitous drawdown of dissolved oxygen (DO) in the water column. While many past studies have addressed the dependence of SOD on near-bed velocity and bed shear stress prior to the point of sediment resuspension, there has been limited research that has attempted to characterize the complex and dynamic phenomenon of resuspended-sediment oxygen demand. To address this issue, a new in situ experimental apparatus referred to as the U of I Hydrodynamic SOD Sampler was designed to achieve a broad range of velocities and associated bed shear stresses. This allowed SOD to be analyzed across the spectrum of no sediment resuspension associated with low velocity/ bed shear stress through full sediment resuspension associated with high velocity / bed shear stress. The current study split SOD into two separate components: (1) SODNR is the sediment oxygen demand associated with non-resuspension conditions and is a surface sink calculated using traditional methods to yield a value with units (g/m2/day); and (2) SODR is the oxygen demand associated with resuspension conditions, which is a volumetric sink most accurately characterized using non-traditional methods and units that reflect suspension in the water column (mg/L/day). In the case of resuspension, the suspended sediment concentration was analyzed as a function of bed shear stress, and a formulation was developed to characterize SODR as a function of suspended sediment concentration in a form similar to first-order biochemical oxygen demand (BOD) kinetics with Monod DO term. The results obtained are intended to be implemented into a numerical model containing hydrodynamic, sediment transport, and water quality components to yield oxygen demand varying in both space and time for specific flow events. Such implementation will allow evaluation of proposed Bubbly Creek water quality improvement alternatives which take into account the impact of SOD under various flow conditions. Although the findings were based on experiments specific to the conditions in Bubbly Creek, the techniques and formulations developed in this study should be applicable to similar sites.

Comparison of Intraocular Pressure before and after Laser In Situ Keratomileusis Refractive Surgery Measured with Perkins Tonometry, Noncontact Tonometry, and Transpalpebral Tonometry

Purpose. To compare the intraocular pressure (IOP) before and after Laser In Situ Keratomileusis (LASIK), measured by Diaton, Perkins, and noncontact air pulse tonometers. Methods. Fifty-seven patients with a mean age of 34.88 were scheduled for myopia LASIK treatment. Spherical equivalent refraction (SER), corneal curvature (K), and central corneal thickness (CCT) and superior corneal thickness (SCT) were obtained before and after LASIK surgery. IOP values before and after surgery were measured using Diaton, Perkins, and noncontact air pulse tonometers. Results. The IOP values before and after LASIK surgery using Perkins tonometer and air tonometers were statistically significant (). However, no significant differences were found () for IOP values measured with Diaton tonometer. CCT decreases significantly after surgery () but no statistical differences were found in SCT (). Correlations between pre- and postsurgery were found for all tonometers used, with and for the air pulse tonometer, and for Perkins, and and for Diaton. Conclusion. Transpalpebral tonometry may be useful for measuring postsurgery IOP after myopic LASIK ablation because this technique is not influenced by the treatment.

Hibridación in situ fluorescente (FISH)

Background: At the end of 80s, cloning technologies with the increase of the antibodies’ sensibility made easier the development of technologies based on Fluorescence in situ Hibridation (FISH). Nowadays, It’s widely used in the field of basic investigation as much as clinic diagnostic. Method: FISH is a technique that combines molecular biology with histochemistry way to detect specific nucleotide sequences so that chromosome’s section or even whole chromosome can be marked on metaphases cells (cell in division) and on attached cellular nucleus. This detection is realized using DNA fluorescence probes (marked with fluorophores), that can be different according to the structures manage to detect: large single-locus probes, small unique-sequence probes, chromosome- or region-specific “paints” or repetitive sequence probes and genomic DNA probes. Some of the applications of this technique is that can be so useful in the detection of numerical and structural chromosomal alterations such as polyploidies or genomic rearrangement, to mapping metaphases cells and even to detect bacteria or another type of microorganism. In addition, FISH allows us to monitoring diseases (antitumor therapies, quantification of genomic altered cells…) and the precise location of chromosomic broken spots on tumor searching for new genes involved in cancer and detect and map interested known genes. Conclusion: FISH has many advantages ahead of conventional cytogenetic techniques (bands G karyotype) overall at the time of establish a clinic diagnostic to detect tumors and chromosomic aberration, presenting a higher sensibility and specificity as well as being a relative quick technique (24 hours).

Construção de um aparato experimental para monitoramento in situ da deposição de filmes finos de titânio por magnetron sputtering

The technique of surface coating using magnetron sputtering is one of the most widely used in the surface engineering, for its versatility in obtaining different films as well as in the micro / nanometric thickness control. Among the various process parameters, those related to the active species of the plasma are of the most fundamental importance in the mechanism and kinetics of deposition. In order to identify the active species of the plasma, parameters such as gas flow, pressure and density of electric power were varied during titanium coating on glass substrate. By flowing argon gas of 10, 20, 30, 40 and 50 sccm (cubic centimeters per minute) for each gas flow a sequential scan of the electric current of 0.10, 0.20, 0.30, 0.40 , 0.50 A. The maximum value of 0.50 A was chosen based both on literature data and on limitations of the equipment. The monitoring of plasma species present during the deposition was carried out in situ by the technique of optical emission spectroscopy (OES) through the spectrometer Ocean Optics USB2000 Series. For this purpose, an apparatus was developed to adapt the OES inside the plasma reactor to stay positioned closest to the target. The radiations emitted by the species were detected by an optical fiber placed behind the glass substrate and their intensities as a function of wavelength were, displayed on a monitor screen. The acquisition time for each condition of the plain parameters was related to the minima of spectral lines intensities due to the film formed on the substrate. The intensities of different emission lines of argon and titanium were then analyzed as a function of time, to determine the active species and estimate the thickness of the deposited films. After the deposition, the coated glasses thin films were characterized by optical transmittance through an infrared laser. It was found that the thickness and deposition rate determined by in situ analysis were consistent with the results obtained by laser transmittance