During inflammatory responses, macrophages make and secrete cytokines, including the proinflammatory cytokine TNF-alpha (TNF). TNF is a highly potent activator of immune responses with pleiomorphic effects throughout the body. TNF is a key causative agent of chronic inflammatory diseases and is of an intense clinical interest as a therapeutic target. At the outset of this thesis, little was known about how macrophages secrete TNF. Notably, the pathways, carriers and molecules that regulate TNF secretion had not been characterised. A main goal of this work was to identify compartment and molecules involved in the intracellular trafficking of TNF. Live cell imaging of GFP-TNF was established and this provided novel and important new insights into trafficking. Both endogenous and GFP-tagged TNF were followed in macrophages using fluorescence microscopy. The trafficking of other molecules in macrophages was also studied. The major findings of this work include the identification of a new two-step secretory pathway for TNF and other proteins from the trans-Golgi Network (TGN) to the cell surface. This pathway goes via the recycling endosome as an intermediate station. Pleiotrophic tubular-vesicular carriers containing TNF bud off the TGN for the post-Golgi trafficking of TNF and their characterization both in live cell imaging and in biochemical analysis of isolated vesicles constituted the main parts of this work. Functional studies, including endosome inactivation and overexpression of Rab11 mutants (proteins functioning at the level of the recycling endosome) revealed that recycling endosomes have indeed an essential role in the exocytic trafficking of TNF in macrophages. This thesis also provides further insight into recycling endosomes as a possible intermediate step in the exocytic trafficking of several other proteins including the adhesion protein E-Cadherin, that function at the cell surface. Finally, the last chapter of this thesis examines endocytic pathways in activated macrophages. Assays for fluid phase endocytosis and receptor-mediated endocytosis were established and the regulation of both pathways was compared. The results show that LPS has opposite effects on fluid phase and receptor mediated endocytosis, decreasing and increasing their activity respectively. Recycling of transferrin through the recycling endosome was also measured, providing a link with studies on TNF exocytosis. Overall, the work in this thesis has made a major contribution to our understanding of TNF trafficking in macrophages, of macrophage pathways more generally and of trafficking at a fundamental level. The findings herein set the stage for more in depth analysis at a single molecular level to explore TNF regulation in normal and disease cells.

Chaperonin 10 (Cpn10) is a mitochondrial protein with protein folding function. There is substantial evidence that extracellular Cpn10 regulates the immune response. Prior research has shown that Cpn10 binds to T cells, inhibits LPS-induced RAW264.7 macrophage cell- and healthy donor peripheral blood mononuclear cell (PBMC)-activation, and downregulates lipopolysaccharide (LPS)-induced membrane distribution of the MHC II molecule on dendritic cells (DC). Recent Phase IIa rheumatoid arthritis (RA), psoriasis and multiple sclerosis (MS) clinical trials demonstrate improved disease amelioration with Cpn10. Despite compelling evidence of the anti-inflammatory properties of Cpn10, the precise mechanisms of action are unknown. The principal aim was to characterise the modulation of inflammation by Cpn10 and in the process create a bioassay that would allow for the reliable assessment of batch-to-batch variability of Cpn10 preparations. For this purpose, a Cpn10 bioassay was performed in the RAW264.7 cell line and expanded to DC and T cell lines. Furthermore, the analysis of gene expression in healthy donor PBMC was performed, as a mixed cell population experiment, to reflect possible involvement of cell-to-cell communication pathways. Initial data showed that Cpn10 reduced LPS-induced tumour necrosis factor ?? (TNF??) expression in RAW264.7 cells. However, the Cpn10 preparation was shown to contain trace lipid contaminants, which induced cellular tolerance, resulting in the observed reduction in TNF??. Experiments with a second batch of Cpn10 showed no reduction of LPS-induced TNF?? in the RAW264.7 cells, seen with the primary batch of Cpn10 and previously reported characterisation of Cpn10. The Cpn10 bioassay conducted in DC and T cell lines was shown to have the potential to decrease toll-like receptor 9 (TLR9) expression, suggesting that Cpn10 may attenuate immune responses by downregulating receptor recognition of bacterial components. The Cpn10 bioassay conducted in LPS-stimulated PBMCs revealed that Cpn10 downregulates gene expression of Th1 related genes including the polarising cytokines IL-7, IL-12B and IL-23A and Th2 related genes including the transcriptions factors GATA3, GFI1 and CEBPB. The downregulation of these genes may play an immuno-modulatory role, having improved efficacy of Cpn10 in T cell mediated autoimmune diseases, with possible therapeutic implications in Th2 mediated diseases such as asthma. The research carried out in this thesis provides insight into the success of Cpn10 in the RA, MS and psoriasis clinical trials. These results have also supported previously published data and provide additional insight into the mechanism of action of Cpn10. In addition, a Cpn10 bioassay has been established using healthy donor PBMCs stimulated with LPS and results show a reduced expression of Th1 and Th2 associated genes. The findings that in mixed cell populations, Cpn10 downregulates not only genes involved in Th1 polarisation mainly at the signal 3 level, but is also capable of downregulating Th2 polarising genes at the signal 1 level of TCR mediated transcription factors, are of particular interest. Ultimately, research from this project has confirmed the anti-inflammatory action of Cpn10 and given useful insight into how Cpn10 acts to modulate the inflammatory response.

This thesis was focused on the pro-inflammatory cytokines IL-1beta and IFN-gamma. Absolute and differential leukocyte count was also monitored. The experiment was conducted at 8 clinically healthy heifers, hybrids of Holstein and Czech Pied that have been housed by tethering in stalls and fed with a standard diet. The inflammatory reaction was induced by lipopolysaccharide (LPS; 5 ug in 20 ml PBS), as a control a phosphate buff-ered saline (PBS) was used. Results were measured at 1, 2, 3 and 7 days after stimulation of mammary gland by above-mentioned factors. Concentration of each cytokine was detected by a sandwich ELISA using commercially available kits. At 1 day after stimulation of mammary gland by LPS and PBS an average number of leukocytes, which was statistically significantly higher in the case of stimulation by LPS (P <0.01), was detected. After 7 days there was a significant decrease in the total number of leukocytes. There has also been a shift in the differential leukocyte count. Most abundant cell type were neutrophils, whose number was higher in the case of stimulation by LPS. Between day 1 and day 7 after challenge, there was a gradual reduction in the proportion of neutrophils. In the same period an increase in the proportion of macrophages and lymphocytes was detected. Concentration of IL-1beta also increased, 1 day after the activation a striking increase has been detected. In following days there was gradual decline of IL-1beta concentration almost to the level prior to treatment of the mammary gland. In the case of IFN-gamma similar pattern in the form of strong growth and a subsequent gradual decline in concentration to the original values was detected. There was found positive correlation between the increase in IL-1beta and IFN-gamma concentration and a shift in the differential leukocyte count in favor of neutrophils, which confirmed the important role of these pro-inflammatory cytokines in the establishment of inflammatory response and the mobilization of the components of natural and specific immunity.

A large number of natural disasters affects hundreds of thousands of people each year in their housing around the world. Therefore, there is a call to find more appropriate strategies for housing reconstruction following a disaster. This study aims to reduce the construction time and cost of housing affected by such disasters. The academic literature on the 3 Dimensional Lightweight Panels construction system (3D-LPs), Building Information Modelling system (BIM) and experiences gained in post-disaster housing reconstruction strengthens the argument that here is an opportunity to contribute to solve the housing reconstruction problem. The study points out that the combination of these systems and community participation presents an option to produce both affordable and sustainable housing in the shortest time on a large scale by the affected people after overcoming the emergence phase of a disaster. A holistic philosophy was used to study the housing reconstruction problem as a whole to understand all parts of the problem and three research questions were set up to explore the possible solution to this problem. The research strategy to address the problem was based on a survey of worldwide experts, interviewing a forum of lightweight concrete panel manufacturers and the modelling of a basic housing prototype in BIM. Research question (1) How can displaced people use their own labour to save money and time? and research question (2) How does the 3D-LPs construction system contribute to housing recovery after natural disasters? Research questions (1) and (2) were answered by 17 open-ended questions conducted with 22 housing experts from 11 countries and 7 semi-structured interviews composed of 14 questions with 7 manufacturers of construction materials respectively which collected rich qualitative data (15,419 words) that were analysed in Nvivo 10 through pattern matching and validated by triangulation techniques to give reliability to the study. The housing prototype modelling was used to answer the research question (3) Can the BIM model show the cost-benefit in building housing with the 3D-LPs construction system and displaced people's own labour?The main findings of this study are that a housing prototype built with 3D-LPs is 36.82% cheaper in comparison to houses built with bricks and reinforcement elements and could be built by unskilled people in 90 days. The study provides novel in-depth knowledge of how unskilled people from communities affected should participate in housing reconstruction and how new construction systems can be implemented after disasters, which contributes to the body of knowledge. In addition, the study provides guidelines to implement a system directed at unskilled people and also Non-Governmental Organizations (NGOs) in a novel way, to help to solve the housing reconstruction problem and engage the displaced people in the housing reconstruction.

The aim of this thesis was an analyse of individual didactic situations and their valuation based on selected strategies. There was used video material from 20 lessons on linear equation collected from one basic and one grammar school. The conclusion of the analysis is supported by theories from available sources. This thesis might be useful for students of pedagogical faculties to deepen their knowledge and to help them avoid possible mistakes. It might be used also as a self-study material for teachers.

Esse trabalho teve por objetivo contribuir para o entendimento das novas dinâmicas de criação, difusão e exploração de conhecimento a partir da análise de dois SLPs de cerâmica de revestimento, um Santa Gertrudes/SP e outro em Criciúma/SC. Para compreender este fenômeno foi realizado um estudo exploratório em que foram identificadas a estrutura produtiva e de comercialização de algumas empresas, as formas de governança e o arcabouço institucional dos SLPs, bem como os processos de aprendizagem ocorridos entre os agentes ao longo do tempo. Apesar do escopo do trabalho estar mais relacionado com a geração de conhecimento via interação, a pesquisa também buscou investigar os processos de aprendizagem gerados a partir de fontes internas de conhecimento, isto é, aquelas em que o conhecimento pode ser gerado dentro da firma. O modelo interativo de inovação ressalta a relevância da cooperação entre firmas e demais instituições e, portanto, o papel dos vínculos e redes envolvendo diferentes organizações. Assim, ficou evidente que o conhecimento externo é um input essencial ao processo de geração de novos conhecimentos. O conhecimento nesses SLPs resulta das interações entre agentes heterogêneos capazes de aprender e estabelecer redes de relações, embora em um limitado espectro de atividades, mas enraizada em um espaço técnico e de produção limitados onde cada agente acumula competência por meio de processos de aprendizagem através de rotinas e de uso (learning-by-doing e learning-byusing). As chances de que seja gerado um novo conhecimento depende dos níveis de acumulação de habilidades e competências, educação e acesso à informação de outros agentes tanto de fora como internos aos SLPs. Esses agentes estão espalhados no espaço e cada um possui partes de conhecimento que podem se complementar. / The aim of this work is to understand the new dynamics on creation, diffusion and exploration of knowledge concerning SLPs, assuming that geography and innovation are deeply connected. This environment was studied taking an analysis of two local system of ceramic tile located in Criciúma/SC and Santa Gertrudes/SP. It was developed an exploratory study to understand this phenomena in which were identified the sale and productive structure of some firms, their governance and SLP’s institutional range, with a special focus on the learning processes occurring among the agents. The way the clusters interact with each other and with different agents reveals the importance of the cooperation as well the role of good connections among them. Thus, it became clear that external knowledge is an essential ingredient in the process of acquiring new methods. The union of all knowledge accumulated results in interactions among all the parts involved. They all are able to learn, to stabilish networks in a spectrum limited according to the technical space of each firm competence which is produced by means of learning processes derived from learning-by-doing and learning-by-using. The probabilities of generating a new knowledge depends on the levels of acquiring competence, abilities, educational level as well as the access to information of external and internal SLPs agents. These same agents are distributed in a certain space and each one of them possesses parts of the knowledge that can be complementary.

No description available.

The binding of CD80/CD86 on the APC to CD28 on the T cell surface provides a second signal for T cell activation. While it was once believed that this interaction represented a one-way signal, resulting in T cell activation, recently, it has been investigated as a bidirectional signaling process. CD80/86 activation produces IL-6 in DCs, but its role in macrophage activation is unknown. Dysregulation of CD80/86 expression has been observed in autoimmune disorders and cancer, and may also influence the development of immune responses including production of cytokines in response to stimulation with TLR-4 ligand, LPS. Therefore, the focus of my project was twofold: 1) to investigate the role of CD80/86 as signaling receptors capable of transmitting extracellular signals, and 2) to determine the TLR-4 activated pathways that regulate CD80/86 expression in human monocyte-derived macrophages (MDMs). Since I demonstrated that activation of CD80/86 alone did not induce expression of the four cytokines investigated, I hypothesized that CD80/86 synergizes with other signaling pathways. I show for the first time that CD80/86 activation synergizes with TLR-4 signaling to produce IL-27 and IL-10 in human MDMs. Since cIAPs play a key role in TLR-4-mediated signaling, I investigated their role in TLR-4- and CD80/86-activated production of IL-10 and IL-27. Degradation of IAPs by SMAC mimetics inhibited LPS-induced IL-10 and IL-27 production in MDMs. However, it did not alter the TLR-4 and CD80/86 synergistic effect on IL-10 and IL-27 production suggesting that IAPs may not play a role in CD80/86 activation of macrophages. Since I have demonstrated this role for IAPs, I extended my studies by examining the involvement of IAPs and other upstream signaling molecules such as SHP-1, RIP1, TRAF2, in modulating the LPS-induced CD80/86 expression. I showed that cIAP2, SHP-1, RIP1, TRAF2 co-localize to form a complex that regulates the LPS-induced CD80 and CD86 expression through AKT-activated p38 MAPK in human macrophages. These findings may lead to the development of novel therapeutic interventions in the treatment of autoimmune diseases.

Background: COPD is characterised by increased neutrophilic inflammation which further increases during exacerbations. Corticosteroids are currently one of the mainstays of treatment but they have limited effectiveness; there is a great need to develop new anti-inflammatory pharmacotherapies for use in COPD. Inhaled LPS has been used as a model of increased neutrophilic inflammation in healthy patients, smokers and asthmatics. Its use in patients with COPD as a model of exacerbations has not yet been evaluated. PI3 kinase is a vital intracellular enzyme, which upon activation leads to a number of cellular processes; the γ and δ isoforms of the enzyme are of particular importance in leucocyte migration, development and activation. There is increasing evidence for upregulation of this pathway in COPD.Aims: (1) To test the safety of the use of inhaled LPS in patients with COPD for use as a model of exacerbation and to investigate the systemic and airway inflammatory response in vivo. (2) To investigate the action of PI3 kinase enzyme inhibitors and dexamethasone in vitro on neutrophilic inflammation in COPD patients during the stable state and exacerbations. Methods: (1) 12 patients with mild to moderate COPD inhaled 5µg LPS; safety measurements and airway and systemic biomarkers were collected up to 24 hours post inhalation. (2) The effect of PI3 kinase enzyme inhibitors and dexamethasone on MMP-9 and ROS release from peripheral and airway neutrophils from stable COPD and exacerbations was examined in vitro. The effect of PI3 kinase enzyme inhibitors and dexamethasone on cytokine release from peripheral neutrophils from stable COPD patients was also investigated. Results: (1) Inhaled LPS (5µg) caused a significant fall in FEV1 and increase in sputum neutrophil numbers. There was an associated increase in systemic IL-6, CRP and CC-16, all with differing temporal patterns. No patients reported any significant symptoms. (2) PI3 kinase enzyme inhibitors significantly reduced MMP-9 and ROS release from airway and peripheral neutrophils from COPD patients in the stable state and during exacerbations; dexamethasone had minimal effect. Cytokine release from peripheral neutrophils from COPD patients in the stable state was also significantly inhibited by PI3 kinase enzyme inhibitors and dexamethasone. Conclusions: (1) Inhaled LPS in patients with COPD is a safe model to induce acute on chronic neutrophilic inflammation and therefore could be used as a model to study COPD exacerbations. (2) PI3 kinase enzyme inhibitors reduce COPD neutrophil MMP-9, ROS and cytokine release in vitro and are therefore are a promising new anti-inflammatory pharmacotherapy.

Doctor en Ciencias, Mención Computación / Una malla es una discretización de la geometría de un cierto dominio. Las mallas pueden estar compuestas de diversos elementos: triángulos, cuadriláteros, tetraedros, etc. Una herramienta para la generación de mallas es un aplicación que permite crear, refinar, desrefinar, mejorar, suavizar, visualizar y posprocesar mallas y/o una región particular de ella, como también asignar valores físicos a los elementos de la malla (temperatura, concentración, etc.). Las herramientas para la generación de mallas son complejas y sofisticadas, y construir una herramienta nueva desde cero o mantener una existente, demanda un esfuerzo enorme. Existe una necesidad y oportunidad para usar enfoques nuevos en el desarrollo de estas herramientas, de manera de reducir tanto el tiempo como los costos de desarrollo, sin comprometer la calidad. La experiencia en el desarrollo de estas herramientas provee la motivación para la construcción de otras nuevas mediante la reutilización del trabajo realizado durante los desarrollos previos. Estas herramientas comparten varias características y sus variaciones pueden ser manejadas sistemáticamente. Esto hace que el desarrollo de estas herramientas sea una buena oportunidad para aplicar el enfoque de Línea de Productos de Software (LPS). Los procesos existentes de LPS son generales y requieren usualmente una serie de pasos y documentación innecesaria en el dominio de las herramientas para la generación de mallas. Así, esta tesis propone un modelo de proceso de LPS específico para este tipo de herramientas. Un proceso de desarrollo de LPS está centrado en la reutilización de software, e involucra principalmente dos fases: la ingeniería del dominio (ID) y la ingeniería de la aplicación (IA). El proceso presentado en este trabajo está centrado en dos etapas de la ID: el análisis del dominio (AD) y el diseño del dominio (DD). En el AD se define el modelo del dominio y el alcance de la LPS. En el DD la arquitectura de la línea de productos (ALP) es creada; esta arquitectura es válida y compartida por todos los productos en la LPS. Un modelo de características es comúnmente usado para modelar el dominio. En este trabajo, el AD también ocupa un diccionario, escenarios, acciones y metas para proveer el razonamiento utilizado para la construcción del modelo de características. Esta tesis presenta un proceso riguroso para obtener el modelo del dominio. Este modelo es formalizado mediante condiciones de consistencia y completitud. El proceso de definición del alcance es presentado a través de un diagrama de actividad. Además, el enfoque presentado en esta tesis presenta explícitamente los diferentes productos de la LPS, estableciendo relaciones entre productos y las características de la LPS, lo que permite administrar el desarrollo del producto. La etapa de DD se centra en la creación de la ALP, artefacto esencial para la construcción de productos de la LPS. Para ello, este trabajo provee un proceso deductivo y otro transformacional. En el primero, una ALP explícita es desarrollada, usando los artefactos producidos en el AD. Además, tanto la vista arquitectónica estructural como la de comportamiento son establecidas. Ambas vistas son generales y permiten la representación de cualquier producto dentro del alcance de la LPS. En el proceso transformacional, una ALP implícita es desarrollada usando reglas de transformación, las que han sido creadas usando artefactos producidos en el AD. En este proceso se produce la arquitectura para productos específicos, y la ALP es definida como la suma de todas las arquitecturas de los productos. Tanto el AD como el DD son descritos en detalle, y la aplicación del modelo de la LPS es ilustrado a través de un ejemplo bien documentado en el dominio de las herramientas para la generación de mallas, el que tiene un grado relativamente alto de complejidad. En este ejemplo, un modelo del dominio formalizado es introducido, y la arquitectura es definida tanto para el proceso deductivo como para el transformacional.