Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg -1. © 2002 Elsevier Science (USA). All rights reserved.

O contexto afetivo-familiar de relações incestuosas entre irmãos

Pós-graduação em Psicologia - FCLAS

TOWARDS THE JOINT USE OF ICD AND ICF: A CALL FOR CONTRIBUTION

Background: To optimize patient functioning, rehabilitation professionals often rely on measurements of functioning as well as on classifications. Although the International Classification of Diseases (ICD) and the International Classification of Functioning, Disability and Health (ICF) are used, their joint use has yet to become an established practice. To encourage their joint use in daily practice, the World Health Organization (WHO) has invited all rehabilitation practitioners worldwide to support the ICD-11 revision process by identifying the ICF categories that correspond to specific rehabilitation-relevant health conditions. The first step in completing this task, generating the list of these health conditions, was taken at a February 2012 workshop in Sao Paulo, Brazil. Objectives: The objectives of this paper are to present the results of the Sao Paulo workshop, and to invite practitioners to participate in the ICD-ICF joint use initiative. Discussion: Alternating plenary and small working group sessions were held and 103 rehabilitation-relevant health conditions were identified. With this list available, WHO together with the International Society of Physical and Rehabilitation Medicine (ISPRM), is reaching out to clinicians of all rehabilitation disciplines to take on the challenge of identifying the ICF categories for at least one of the health conditions listed.

Safety by design: production of engineering surface modified nanomaterials

This PhD thesis focused on nanomaterial (NM) engineering for occupational health and safety, in the frame of the EU project “Safe Nano Worker Exposure Scenarios (SANOWORK)”. Following a safety by design approach, surface engineering (surface coating, purification process, colloidal force control, wet milling, film coating deposition and granulation) were proposed as risk remediation strategies (RRS) to decrease toxicity and emission potential of NMs within real processing lines. In the first case investigated, the PlasmaChem ZrO2 manufacturing, the colloidal force control applied to the washing of synthesis rector, allowed to reduce ZrO2 contamination in wastewater, performing an efficient recycling procedure of ZrO2 recovered. Furthermore, ZrO2 NM was investigated in the ceramic process owned by CNR-ISTEC and GEA-Niro; the spray drying and freeze drying techniques were employed decreasing NM emissivity, but maintaining a reactive surface in dried NM. Considering the handling operation of nanofibers (NFs) obtained through Elmarco electrospinning procedure, the film coating deposition was applied on polyamide non-woven to avoid free fiber release. For TiO2 NF the wet milling was applied to reduce and homogenize the aspect ratio, leading to a significant mitigation of fiber toxicity. In the Colorobbia spray coating line, Ag and TiO2 nanosols, employed to transfer respectively antibacterial or depolluting properties to different substrates, were investigated. Ag was subjected to surface coating and purification, decreasing NM toxicity. TiO2 was modified by surface coating, spray drying and blending with colloidal SiO2, improving its technological performance. In the extrusion of polymeric matrix charged with carbon nanotube (CNTs) owned by Leitat, the CNTs used as filler were granulated by spray drying and freeze spray drying techniques, allowing to reduce their exposure potential. Engineered NMs tested by biologists were further investigated in relevant biological conditions, to improve the knowledge of structure/toxicity mechanisms and obtain new insights for the design of safest NMs.

Electromagnetic Instability Studies in Fusion Plasmas Edge

Este trabajo esta dedicado al estudio de las estructuras macroscópicas conocidas en la literatura como filamentos o blobs que han sido observadas de manera universal en el borde de todo tipo de dispositivos de fusión por confinamiento magnético. Estos filamentos, celdas convectivas elongadas a lo largo de las líneas de campo que surgen en el plasma fuertemente turbulento que existe en este tipo de dispositivos, parecen dominar el transporte radial de partículas y energía en la región conocida como Scrape-off Layer, en la que las líneas de campo dejan de estar cerradas y el plasma es dirigido hacia la pared sólida que forma la cámara de vacío. Aunque el comportamiento y las leyes de escala de estas estructuras son relativamente bien conocidos, no existe aún una teoría generalmente aceptada acerca del mecanismo físico responsable de su formación, que constituye una de las principales incógnitas de la teoría de transporte del borde en plasmas de fusión y una cuestión de gran importancia práctica en el desarrollo de la siguiente generación de reactores de fusión (incluyendo dispositivos como ITER y DEMO), puesto que la eficiencia del confinamiento y la cantidad de energía depositadas en la pared dependen directamente de las características del transporte en el borde. El trabajo ha sido realizado desde una perspectiva eminentemente experimental, incluyendo la observación y el análisis de este tipo de estructuras en el stellarator tipo heliotrón LHD (un dispositivo de gran tamaño, capaz de generar plasmas de características cercanas a las necesarias en un reactor de fusión) y en el stellarator tipo heliac TJ-II (un dispositivo de medio tamaño, capaz de generar plasmas relativamente más fríos pero con una accesibilidad y disponibilidad de diagnósticos mayor). En particular, en LHD se observó la generación de filamentos durante las descargas realizadas en configuración de alta _ (alta presión cinética frente a magnética) mediante una cámara visible ultrarrápida, se caracterizó su comportamiento y se investigó, mediante el análisis estadístico y la comparación con modelos teóricos, el posible papel de la Criticalidad Autoorganizada en la formación de este tipo de estructuras. En TJ-II se diseñó y construyó una cabeza de sonda capaz de medir simultáneamente las fluctuaciones electrostáticas y electromagnéticas del plasma. Gracias a este nuevo diagnóstico se pudieron realizar experimentos con el fin de determinar la presencia de corriente paralela a través de los filamentos (un parámetro de gran importancia en su modelización) y relacionar los dos tipos de fluctuaciones por primera vez en un stellarator. Así mismo, también por primera vez en este tipo de dispositivo, fue posible realizar mediciones simultáneas de los tensores viscoso y magnético (Reynolds y Maxwell) de transporte de cantidad de movimiento. ABSTRACT This work has been devoted to the study of the macroscopic structures known in the literature as filaments or blobs, which have been observed universally in the edge of all kind of magnetic confinement fusion devices. These filaments, convective cells stretching along the magnetic field lines, arise from the highly turbulent plasma present in this kind of machines and seem to dominate radial transport of particles and energy in the region known as Scrapeoff Layer, in which field lines become open and plasma is directed towards the solid wall of the vacuum vessel. Although the behavior and scale laws of these structures are relatively well known, there is no generally accepted theory about the physical mechanism involved in their formation yet, which remains one of the main unsolved questions in the fusion plasmas edge transport theory and a matter of great practical importance for the development of the next generation of fusion reactors (including ITER and DEMO), since efficiency of confinement and the energy deposition levels on the wall are directly dependent of the characteristics of edge transport. This work has been realized mainly from an experimental perspective, including the observation and analysis of this kind of structures in the heliotron stellarator LHD (a large device capable of generating reactor-relevant plasma conditions) and in the heliac stellarator TJ-II (a medium-sized device, capable of relatively colder plasmas, but with greater ease of access and diagnostics availability). In particular, in LHD, the generation of filaments during high _ discharges (with high kinetic to magnetic pressure ratio) was observed by means of an ultrafast visible camera, and the behavior of this structures was characterized. Finally, the potential role of Self-Organized Criticality in the generation of filaments was investigated. In TJ-II, a probe head capable of measuring simultaneously electrostatic and electromagnetic fluctuations in the plasma was designed and built. Thanks to this new diagnostic, experiments were carried out in order to determine the presence of parallel current through filaments (one of the most important parameters in their modelization) and to related electromagnetic (EM) and electrostatic (ES) fluctuations for the first time in an stellarator. As well, also for the first time in this kind of device, measurements of the viscous and magnetic momentum transfer tensors (Reynolds and Maxwell) were performed.

Small molecule dual-inhibitors of TRPV4 and TRPA1 for attenuation of inflammation and pain.

TRPV4 ion channels represent osmo-mechano-TRP channels with pleiotropic function and wide-spread expression. One of the critical functions of TRPV4 in this spectrum is its involvement in pain and inflammation. However, few small-molecule inhibitors of TRPV4 are available. Here we developed TRPV4-inhibitory molecules based on modifications of a known TRPV4-selective tool-compound, GSK205. We not only increased TRPV4-inhibitory potency, but surprisingly also generated two compounds that potently co-inhibit TRPA1, known to function as chemical sensor of noxious and irritant signaling. We demonstrate TRPV4 inhibition by these compounds in primary cells with known TRPV4 expression - articular chondrocytes and astrocytes. Importantly, our novel compounds attenuate pain behavior in a trigeminal irritant pain model that is known to rely on TRPV4 and TRPA1. Furthermore, our novel dual-channel blocker inhibited inflammation and pain-associated behavior in a model of acute pancreatitis - known to also rely on TRPV4 and TRPA1. Our results illustrate proof of a novel concept inherent in our prototype compounds of a drug that targets two functionally-related TRP channels, and thus can be used to combat isoforms of pain and inflammation in-vivo that involve more than one TRP channel. This approach could provide a novel paradigm for treating other relevant health conditions.

Diaphragm muscle adaptation to sustained hypoxia: lessons from animal models with relevance to high altitude and chronic respiratory diseases

The diaphragm is the primary inspiratory pump muscle of breathing. Notwithstanding its critical role in pulmonary ventilation, the diaphragm like other striated muscles is malleable in response to physiological and pathophysiological stressors, with potential implications for the maintenance of respiratory homeostasis. This review considers hypoxic adaptation of the diaphragm muscle, with a focus on functional, structural, and metabolic remodeling relevant to conditions such as high altitude and chronic respiratory disease. On the basis of emerging data in animal models, we posit that hypoxia is a significant driver of respiratory muscle plasticity, with evidence suggestive of both compensatory and deleterious adaptations in conditions of sustained exposure to low oxygen. Cellular strategies driving diaphragm remodeling during exposure to sustained hypoxia appear to confer hypoxic tolerance at the expense of peak force-generating capacity, a key functional parameter that correlates with patient morbidity and mortality. Changes include, but are not limited to: redox-dependent activation of hypoxia-inducible factor (HIF) and MAP kinases; time-dependent carbonylation of key metabolic and functional proteins; decreased mitochondrial respiration; activation of atrophic signaling and increased proteolysis; and altered functional performance. Diaphragm muscle weakness may be a signature effect of sustained hypoxic exposure. We discuss the putative role of reactive oxygen species as mediators of both advantageous and disadvantageous adaptations of diaphragm muscle to sustained hypoxia, and the role of antioxidants in mitigating adverse effects of chronic hypoxic stress on respiratory muscle function.

THE INFLUENCE OF MOTIVATION ON EMOTION REGULATION AND MOTOR PERFORMANCE: EXAMINATION OF A NEURO-AFFECTIVE MODEL

Mental stress is known to disrupt the execution of motor performance and can lead to decrements in the quality of performance, however, individuals have shown significant differences regarding how fast and well they can perform a skilled task according to how well they can manage stress and emotion. The purpose of this study was to advance our understanding of how the brain modulates emotional reactivity under different motivational states to achieve differential performance in a target shooting task that requires precision visuomotor coordination. In order to study the interactions in emotion regulatory brain areas (i.e. the ventral striatum, amygdala, prefrontal cortex) and the autonomic nervous system, reward and punishment interventions were employed and the resulting behavioral and physiological responses contrasted to observe the changes in shooting performance (i.e. shooting accuracy and stability of aim) and neuro-cognitive processes (i.e. cognitive load and reserve) during the shooting task. Thirty-five participants, aged 18 to 38 years, from the Reserve Officers’ Training Corp (ROTC) at the University of Maryland were recruited to take 30 shots at a bullseye target in three different experimental conditions. In the reward condition, $1 was added to their total balance for every 10-point shot. In the punishment condition, $1 was deducted from their total balance if they did not hit the 10-point area. In the neutral condition, no money was added or deducted from their total balance. When in the reward condition, which was reportedly most enjoyable and least stressful of the conditions, heart rate variability was found to be positively related to shooting scores, inversely related to variability in shooting performance and positively related to alpha power (i.e. less activation) in the left temporal region. In the punishment (and most stressful) condition, an increase in sympathetic response (i.e. increased LF/HF ratio) was positively related to jerking movements as well as variability of placement (on the target) in the shots taken. This, coupled with error monitoring activity in the anterior cingulate cortex, suggests evaluation of self-efficacy might be driving arousal regulation, thus affecting shooting performance. Better performers showed variable, increasing high-alpha power in the temporal region during the aiming period towards taking the shot which could indicate an adaptive strategy of engagement. They also showed lower coherence during hit shots than missed shots which was coupled with reduced jerking movements and better precision and accuracy. Frontal asymmetry measures revealed possible influence of the prefrontal lobe in driving this effect in reward and neutral conditions. The possible interactions, reasons behind these findings and implications are discussed.

Work motivation as a determinant of organizational and professional commitment in case of temporary organizations : theoretical perspectives

The purpose of this paper is to present a theoretical framework to investigate the relationship between work motivation, organizational commitment, and professional commitment in temporary organizations. Through a review of theory, we contend that work motivation has two major patterns- internal motivation (that includes intrinsic, need-based, and self-deterministic theories), and external motivation (that includes cognitive or process-based theories of motivation) through which it has been investigated. We also subsume the nature of employee commitment to be of three types- affective, continuance, and normative. This commitment may either be towards organization or profession. A literature review reveals that the characteristics of the temporary organization - specifically tenure, and task - regulate the relationship between work motivation, and organizational commitment, and professional commitment. Relevant propositions are presented.

Multimedia psycho-educational interventions to support patient self-care in degenerative conditions: a realist review

Objective: Multimedia interventions are increasingly used to deliver information in order to promote self-care among patients with degenerative conditions. We carried out a realist review of the literature to investigate how the characteristics of multimedia psychoeducational interventions combine with the contexts in which they are introduced to help or hinder their effectiveness in supporting self-care for patients with degenerative conditions.

Method: Electronic databases (Medline, Science Direct, PSYCHinfo, EBSCO, and Embase) were searched in order to identify papers containing information on multimedia psychoeducational interventions. Using a realist review approach, we reviewed all relevant studies to identify theories that explained how the interventions work.

Results: Ten papers were included in the review. All interventions sought to promote self-care behaviors among participants. We examined the development and content of the multimedia interventions and the impact of patient motivation and of the organizational context of implementation. We judged seven studies to be methodologically weak. All completed studies showed small effects in favor of the intervention.

Significance of Results: Multimedia interventions may provide high-quality information in an accessible format, with the potential to promote self-care among patients with degenerative conditions, if the patient perceives the information as important and develops confidence about self-care. The evidence base is weak, so that research is needed to investigate effective modes of delivery at different resource levels. We recommend that developers consider how an intervention will reduce uncertainty and increase confidence in self-care, as well as the impact of the context in which it will be employed.

The Relationship between athlete motivation, strategies used to cope with stress and affective outcomes in Canadian University athletes

Motivation to perform and coping with stress during performance are key factors in determining numerous outcomes of sporting performance. However, less evidence is in place assessing their relationship. The aim of this investigation was to assess the relationship between athlete motivation and the coping strategies used to deal with stress during their sporting performance, as well as the relationship between motivation and affect and coping and affect. One hundred and forty five university athletes completed questionnaires. Regressions revealed that two of the three self determined levels of motivation, identified and integrated regulation, predicted increased task-oriented coping strategies. Two of the three non-self determined levels of motivation, amotivation and external regulation, significantly predicted disengagement-oriented coping. Additionally, intrinsic motivation and task-oriented coping predicted increase positive affect. Increased disengagement-oriented coping predicted decreased positive affect. Disengagement-oriented coping significantly predicted increased negative affect. These findings increase understanding of motivations role in predicting athletes coping.

Les facteurs et les conditions organisationnelles favorisant la motivation des enseignants du primaire et du secondaire dans leur développement professionnel lié à l’intégration des TIC

Cette recherche se situe dans le contexte de la société du savoir et de la nécessité de l’apprentissage et du développement professionnel tout au long de la vie. Elle porte sur la dynamique motivationnelle qui anime les enseignants en processus d’apprentissage professionnel, c’est-à-dire lorsqu’ils doivent accomplir des activités dans un programme de formation continue. Les objectifs de cette recherche sont les suivants : développer et valider une échelle qui mesure la satisfaction des besoins psychologiques fondamentaux dans le contexte du développement professionnel lié à l’intégration des TIC chez les enseignants du primaire et du secondaire; déterminer le degré de satisfaction des besoins psychologiques fondamentaux des enseignants dans le contexte de la formation continue concernant l’intégration des TIC; déterminer la perception des enseignants à l’égard de la pertinence des activités de formation continue; identifier les activités et les conditions organisationnelles favorisant la motivation des enseignants du primaire dans leur développement professionnel concernant l’intégration des TIC. Pour atteindre nos objectifs de recherche, nous avons adopté une méthodologie de type mixte, comprenant l’analyse des données quantitatives recueillies à l’aide d’un questionnaire à question fermées (64 participants) et des données qualitatives collectées à l’aide d’entrevues individuelles (5 participants). Malgré certaines limites méthodologiques et conceptuelles, les résultats de la présente recherche indiquent que le soutien à l’autonomie, au sentiment de compétence et à l’appartenance sociale, de même que la pertinence des activités de formation sont proposés comme déterminants principaux de la satisfaction et de la motivation dans le développement professionnel des enseignants. Les résultats de cette recherche ouvrent également la voie à des pistes pour favoriser la motivation dans le développement professionnel et proposent des idées de recherche prospectives.

Anticancer activity of organometallic compounds. 3. The reaction of dimethyltin dichloride with nucleosides under biologically relevant conditions

This paper reports the reaction of SnMe2Cl2 with adenosine, guanosine and inosine in aqueous solution at pH 4.5. The nucleosides give probably polymeric species in which there is monodentate coordination to O2′ of the ribose ring as indicated by 80 MHz PMR.

Mechanisms of motivation-cognition interaction: challenges and opportunities

There has been a recent rejuvenation of interest in studies of motivation-cognition interactions arising from many different areas of psychology and neuroscience. The current issue of Cognitive, Affective, and Behavioral Neuroscience provides a sampling of some of the latest research from a number of these different areas. In this introductory paper, we provide an overview of the current state of the field, in terms of key research developments and candidate neural mechanisms receiving focused investigation as potential sources of motivation-cognition interaction. However, our primary goal is conceptual: to highlight the distinct perspectives taken by different research areas in terms of how motivation is defined, the relevant dimensions and dissociations that are emphasized, and the theoretical questions being targeted. Together, these distinctions present both challenges and opportunities for efforts aiming towards a more unified and cross-disciplinary approach. We identify a set of pressing research questions calling out for this sort of cross-disciplinary approach, with the explicit goal of encouraging integrative and collaborative investigations directed towards them.

Process in skill acquisition : motivation, interruptions, memory, affective states, and metacognition

Models of skill acquisition, such as Ackerman's (1988) theory of skill acquisition, largely ignore the experiences and dynamic internal processes of a person while learning a skill (e.g., how they feel, what strategies they are using, the role of the external environment). Riding and Powell (1993) suggested that the information-processing model (a common conceptualisation of learning and thinking) is an inadequate model to explain the complexities of human brain functions. These models are largely holistic, and do not provide detail about the qualitative aspects of a person's learning experience. This paper attempts to highlight the importance of a dynamic description of skill acquisition and to “unpack” the skill acquisition stages, with a focus given to process rather than performance or outcome. Learning more about what is going on “inside people's heads” whilst learning a skill, and what feelings or affective states are present, can inform both skill acquisition theory and various practical issues, such as why some training programs fail and some succeed. The role of emotions, motivation, environment, and other factors; the concepts of skill and ability; and various theories of skill acquisition are discussed. This highlights missing elements in the typical cognitive experimental focus. The concept of process itself is explored, with a focus on learning in general. Process-oriented factors such as motivation, memory, interruptions, emotion, and metacognition are investigated in relation to skilled performance. Areas for future research and some practical implications for training are outlined.