Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis sigma/anti-sigma factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the sigma factor/anti-sigma factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of sigma factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems. (C) 2010 Elsevier Inc. All rights reserved.

Therapeutic work with the present moment: A conversation analytical study of guidance into immediacy

Therapeutic work with the client’s present moment experience in existential therapy was studied by means of conversation analysis. Using publicly available video recordings of therapy sessions as data, an existential therapist’s practice of guiding a client into immediacy, or refocusing the talk on a client’s immediate experience, was described and compared with a therapist’s corresponding action in cognitive therapy. The study contributes to the description of interactional practice of existential therapy, and involves the first application of conversation analysis to a comparative study of psychotherapy process. The potential utility of this approach and the clinical and empirical implications of the present findings are discussed.

An algebraic formulation of static isotropy and design of statically isotropic 6–6 Stewart platform manipulators

In this paper, we present an algebraic method to study and design spatial parallel manipulators that demonstrate isotropy in the force and moment distributions. We use the force and moment transformation matrices separately, and derive conditions for their isotropy individually as well as in combination. The isotropy conditions are derived in closed-form in terms of the invariants of the quadratic forms associated with these matrices. The formulation is applied to a class of Stewart platform manipulator, and a multi-parameter family of isotropic manipulators is identified analytically. We show that it is impossible to obtain a spatially isotropic configuration within this family. We also compute the isotropic configurations of an existing manipulator and demonstrate a procedure for designing the manipulator for isotropy at a given configuration.

Change detection and controlling forest information using multi-temporal Landsat TM imagery.

A method was developed for relative radiometric calibration of single multitemporal Landsat TM image, several multitemporal images covering each others, and several multitemporal images covering different geographic locations. The radiometricly calibrated difference images were used for detecting rapid changes on forest stands. The nonparametric Kernel method was applied for change detection. The accuracy of the change detection was estimated by inspecting the image analysis results in field. The change classification was applied for controlling the quality of the continuously updated forest stand information. The aim was to ensure that all the manmade changes and any forest damages were correctly updated including the attribute and stand delineation information. The image analysis results were compared with the registered treatments and the stand information base. The stands with discrepancies between these two information sources were recommended to be field inspected.

The challenge of LC/MS/MS multi-mycotoxin analysis - Heracles battling the Hydra?

Mycotoxins are secondary metabolites of filamentous fungi. They pose a health risk to humans and animals due to their harmful biological properties and common occurrence in food and feed. Liquid chromatography/mass spectrometry (LC/MS) has gained popularity in the trace analysis of food contaminants. In this study, the applicability of the technique was evaluated in multi-residue methods of mycotoxins aiming at simultaneous detection of chemically diverse compounds. Methods were developed for rapid determination of toxins produced by fungal genera of Aspergillus, Fusarium, Penicillium and Claviceps from cheese, cereal based agar matrices and grains. Analytes were extracted from these matrices with organic solvents. Minimal sample clean-up was carried out before the analysis of the mycotoxins with reversed phase LC coupled to tandem MS (MS/MS). The methods were validated and applied for investigating mycotoxins in cheese and ergot alkaloid occurrence in Finnish grains. Additionally, the toxin production of two Fusarium species predominant in northern Europe was studied. Nine mycotoxins could be determined from cheese with the method developed. The limits of quantification (LOQ) allowed the quantification at concentrations varying from 0.6 to 5.0 µg/kg. The recoveries ranged between 96 and 143 %, and the within-day repeatability (as relative standard deviation, RSDr) between 2.3 and 12.1 %. Roquefortine C and mycophenolic acid could be detected at levels of 300 up to 12000 µg/kg in the mould cheese samples analysed. A total of 29 or 31 toxins could be analysed with the method developed for agar matrices and grains, with the LOQs ranging overall from 0.1 to 1250 µg/kg. The recoveries ranged generally between 44 and 139 %, and the RSDr between 2.0 and 38 %. Type-A trichothecenes and beauvericin were determined from the cereal based agar and grain cultures of F. sporotrichioides and F. langsethiae. T-2 toxin was the main metabolite, the average levels reaching 22000 µg/kg in the grain cultures after 28 days of incubation. The method developed for ten ergot alkaloids from grains allowed their quantification at levels varying from 0.01 to 10 µg/kg. The recoveries ranged from 51 to 139 %, and the RSDr from 0.6 to 13.9 %. Ergot alkaloids were measured in barley and rye at average levels of 59 and 720 µg/kg, respectively. The two most prevalent alkaloids were ergocornine and ergocristine. The LC/MS methods developed enabled rapid detection of mycotoxins in such applications where several toxins co-occurred. Generally, the performance of the methods was good, allowing reliable analysis of the mycotoxins of interest with sufficiently low quantification limits. However, the variation in validation results highlighted the challenges related to optimising this type of multi-residue methods. New data was obtained about the occurrence of mycotoxins in mould cheeses and of ergot alkaloids in Finnish grains. In addition, the study revealed the high mycotoxin-producing potential of two common fungi in Finnish crops. The information can be useful when risks related to fungal and mycotoxin contamination will be assessed.

Computer simulation of multi-elemental fusion reactor materials

Thermonuclear fusion is a sustainable energy solution, in which energy is produced using similar processes as in the sun. In this technology hydrogen isotopes are fused to gain energy and consequently to produce electricity. In a fusion reactor hydrogen isotopes are confined by magnetic fields as ionized gas, the plasma. Since the core plasma is millions of degrees hot, there are special needs for the plasma-facing materials. Moreover, in the plasma the fusion of hydrogen isotopes leads to the production of high energetic neutrons which sets demanding abilities for the structural materials of the reactor. This thesis investigates the irradiation response of materials to be used in future fusion reactors. Interactions of the plasma with the reactor wall leads to the removal of surface atoms, migration of them, and formation of co-deposited layers such as tungsten carbide. Sputtering of tungsten carbide and deuterium trapping in tungsten carbide was investigated in this thesis. As the second topic the primary interaction of the neutrons in the structural material steel was examined. As model materials for steel iron chromium and iron nickel were used. This study was performed theoretically by the means of computer simulations on the atomic level. In contrast to previous studies in the field, in which simulations were limited to pure elements, in this work more complex materials were used, i.e. they were multi-elemental including two or more atom species. The results of this thesis are in the microscale. One of the results is a catalogue of atom species, which were removed from tungsten carbide by the plasma. Another result is e.g. the atomic distributions of defects in iron chromium caused by the energetic neutrons. These microscopic results are used in data bases for multiscale modelling of fusion reactor materials, which has the aim to explain the macroscopic degradation in the materials. This thesis is therefore a relevant contribution to investigate the connection of microscopic and macroscopic radiation effects, which is one objective in fusion reactor materials research.

A multi-stage model for drop breakage in stirred vessels

Existing models for dmax predict that, in the limit of μd → ∞, dmax increases with 3/4 power of μd. Further, at low values of interfacial tension, dmax becomes independent of σ even at moderate values of μd. However, experiments contradict both the predictions show that dmax dependence on μd is much weaker, and that, even at very low values of σ,dmax does not become independent of it. A model is proposed to explain these results. The model assumes that a drop circulates in a stirred vessel along with the bulk fluid and repeatedly passes through a deformation zone followed by a relaxation zone. In the deformation zone, the turbulent inertial stress tends to deform the drop, while the viscous stress generated in the drop and the interfacial stress resist deformation. The relaxation zone is characterized by absence of turbulent stress and hence the drop tends to relax back to undeformed state. It is shown that a circulating drop, starting with some initial deformation, either reaches a steady state or breaks in one or several cycles. dmax is defined as the maximum size of a drop which, starting with an undeformed initial state for the first cycle, passes through deformation zone infinite number of times without breaking. The model predictions reduce to that of Lagisetty. (1986) for moderate values of μd and σ. The model successfully predicts the reduced dependence of dmax on μd at high values of μd as well as the dependence of dmax on σ at low values of σ. The data available in literature on dmax could be predicted to a greater accuracy by the model in comparison with existing models and correlations.

A multi-stage model for drop breakage in stirred vessels

Existing models for dmax predict that, in the limit of μd → ∞, dmax increases with 3/4 power of μd. Further, at low values of interfacial tension, dmax becomes independent of σ even at moderate values of μd. However, experiments contradict both the predictions show that dmax dependence on μd is much weaker, and that, even at very low values of σ,dmax does not become independent of it. A model is proposed to explain these results. The model assumes that a drop circulates in a stirred vessel along with the bulk fluid and repeatedly passes through a deformation zone followed by a relaxation zone. In the deformation zone, the turbulent inertial stress tends to deform the drop, while the viscous stress generated in the drop and the interfacial stress resist deformation. The relaxation zone is characterized by absence of turbulent stress and hence the drop tends to relax back to undeformed state. It is shown that a circulating drop, starting with some initial deformation, either reaches a steady state or breaks in one or several cycles. dmax is defined as the maximum size of a drop which, starting with an undeformed initial state for the first cycle, passes through deformation zone infinite number of times without breaking. The model predictions reduce to that of Lagisetty. (1986) for moderate values of μd and σ. The model successfully predicts the reduced dependence of dmax on μd at high values of μd as well as the dependence of dmax on σ at low values of σ. The data available in literature on dmax could be predicted to a greater accuracy by the model in comparison with existing models and correlations.

A Multi frequency study of Radio Intensity Variations For Active Galactic Nuclei of Different Optical Classes

A study of radio intensity variations at seven frequencies in the range 0.3 to 90 GHz for compact extragalactic radio sources classified as BL Lacs and high- and low-optical polarization quasars (HPQs and LPQs) is presented. This include the results of flux-density monitoring of 33 compact sources for three years at 327 MHz with the Ooty Synthesis Radio Telescope. The degrees of 'short-term' (tau less than about 1 yr) variability for the three optical types are found to be indistinguishable at low frequencies (less than 1 GHz), pointing to an extrinsic origin for the low-frequency variability. At high frequencies, a distinct dependence on optical type is present, the variability increasing from LPQs, through HPQs to BL Lacs. This trend persists even when only sources with ultra-flat radio spectra (alpha greater than -0.2) are considered. Implications of this for the phenomenon of high-frequency variability and the proposed unification schemes for different optical types of active galactic nuclei are discussed.

A fault-tolerant multi-transputer architecture

A new fault-tolerant multi-transputer architecture capable of tolerating failure of any one component in the system is described. In the proposed architecture the processing nodes are automatically reconfigured in the event of a fault and the computations continue from the stage where the fault occurred. The process of reconfiguration is transparent to the user, and the identity of the failed component is communicated to the user along with the results of computations. Parallel solution of a typical engineering problem involving solution of Laplace's equation by the boundary element method has been implemented. The performance of the architecture in the event of faults has been investigated.

Stability of synchronization in a multi-cellular system

Networks of biochemical reactions regulated by positive-and negative-feedback processes underlie functional dynamics in single cells. Synchronization of dynamics in the constituent cells is a hallmark of collective behavior in multi-cellular biological systems. Stability of the synchronized state is required for robust functioning of the multi-cell system in the face of noise and perturbation. Yet, the ability to respond to signals and change functional dynamics are also important features during development, disease, and evolution in living systems. In this paper, using a coupled multi-cell system model, we investigate the role of system size, coupling strength and its topology on the synchronization of the collective dynamics and its stability. Even though different coupling topologies lead to synchronization of collective dynamics, diffusive coupling through the end product of the pathway does not confer stability to the synchronized state. The results are discussed with a view to their prevalence in biological systems. Copyright (C) EPLA, 2010

Attitude estimation using moment invariants

The problem of estimating the three-dimensional rotational parameters of a rigid body from its monocular image data has been considered using the method of moment invariants. Second- and third-order moment invariants are used to construct the feature vector for the scale and orientation independent identification of the camera view axis direction in the body-fixed reference frame. The camera rotation angle about the view axis is derived from second-order central moments. The relative attitude of the rigid body is then expressed in terms of quaternion parameters to model the outputs of a video sensor in attitude control simulations. Experimental results and simulation outputs are presented using the mathematical model of a spacecraft.

Young Actors in Transnational Agoras : Multi-sited Ethnography of Cosmopolitan Micropolitical Orientations

This study examines young people s political participation in transnational meetings. Methodologically the study aims to shed light on multi-sited global ethnography. Young people are viewed here as a social age group sensitive to critical, alternative and even radical political participation. The diversity of the young actors and their actions is captured by using several different methods. What is more, the study spurs us coming from the Global North to develop social science research towards methodological cosmopolitanism and to consider our research practices from a moral cosmopolitan perspective. The research sites are the EU Presidency Youth Event (2006 Hyvinkää, Finland), the Global Young Greens Founding Conference (2007 Nairobi, Kenya), the European Social Forum (2008 Malmö, Sweden) and three World Social Forums (2006 Bamako, Mali; 2007 Nairobi Kenya and 2009 Belém, Brazil). The data consists of participant observation, documents and media articles of the meetings, interviews, photos, video, and internet data. This multidisciplinary study combines youth research, development studies, performative social science and political sociology. In this research the diverse field of youth political participation in transnational agoras is studied by using a cross-table of cosmopolitan resources (or the lack of them) and everydaymakers expert citizen dichotomy. First, the young participants of the EU Presidency youth event are studied as an example of expert citizens with cosmopolitan resources (these resources include, for example, language skills, higher education and international social network). Second, the study analyses those everyday-makers who use performative politics to demonstrate their political missions here and now. But in order to make the social movement global they need cosmopolitan resources to be able to use the social media tools and work globally. Third, the study reflects upon the difficulties of reaching those actors who lack cosmopolitan resources, either everyday-makers or expert citizens. The go-along method and the use of the interpreters are shown as ways to reach these young people s political missions. Fourth, the research underlines the importance of contact zones (i.e. spaces or situations where the aforementioned orientations and their differences temporarily disappear or weaken) for deeper democracy and for boosted dialogue between different kinds of participants. Keywords: political participation, young people, multi-sited ethnography, youth research, political sociology, development studies, performative social science