Primary immunodeficiencies unravel critical aspects of the pathophysiology of autoimmunity and of the genetics of autoimmune disease

Background Primary Immunodeficiencies (PIDs) represent unique opportunities to understand the operation of the human immune system. Accordingly, PIDs associated with autoimmune manifestations provide insights into the pathophysiology of autoimmunity as well as into the genetics of autoimmune diseases (AID). Epidemiological data show that there are PIDs systematically associated with AID, such as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), Omenn syndrome, autoinunune polyendocrinopathy-candidiasis-ectodertnal dystrophy (APECED), autoinumine lymphoproliferative syndrome (ALPS), and C1q deficiency, while strong associations are seen with a handful of other deficits. Conclusion We interpret such stringent disease associations, together with a wealth of observations in experimental systems, as indicating first of all that natural tolerance to body components is an active, dominant process involving many of the components that ensure responsiveness, rather than, as previously believed, the result of the mere purge of autoreactivities. More precisely, it seems that deficits of Treg cell development, functions, numbers, and T cell receptor repertoire are among the main factors for autoimmunity pathogenesis in many (if not all) PIDs most frequently presenting with autoimmune features. Clearly, other pathophysiological mechanisms are also involved in autoimmunity, but these seem less critical in the process of self-tolerance. Comparing the clinical picture of IPEX cases with those, much less severe, of ALPS or APECED, provides some assessment of the relative importance of each set of mechanisms.

DIFFERENTIATION BETWEEN Nocardia spp. AND Mycobacterium spp.: CRITICAL ASPECTS FOR BACTERIOLOGICAL DIAGNOSIS

New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT™) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients.

Critical aspects on the behavior of material from the mechanical tool-workpiece interaction in single point diamond turning

Some material aspects such as grain size, purity and anisotropy exert an important influence on surface quality, especially in single point diamond turning. The aim of this paper is to present and discuss some critical factors that can limit the accuracy of ultraprecision machining of non-ferrous metals and to identify the effects of them on the cutting mechanism with single point diamond tools. This will be carried out through observations of machined surfaces and chips produced using optical and scanning electron microscopy. Solutions to reduce the influence of some of these limiting factors related with the mechanism of generation of mirror-like surfaces will be discussed.

Equine and human mutual welfare: a whole subject? Critical aspects and possible strategies in equine-assisted activities and therapies

General aim of the study is equine welfare, particularly concerning different husbandry methodic and inter-specific relational factors. Specific aim is the evaluation of possible mutual (to humans and to equines) benefits and the analysis of critical factors/strength points, of human-horse relationship within Therapeutic Riding context (TR). The peculiarities of human-horse relationship (compared to the bond with “Pet”) are analyzed, concerning their socio-anthropological, psychological, psycho-dynamic distinctive characteristics. 8 European representative therapeutic riding centers (TRC) were therefore selected (on the basis of their different animals’ husbandry criteria, and of the different rehabilitative methodologies adopted). TRC were investigated through 2 different questionnaires, specifically settled to access objective/subjective animal welfare parameters; the quality of human-horse relationship; technicians’ emotional experienced. 3 Centers were further selected, and behavioral (145 hours of behavioral recording) and physiological parameters (heart rate and heart rate variability) were evaluated, aimed to access equine welfare and horses’ adaptive responses/coping (towards general environment and towards TR job). Moreover a specific “handling-task” was ideated and experimented, aimed to measure the quality of TR technicians-horses relationship. We did therefore evaluate both the individual horses’ responses and the possible differences among Centers. Data collected highlight the lack of univocal standardized methodic, concerning the general animals’ management and the specific methodologies (aimed to improve animal welfare and to empower TR efficacy). Some positive and some critical aspects were detected concerning TR personnel-horse relationship. Another experimental approach did evaluate the efficacy (concerning the mutual benefits’ empowerment) of an “ethologically-fitted” TR intervention, aimed to educate children to and through the relationship with horses. Our data evidenced that the improvement of human horse relationship, through structured educational programs for TR personnel might have important consequences both to human and equine welfare.

Critical aspects of substrate nanopatterning for the ordered growth of GaN nanocolumns.

Precise and reproducible surface nanopatterning is the key for a successful ordered growth of GaN nanocolumns. In this work, we point out the main technological issues related to the patterning process, mainly surface roughness and cleaning, and mask adhesion to the substrate. We found that each of these factors, process-related, has a dramatic impact on the subsequent selective growth of the columns inside the patterned holes. We compare the performance of e-beam lithography, colloidal lithography, and focused ion beam in the fabrication of hole-patterned masks for ordered columnar growth. These results are applicable to the ordered growth of nanocolumns of different materials.

Investigation of some critical aspects of on-line surface measurement by a wavelength-division-multiplexing technique

Some critical aspects of a new kind of on-line measurement technique for micro and nanoscale surface measurements are described. This attempts to use spatial light-wave scanning to replace mechanical stylus scanning, and an optical fibre interferometer to replace optically bulky interferometers for measuring the surfaces. The basic principle is based on measuring the phase shift of a reflected optical signal. Wavelength-division-multiplexing and fibre Bragg grating techniques are used to carry out wavelength-to-field transformation and phase-to-depth detection, allowing a large dynamic measurement ratio (range/resolution) and high signal-to-noise ratio with remote access. In effect the paper consists of two parts: multiplexed fibre interferometry and remote on-machine surface detection sensor (an optical dispersive probe). This paper aims to investigate the metrology properties of a multiplexed fibre interferometer and to verify its feasibility by both theoretical and experimental studies. Two types of optical probes, using a dispersive prism and a blazed grating, respectively, are introduced to realize wavelength-to-spatial scanning.

Investigation of some critical aspects of on-line surface measurement by a wavelength-division-multiplexing technique

Some critical aspects of a new kind of on-line measurement technique for micro and nanoscale surface measurements are described. This attempts to use spatial light-wave scanning to replace mechanical stylus scanning, and an optical fibre interferometer to replace optically bulky interferometers for measuring the surfaces. The basic principle is based on measuring the phase shift of a reflected optical signal. Wavelength-division-multiplexing and fibre Bragg grating techniques are used to carry out wavelength-to-field transformation and phase-to-depth detection, allowing a large dynamic measurement ratio (range/resolution) and high signal-to-noise ratio with remote access. In effect the paper consists of two parts: multiplexed fibre interferometry and remote on-machine surface detection sensor (an optical dispersive probe). This paper aims to investigate the metrology properties of a multiplexed fibre interferometer and to verify its feasibility by both theoretical and experimental studies. Two types of optical probes, using a dispersive prism and a blazed grating, respectively, are introduced to realize wavelength-to-spatial scanning.

Aspectos bioquímicos y celulares de la reproducción de los vectores de la enfermedad de Chagas: regulación e importancia fisiológica. Biochemical and cellular aspects of the reproduction of Chagas’ disease vectors: regulation and physiologycal impact

En la hipótesis de trabajo del presente proyecto se considera la importancia del metabolismo de lípidos y proteínas en los insectos hematófagos, en particular en los vectores de la enfermedad de Chagas, para afrontar exitosamente la demanda energética de la reproducción. Las hembras de estas especies pueden ingerir una comida de sangre abundante en lípidos y proteínas, los que son modificados en el intestino para su utilización y posterior almacenamiento en estructuras organizadas en el tejido ovárico, sustentando así el rápido crecimiento de los ovocitos. Estos aspectos resultan críticos para el ciclo de vida del insecto y para el mantenimiento de la cadena epidemiológica de la enfermedad. En estas especies, recientemente hemos caracterizado a nivel bioquímico y celular la interacción entre lipoproteínas y tejidos [Fruttero y col., Insect Biochem. Mol. Biol. 39: 322-331 (2009); Fruttero y col. Biocel 33 (3): 260 (2009)] y las fases del ciclo reproductivo [Aguirre y col., J. Insect Physiol. 54: 393-402 (2008)]. No obstante, los factores que participan en su regulación son aún escasamente conocidos. En este contexto, el estudio propone emplear dos especies de triatominos con el objeto de: (1) caracterizar los factores involucrados en la formación y regulación de reservas nutricionales en los ovocitos; (2) analizar los eventos que participan en la regresión del tejido ovárico: atresia folicular y mecanismos de muerte celular. (3) evaluar el impacto de productos naturales (ureasas vegetales y péptidos derivados) en el desarrollo del tejido ovárico. Para la ejecución de los objetivos se llevarán a cabo ensayos in vivo e in vitro con trazadores fluorescentes, fraccionamiento subcelular, estudios de expresión de proteínas (mRNA y proteína), estudios histo-morfológicos, ultraestructurales e inmunocitoquímicos, microscopía láser confocalizada, ensayos de actividad enzimática, ELISA, western-blot, electroforesis bidimensional, espectrometria de masas en tándem, etc. También se evaluarán los mecanismos de muerte celular (apoptosis/autofagia) mediante microscopía electrónica, detección de apoptosis in situ (TUNEL), inmunofluorescencia, etc. Los resultados obtenidos permitirán un mejor conocimiento sobre la fisiología y bioquímica de estos vectores, los que resultan indispensables en el diseño de nuevas estrategias para su control. Debido a la carencia de un tratamiento específico para la enfermedad y a la falta de métodos preventivos (vacuna), el control del vector es una de las vías más importantes para reducir la incidencia de la enfermedad. Actualmente, la situación socio-económica que sufren amplios núcleos de nuestra población propicia condiciones de vida que facilitan la reproducción de los vectores y la transmisión vectorial del parásito. El estudio permitirá además explorar aspectos bioquímicos y celulares básicos, generando conocimientos que podrían ser extensivos a otros insectos de importancia económica en la ganadería y/o agricultura. The aim of this project is to analyze the biochemical and cellular events involved in the lipid and protein metabolism in Chagas' disease vectors, and to evaluate their impact on the physiology of reproduction, particularly in the formation of nutritional resources in developing oocytes. At present, little is known about these critical aspects for the life cycle of the insect and for the epidemiology of the disease. The experimental approaches, which will be carried out using two species of triatomines, were designed: (1) to characterize factors involved in the formation and regulation of nutritional resources in developing oocytes; (2) to analyze the biochemical and cellular events that play a role during the regression of ovarian tissue, including the processes of oocyte resorption and programmed cell death. (3) to evaluate the impact of natural products (ureases from jackbean and related peptides) in the development of ovarian tissue. Methods and techniques involved in the project are: in vivo and in vitro assays with fluorescent tracers, ELISA, chemical assays, enzyme activities, western-blot; protein expression (mRNA), histological techniques, immunohistochemical and ultrastructural studies. Cell death will be analyzed by detection of apoptosis in situ (TUNEL), immunofluorescence (for autophagy), among others. The results obtained from the study will offer the opportunity to explore important aspects in the biology and physiology of Chagas' disease vectors that could be of potential utility in designing alternative strategies for the control of the insect.

Identification of a conserved cluster in the RH domain of GRK critical for activation by GPCRs

One of the most critical aspects of G Protein Coupled Receptors (GPCRs) regulation is their rapid and acute desensitization following agonist stimulation. Phosphorylation of these receptors by GPCR kinases (GRK) is a major mechanism of desensitization. Considerable evidence from studies of rhodopsin kinase and GRK2 suggests there is an allosteric docking site for the receptor distinct from the GRK catalytic site. While the agonist-activated GPCR appears crucial for GRK activation, the molecular details of this interaction remain unclear. Recent studies suggested an important role for the N- and C-termini and domains in the small lobe of the kinase domain in allosteric activation; however, neither the mechanism of action of that site nor the RH domain contributions have been elucidated. To search for the allosteric site, we first indentified evolutionarily conserved sites within the RH and kinase domains presumably deterministic of protein function employing evolutionary trace (ET) methodology and crystal structures of GRK6. Focusing on a conserved cluster centered on helices 3, 9, and 10 in the RH domain, key residues of GRK5 and 6 were targeted for mutagenesis and functional assays. We found that a number of double mutations within helices 3, 9, and 10 and the N-terminus markedly reduced (50–90%) the constitutive phosphorylation of the β-2 Adrenergic Receptor (β2AR) in intact cells and phosphorylation of light-activated rhodopsin (Rho*) in vitro as compared to wild type (WT) GRK5 or 6. Based on these results, we designed peptide mimetics of GRK5 helix 9 both computationally and through chemical modifications with the goal of both confirming the importance of helix 9 and developing a useful inhibitor to disrupt the GPCR-GRK interaction. Several peptides were found to block Rho* phosphorylation by GRK5 including the native helix 9 sequence, Peptide Builder designed-peptide preserving only the key ET residues, and chemically locked helices. Most peptidomimetics showed inhibition of GRK5 activity greater than 80 % with an IC50 of ∼ 30 µM. Alanine scanning of helix 9 has further revealed both essential and non-essential residues for inhibition. Importantly, substitution of Arg 169 by an alanine in the native helix 9-based peptide gave an almost complete inhibition at 30 µM with an IC50 of ∼ 10 µM. In summary we report a previously unrecognized crucial role for the RH domain of GRK5 and 6, and the subsequent identification of a lead peptide inhibitor of protein-protein interaction with potential for specific blockade of GPCR desensitization. ^

Analyses of Individual and Organizational Competences Associated with Knowledge Management Practice

The research analyzed critical aspects of the knowledge management process based on the analyses of knowledge, abilities and attitudes required to individual knowledge workers and to organizations responsible for the management process. In the present work a characterization of the knowledge management process was developed and information and knowledge wokers defined. Competence concept was discussed and specialists gave opinions about critical competences to knowledge management process. The opinions were organized and analyzed by the Delphi method. The results aggregate to the management context by discussing an extremely important resource to organizations - knowledge - and because they support its management process. The research identified wide critical aspects that are compatible with current organizational challenges, directing the process management to important themes as: the worker able to create, the organization able to convert individual knowledge into organizational knowledge, knowledge sharing while still tacit, the maximization organizational knowledge use, information and knowledge generation and preservation, among others important topics to be observed by knowledge workers and by administrators responsible for the knowledge management process.

Balanced scorecard: estudo de caso de uma empresa de serviços de gestão

Dissertação para a obtenção do Grau de Mestre em Contabilidade e Finanças Orientadora: Doutora Cláudia Lopes

Modelo de avaliação do relacionamento com o cliente em serviços da Administração Pública

Dissertação de Mestrado em Gestão de Empresas/MBA.