p53-dependent growth arrest of REF52 cells containing newly amplified DNA.

The rat cell line REF52 is not permissive for gene amplification. Simian virus 40 tumor (T) antigen converts these cells to a permissive state, as do dominant negative mutants of p53, suggesting that the effect of T antigen is due mainly to its ability to bind to p53. To manipulate permissivity, we introduced a temperature-sensitive mutant of T antigen (tsA58) into REF52 cells and selected for resistance to N-(phosphonacetyl)-L-aspartate (PALA). Most freshly isolated PALA-resistant colonies, each of approximately 200 cells, selected at a permissive temperature, arrested when shifted to a nonpermissive temperature. Growth arrest was stable, with no evidence of apoptosis, as long as T antigen was absent but was reversed when T antigen was restored. In contrast, PALA-resistant clones grown to approximately 10(7) cells at a permissive temperature did not arrest when shifted to a nonpermissive temperature. All PALA-resistant clones examined had amplified carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) genes, present in structures consistent with a mechanism involving bridge-breakage-fusion (BBF) cycles. We propose that p53-mediated growth arrest operates only early during the complex process of gene amplification, when newly formed PALA-resistant cells contain broken DNA, generated in BBF cycles. During propagation under permissive conditions, the broken DNA ends are healed, and, even though the p53-mediated pathway is still intact at a nonpermissive temperature and the cells contain amplified DNA, they are not arrested in the absence of broken DNA. The data support the hypothesis that BBF cycles are an important mechanism of amplification and that the broken DNA generated in each cycle is a key signal that regulates permissivity for gene amplification.

Complexos de dirutênio com os fármacos ibuprofeno, ácido acetilsalicílico, naproxeno, indometacina, e com ácido y-linolêncio: Síntese, caracterização, avaliação da ação antiproliferativa sobre células tumorais e estudo da interação da unidade dimetálica com adenina e adenosina

Este trabalho tem como principal objetivo contribuir para o desenvolvimento de novos potenciais metalofármacos de rutênio. Nele são descritas a síntese, a caracterização e a avaliação da ação antiproliferativa de alguns complexos de dirutênio (II,III) com os fármacos antiinflamatórios não-esteróides (AINEs): ibuprofeno (ibp), ácido acetilsalicílico (aas), naproxeno (npx) e indometacina (ind) e também com o ácido γ-linolênico (lin), sobre células cancerígenas. Os compostos obtidos foram caracterizados por análise elementar, espectroscopia de absorção eletrônica, medidas de susceptibilidade magnética, espectroscopia vibracional FTIR e Raman, difratometria de raios X de pó, medidas de condutividade molar e análise térmica (TG, OTAe OSC). Todos os complexos sintetizados apresentam estrutura em gaiola, com os carboxilatos derivados dos fármacos AINEs coordenados à unidade dimetálica Ru2( (II,III), em ponte equatorial, estabilizando assim a ligação direta rutênio-rutênio. As posições axiais são ocupadas por íons cloreto, no caso dos complexos [Ru2(O2(CR)4(Cl] (O2(CR = ibp, aas, npx ou ind), ou por moléculas de água, nas espécies do tipo [Ru2(O2(CR)4(H2O)2]PF6(O2CR =npx e ind). Ensaios biológicos demonstraram que os compostos [Ru2(ibp) 4Cl]•½H2O e [Ru2(npx)4(H2O)2]PF6 apresentam ação antiproliferativa sobre células de glioma de rato C6 in vitro, dependendo do tempo de exposição do meio celular ao complexo. O complexo [Ru2 (lin)4Cl] também apresenta efeito sobre a proliferação de células C6; entretanto, nesse caso, efeitos significativos são observaçlos já nas primeiras 24 h de exposição. Estudos mostraram que as bases adenina e adenosina reagem com o complexo [Ru2(OAc)4(H2O)2]PF6 sem que ocorra quebra da estrutura em gaiola. As bases nitrogenadas substituem axialmente as moléculas de água, formando pontes axiais entre duas unidades de dirutênio (II,III) no estado sólido.

The Effects of Molecular Chaperones on Tau Fibril Assembly

The accumulation of microtubule-associated protein tau into fibrillar aggregates is the hallmark of Alzheimer’s disease and other neurodegenerative disorders, collectively referred to as tauopathies. Fibrils can propagate from one cell to the next and spread throughout the brain. However, a study shows that only small aggregates can be taken up by cultured neuronal cells. The mechanisms that lead to the breakage of fibrils into smaller fragments remain unknown. In yeast, the AAA+ chaperone HSP104 processes the reactivation of protein aggregates and is responsible for fragmentation of fibrils. This study focused on investigating the effects of molecular chaperones on tau fibrils and using HSP104 as a model system to test whether we can monitor fibril fracturing. The assays used to detect the chaperone’s actions on tau utilized acrylodan fluorescence, thioflavin T fluorescence, and sedimentation. Tau fibrils were either formed with a cofactor, heparin, to accelerate assembly or without a cofactor. In the process of investigating the effects of HSP104 on tau fibrils, this study established an assay to determine the effects of breakage on the seeding properties of tau fibrils. Our findings demonstrated that the sonication of tau fibrils produces smaller fragments (seeds) that accelerate the conversion of monomeric tau into fibrils. The use of this assay with HSP104 provided evidence that HSP104 inhibits the elongation of tau fibrils. Indeed, HSP104 inhibits the aggregation of soluble tau into aggregates. However, tau fibril breakage and dissociation were not observed with HSP104, either alone or in combination with co-chaperones (HSP70 and HSP40). Our findings provide insights into the seeding properties of tau fibrils, and suggest that fragmentation is a critical part of tau assembly. This knowledge should be valuable for understanding tau fibril aggregation and propagation in the brain, which is necessary to identify new treatments for neurodegenerative diseases.

Centromere identification and inactivation on a neo-Y chromosome fusion in threespine stickleback fish (Gasterosteus aculeatus)

Thesis (Ph.D.)--University of Washington, 2016-05

Modelling the effect of shear history on activated sludge flocculation

The aim of this paper is to investigate the effect of shear history on activated sludge flocculation dynamics and to model the observed relationships using population balances. Activated sludge flocs are exposed to dramatic changes in the shear rate within the treatment process, as they pass through localised high and low mixing intensities within the aeration basin and are cycled through the different unit operations of the treatment process. We will show that shear history is a key factor in determining floc size, and that the floc size varies irreversibly with changes in shear rate. A population balance model of the flocculation process is also introduced and evaluated.

A liberation model for comminution based on probability theory

Mineral processing plants use two main processes; these are comminution and separation. The objective of the comminution process is to break complex particles consisting of numerous minerals into smaller simpler particles where individual particles consist primarily of only one mineral. The process in which the mineral composition distribution in particles changes due to breakage is called 'liberation'. The purpose of separation is to separate particles consisting of valuable mineral from those containing nonvaluable mineral. The energy required to break particles to fine sizes is expensive, and therefore the mineral processing engineer must design the circuit so that the breakage of liberated particles is reduced in favour of breaking composite particles. In order to effectively optimize a circuit through simulation it is necessary to predict how the mineral composition distributions change due to comminution. Such a model is called a 'liberation model for comminution'. It was generally considered that such a model should incorporate information about the ore, such as the texture. However, the relationship between the feed and product particles can be estimated using a probability method, with the probability being defined as the probability that a feed particle of a particular composition and size will form a particular product particle of a particular size and composition. The model is based on maximizing the entropy of the probability subject to mass constraints and composition constraint. Not only does this methodology allow a liberation model to be developed for binary particles, but also for particles consisting of many minerals. Results from applying the model to real plant ore are presented. A laboratory ball mill was used to break particles. The results from this experiment were used to estimate the kernel which represents the relationship between parent and progeny particles. A second feed, consisting primarily of heavy particles subsampled from the main ore was then ground through the same mill. The results from the first experiment were used to predict the product of the second experiment. The agreement between the predicted results and the actual results are very good. It is therefore recommended that more extensive validation is needed to fully evaluate the substance of the method. (C) 2003 Elsevier Ltd. All rights reserved.

Mathematical modelling

The development of a new process model of cement grinding in two-stage mills is discussed. The new model has been used to simulate cement grinding and predicting mill performance in open and closed circuit configuration. The new model considered the two-compartment mill as perfectly mixed slices in series. The breakage rate function uses the back calculation technique to determine offline using drop weight and abrasion tests.

Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein (similar to350 kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function. (C) 2003 Elsevier B.V. All rights reserved.

Simple texture-based liberation modelling of ores

Mineralogical analysis is often used to assess the liberation properties of particles. A direct method of estimating liberation is to actually break particles and then directly obtain liberation information from applying mineralogical analysis to each size-class of the product. Another technique is to artificially apply random breakage to the feed particle sections to estimate the resultant distribution of product particle sections. This technique provides a useful alternative estimation method. Because this technique is applied to particle sections, the actual liberation properties for particles can only be estimated by applying stereological correction. A recent stereological technique has been developed that allows the discrepancy between the linear intercept composition distribution and the particle section composition distribution to be used as guide for estimating the particle composition distribution. The paper will show results validating this new technique using numerical simulation. (C) 2004 Elsevier Ltd. All rights reserved.

Gelatinisation of starch in mixtures of sugars. II. Application of differential scanning calorimetry

Differential scanning calorimetry was used to investigate the effect of mixtures of glucose and fructose, and five types of honeys on starch gelatinisation. At a 1:1 starch:water ratio, glucose generally increased the enthalpy (DeltaH(gel)) and temperatures (T-onset, T-peak and T-end) of gelatinisation more than fructose. Upon mixing, DeltaH(gel) of the low-temperature endotherm decreased in comparison to the sole sugars, but was fairly constant (7.7 +/- 0.33 J/g dry starch). DeltaH(gel) of the high-temperature endotherm increased with the fructose content. For both endotherms, the gelatinisation temperatures were unchanged (CV less than or equal to 3%) for the mixtures. With the honeys (moisture, 14.9-18.0%; fructose, 37.2-44.0%; glucose, 28.3-31.9%) added at 1.1-4.4 g per g dry starch, the enthalpy and temperatures of gelatinisation did not vary significantly (CV less than or equal to 6%). Typical thermograms are presented, and the results are interpreted in the light of the various proposed mechanisms for starch gelatinisation in sugar-water systems, total sugar content and possible sugar-sugar interactions. The thermograms were broader in the presence of the sugars and honeys, and a biphasic character was consistently exhibited. The application of an exponential equation to the gelatinisation temperatures of the starch-honey mixtures revealed an opposing influence of fructose and glucose during gelatinisation. The mechanism of starch gelatinisation may be better understood if techniques could be perfected to quantify breakage and formation of hydrogen bonds in the starch granules, and suggested techniques are discussed. (C) 2004 Elsevier Ltd. All rights reserved.

Analysis of stirred mill performance using DEM simulation: Part 1 - Media motion, energy consumption and collisional environment

Stirred mills are becoming increasingly used for fine and ultra-fine grinding. This technology is still poorly understood when used in the mineral processing context. This makes process optimisation of such devices problematic. 3D DEM simulations of the flow of grinding media in pilot scale tower mills and pin mills are carried out in order to investigate the relative performance of these stirred mills. Media flow patterns and energy absorption rates and distributions are analysed here. In the second part of this paper, coherent flow structures, equipment wear and mixing and transport efficiency are analysed. (C) 2006 Published by Elsevier Ltd.

Modelling comminution patterns within a pilot scale AG/SAG mill

The patterns of rock comminution within tumbling mills, as well as the nature of forces, are of significant practical importance. Discrete element modelling (DEM) has been used to analyse the pattern of specific energy applied to rock, in terms of spatial distribution within a pilot AG/SAG mill. We also analysed in some detail the nature of the forces, which may result in rock comminution. In order to examine the distribution of energy applied within the mill, the DEM models were compared with measured particle mass losses, in small scale AG and SAG mill experiments. The intensity of contact stresses was estimated using the Hertz theory of elastic contacts. The results indicate that in the case of the AG mill, the highest intensity stresses and strains are likely to occur deep within the charge, and close to the base. This effect is probably more pronounced for large AG mills. In the SAG mill case, the impacts of the steel balls on the surface of the charge are likely to be the most potent. In both cases, the spatial pattern of medium-to-high energy collisions is affected by the rotational speed of the mill. Based on an assumed damage threshold for rock, in terms of specific energy introduced per single collision, the spatial pattern of productive collisions within each charge was estimated and compared with rates of mass loss. We also investigated the nature of the comminution process within AG vs. SAG mill, in order to explain the observed differences in energy utilisation efficiency, between two types of milling. All experiments were performed using a laboratory scale mill of 1.19 m diameter and 0.31 m length, equipped with 14 square section lifters of height 40 mm. (C) 2006 Elsevier Ltd. All rights reserved.

Kinetic study of the thermal degradation of high density polyethylene

The thermal degradation of high density polyethylene has been modelled by the random breakage of polymer bonds, using a set of population balance equations. A model was proposed in which the population balances were lumped into representative sizes so that the experimentally determined molecular weight distribution of the original polymer could be used as the initial condition. This model was then compared to two different cases of the unlumped population balance which assumed unimolecular initial distributions of 100 and 500 monomer units, respectively. The model that utilised the experimentally determined molecular weight distribution was found to best describe the experimental data. The model fits suggested a second mechanism in addition to random breakage at slow reaction rates. (c) 2005 Elsevier Ltd. All rights reserved.

The role of advanced DEM based modelling tools in increasing comminution energy efficiency

Particle breakage is an essential part of mineral processing. The aim is to reduce run of mine mineral ore to an optimal size for liberating target minerals and for subsequent recovery by separation processes such as flotation. This size reduction is typically accomplished in a series of stages in a grinding circuit tailored to the properties of the particular mine ore. Commonly this involves two or more classes of equipment starting with crushers, followed by SAG mills and then sometimes ball mills. Occasionally, high pressure grinding rolls or other novel devices are substituted. Broadly, energy consumption increases and energy efficiency decreases with the fineness of the material produced by each piece of equipment.

Genotoxicity investigation of chlorinated degradation products of a cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN), a potent cyanobacterial hepatotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies in many parts of the world and has been associated with the intoxication of humans and livestock.Water treatment via chlorination can degrade the toxin effectively but result in the production of several byproducts. In this study, male and female Balb/c mice were injected via the intraperitoneal (IP) route with a single dose of 10 mg/kg 5-chlorouracil and 10 mg/kg 5-chloro-6-hydroxymethyluracil; these two compounds are the predicted chlorinated degradation products of CYN.DNA was isolated from the mouse livers and examined for strand breakage by alkaline gel electrophoresis (pH 12). The median molecular length (MML) of the DNA distributed in the gel was determined by estimating the midpoint of the DNA size distribution by densitometry. The toxicity of 5-chlorouracil (as measured by DNA strand breakage) was significantly influenced by time from dosing. There was no significant difference in MML between mice dosed with 5-chloro-6-hydroxymethyluracil and the controls. In another experiment, mice were dosed with 0, 0.1, 1, 10 and 100 mg/kg body weight 5-chlorouracil and 0, 0.1, 1, 10 and 20 mg/kg 5-chloro-6-hydroxymethyluracil via IP injection. The heart, liver, kidney, lung and spleen were removed, fixed and examined under electron microscopy. Liver was the main target organ. The EM results revealed marked distortion on the nuclear membrane of liver cells in mice dosed with 1.0 mg/kg 5-chlorouracil or 10 mg/kg 5-chloro-6-hydroxymethyluracil, or higher.