Les opérations géométriques appliquées aux maillages 3D introduisent des dis torsions géométriques qui peuvent être visibles pour un observateur humain. Dans cette thèse, nous étudions l’impact perceptuel de ces distorsions. Plus précisément, notre objectif est de calculer le seuil à partir duquel les distorsions géométriques locales deviennent visibles. Afin d’atteindre notre but, nous définissons tout d’abord des caractéristiques perceptuelles pour les maillages 3D. Nous avons ensuite effectué une étude expérimentale des propriétés du système visuel humain (sensibilité au contraste et effet du masquage visuel) en observant un maillage 3D. Les résultats de ces expériences sont finalement utilisés pour proposer un algorithme qui calcule le seuil de visibilité relatif à une distorsion locale. L’algorithme proposé s’adapte aux différentes conditions d’affichage (résolution et taille de l’écran), d’illumination et au type de rendu. Enfin, nous montrons l’utilité d’un tel algorithme en intégrant le seuil de visibilité dans le pipeline de plusieurs opérations géométriques (ex: simplification, subdivision adaptative) / Geometric operations applied to a 3D mesh introduce geometric distortion in the form of vertex displacement that can be visible to a human observer. In this thesis, we have studied the perceptual impact of these geometric distortions. More precisely, our goal is to compute the threshold beyond which a local geometric distortion becomes visible. In order to reach this goal, we start by evaluating perceptually relevant properties on 3D meshes. We have then performed a series of psychophysical experiments in which we measured the visibility threshold relative to various properties of the Human Visual System (contrast sensitivity and visual masking). The results of these experiments allowed us to propose an algorithm that computes the visibility threshold relative to a local geometric distortion. This algorithm is capable of adapting to the different display condition of 3D meshes (resolution, display size, illumination condition and rendering). Finally, we showcase the utility of our work by integrating the developed perceptual method in several geometric operations such as mesh simplification and adaptive subdivision

O Fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) produzido em Escherichia coli é uma proteína não glicosilada com 175 aminoácidos, de grande importância clínica para o tratamento de neutropenias. O presente trabalho propõe a construção de dois sistemas de expressão em E. coli, um sistema para obtenção do rhG-CSF no citoplasma e outro para secreção da proteína recombinante no meio de cultura utilizando a sequência sinal da L-asparaginase II. Os dois sistemas de expressão foram testados e comparados. A partir desses dados, passou-se para as etapas de obtenção do rhG-CSF com o sistema de expressão sem a sequência sinal. As etapas de renaturação e purificação foram eficientes obtendo-se uma proteína com adequado grau de pureza, integridade estrutural e atividade biológica. Essa proteína também foi utilizada com sucesso para a produção de anticorpos policlonais em camundongos. Com os resultados obtidos, a proteína rhG-CSF mostrou-se viável para estudos posteriores em bioreatores e produção em escala-piloto. / The recombinant human granulocyte colony stimulating factor (rhG-CSF) is a non-glycosylated protein with 175 amino acids. This factor plays an important role in hematopoietic cell proliferation and has been widely used for treating neutropenia. The purpose of this work is to construct two expression systems in E. coli; a system for obtaining rhG-CSF in the cytoplasm and the other for secretion of recombinant protein in the culture medium using the signal sequence of L-asparaginase II. The two expression systems were tested and compared. From these data, the next steps for obtaining the rhG-CSF were done with the expression system without the signal sequence. The refolding and purification steps were efficient, resulting in a protein with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of polyclonal antibodies in mice. With these results, the protein rhG-CSF was feasible for further studies in bioreactors and pilot scale production.

Charakterisierung des Zytokinprofils in Erwachsenen mit einer latenten oder aktiven Tuberkulose in einem hoch endemischen Gebiet Die Tuberkulose (TB) stellt mit rund 2 Milliarden Infizierten weltweit ein globales gesundheitliches Problem dar. Während die große Mehrheit der infizierten Personen in der Lage sind die Krankheit zu kontrollieren, entwickelt sich bei ungefähr 10 % die aktive Form der TB aus. Der zugrunde liegende immunologische Prozess für diese Verteilung ist bis heute nicht bekannt und im Fokus dieser Arbeit. Das adaptive Immunsystem spielt eine entscheidende Rolle in der Immunabwehr gegen Mycobacterium tuberculosis (M. tuberculosis), dem Erreger der TB. Hierbei sind besonders CD4+ T-Zellen für die erfolgreiche Eingrenzung der Erkrankung verantwortlich. Im Vorfeld konnte bereits mehrmals eine Assoziation zwischen polyfunktionalen CD4+ T-Zellen und einem Schutz gegen verschiedenste Krankheitserreger gezeigt werden. Im Rahmen dieser Doktorarbeit wird versucht die Frage zu beantworten, ob eine erhöhte Frequenz von polyfunktionalen CD4+ T-Zellen auch gegen die Ausbildung einer aktiven TB schützen kann. Zur Bearbeitung dieser Fragestellung wurde das TH1 Zytokinprofil von Patienten mit aktiver TB untersucht und mit dem von gesunden latent infizierten Probanden (LTBI) verglichen. Desweiteren wurden die TB Patienten während der antimikrobiellen Therapie begleitet um Änderungen im Zytokinprofil von CD4+ T-Zellen beobachten zu können. Im Rahmen dieser Arbeit wurde zum ersten Mal die simultane Expression der vier TH1 Zytokine IFNg, TNFa, IL-2 und GM-CSF mit Hilfe der multifarben Durchflusszytometrie untersucht. Nach antigenspezifischer Stimulation konnten sowohl in unbehandelten und behandelten Patienten mit aktiver TB ein großer Anteil an multifunktionale Gedächtnis-T-Zellen nachgewiesen werden, die alle vier Zytokine gleichzeitig exprimierten. Bemerkenswerterweise konnte diese Population ebenfalls in LTBI gezeigt werden. Nach den ersten zwei Monaten der Therapie war der Anteil an multifunktionalen T-Zellen signifikant erhöht welches auf einen positiven Einfluss dieser Zellen auf die Behandlung hinweist. Um detaillierte Information über das Expressionspotential von CD4+ T-Zellen zu gewinnen wurden PBMCs mit einem Superantigen inkubiert. Hierbei unterschied sich das Zytokinprofil zwischen den beiden Studiengruppen signifikant und veränderte sich ebenfalls unter Therapie. Während die Expression von IFNg in TB Patienten niedriger war als in LTBI, war die Frequenz von TNFa, IL-2 und GM-CSF-positiver CD4+ T-Zellen signifikant höher in Patienten mit aktiver TB. Zusammenfassend ist zu sagen, dass sowohl in TB Patienten vor und nach Therapie, als auch in LTBI, multifunktionale CD4+ T-Zellen nachgewiesen werden können. Ein Unterschied in der Frequenz konnte dabei nicht festgestellt werden. Daher kann ein Zusammenhang zwischen der Existenz von multifunktionellen CD4+ T-Zellen und einem Schutz gegen eine mögliche Reaktivierung von der latenten zu der aktiven TB nicht beschrieben werden. / Characterization of the cytokine profile in adults with latent and active tuberculosis from a high endemic country Tuberculosis (TB) is a global health problem with ~2 billion infected people worldwide. The vast majority of infected individuals is able to control TB, while only ~10% develop active disease. The immunologic correlates determining the protection against reactivation of the latent form of active TB remain elusive. The adaptive immune system plays an important role in the response against Mycobacterium tuberculosis (M. tuberculosis), especially CD4+ T cells are crucial for efficient containment of the pathogen. Since polyfunctional CD4+ T cells have been associated with protection against various pathogens, the question was raised if higher frequencies of polyfunctional CD4+ T cells can be linked to protection against reactivation of active TB. To address this the TH1 T cell cytokine profile of active TB patients was analyzed and compared with healthy latently infected individuals (LTBI). Furthermore TB patients were followed up under anti-microbial therapy to monitor changes in the cytokine pattern expressed by CD4+ T cells. Hereby, for the first time, the simultaneous expression of four TH1 cytokines, IFNg, TNFa, IL-2 and GM-CSF, was investigated using multi color flow cytometry. After antigen-specific stimulation multifunctional memory T cells (CD45RO+) co-expressing IFNg, TNFa, IL-2 and GM-CSF were strongly represented in both treated and untreated TB patients. Interestingly, this proportion of polyfunctional memory T cells was also found in LTBI. After the first two months of drug treatment the proportion of antigen-specific polyfunctional T cells was significantly increased, indicating a positive impact of these cells during therapy. To gain detailed information about the potential of CD4+ T cells to produce cytokines we incubated PBMCs with a superantigen. In this case the profile was significantly different between these two groups and it changed during therapy. While the expression of IFNg was significantly lower in CD4+ T cell of TB patients in comparison to LTBI, the expression of TNFa, IL2 and GM-CSF showed significant higher frequencies in memory T cells of TB patients. To conclude, upon antigen stimulation, polyfunctional memory T cells are found in TB patients pre- and post therapy as well as in LTBI. A difference in the frequency between active TB patients and LTBI could not be detected and therefore a correlation with protection against reactivation from the latent to the active form of TB cannot be drawn.

Les lésions spinales conduisent à des altérations majeures des fonctions sensorimotrices. Les récupérations fonctionnelles consécutives à ces atteintes sont très limitées, notamment en raison des capacités réduites de réparation des tissus endommagés dans le SNC. En outre, ces récupérations dépendent notamment de plusieurs processus cellulaires tels que l'activation astrogliale qui conduit à la formation de la cicatrice gliale, ou encore l'inflammation dont les cellules microgliales et les mastocytes sont les effecteurs les plus précoces. Cette inflammation est connue pour exacerber les dommages tissulaires et restreindre les possibilités de récupération. Cependant, des études récentes chez l'animal et chez l'Homme montrent que l'inflammation pourrait également avoir des effets favorisant les processus de récupération. Le but de cette thèse était de mieux comprendre les liens qui existent entre neuroinflammation, neuroplasticité et récupérations fonctionnelles après lésion spinale. L’objectif expérimental visait à examiner les réactivités microgliales, mastocytaires et astrocytaires post-lésionnelles, en parallèle avec des restaurations fonctionnelles. Dans ce contexte nous nous sommes plus particulièrement intéressés à l'influence d'une cytokine pro-inflammatoire, le Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) sur ces processus inflammatoires et la plasticité fonctionnelle après une hémisection C4-C5 chez le rat adulte. L’ensemble de nos travaux suggère que le GM-CSF pourrait agir par l’intermédiaire de plusieurs événements cellulaires et moléculaires, en favorisant des phénomènes de plasticité adaptatifs et la récupération partielle de fonctions altérées. / Spinal cord injuries are mostly of traumatic origin and result in major sensorimotor deficits. Postlesion functional recovery is limited, especially because of the reduced capacity of repairing damaged tissues. Moreover, this recovery depends specifically on several cellular processes such as astroglial activation conducting to glial scar formation, or inflammation for which microglial and mast cells are the earliest effectors. This inflammation is known to exacerbate tissue damages and restrain the capacity to recover. However, recent studies in animals and humans show that inflammation may also have beneficial aeffects on recovery processes. The studies conducted during my doctoral research were intended to better understand the links between neuroinflammation, neuroplasticity and functional recovery following spinal cord injury. We aimed at examining microglial, mast cells and astroglial reactivities after the injury, in relation with functional recovery of somatosensory and motor functions. In this context, we were particularly interested in the influence of Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) on inflammatory and plasticity mechanisms after a C4-C5 hemisection in the adult rat. Our doctoral research suggests that GM-CSF could act through several cellular and molecular events promoting adaptive plasticity phenomena underlying partial recovery of impaired functions.

O tráfego de leucócitos é um processo complexo, dependente da ação de inúmeras substâncias químicas, além da perfeita interação celular. Desta forma, este estudo teve como objetivo avaliar a ação dos GCe e da ANXA1 sobre o eixo SDF-1α/CXCR4 e IL-17/IL-23/G-CSF e sobre a expressão de moléculas de adesão CD18, CD49d e CD62L. Foram utilizados camundongos machos Balb/C selvagens (WT) ou ANXA1-/-. As avaliações foram realizadas em condições basais, na presença de altas concentrações de GCe e na vigência de processo inflamatório, induzidos pela administração de ACTH (5 µg/animal, i.p.) ou pela injeção de LPS (100 µg/kg, i.p.), respectivamente, ou na ausência da ação dos GCe, pela ação do RU 38486 (RU, 10 mg/kg, i.p.). A participação da ANXA1 e do receptor FPR2 foi avaliada pelo pré-tratamento com Ac2-26 (1 mg/Kg, i.p.) ou com BOC2 (10 µg/animal, i.p.) durante 4 dias, 1 vez ao dia. A quantificação total e diferencial das células foi realizada em câmara de Neubauer e em esfregaços corados por May-Grunwald ou citometria de fluxo. As quantificações de CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L e maturação granulocítica (CD11b/Ly6G) em células da medula e da circulação foram realizadas por citometria de fluxo. A expressão de ANXA1 nos tecidos do estomago e do baço foi realizada por western blotting e nas células da medula óssea e sangue circulante foi realizada por imunofluorescência. As quantificações de IL-17, IL-23, G-CSF, SDF-1α e corticosterona foram realizadas por ELISA. A quimiotaxia de neutrófilos da medula óssea e sangue periférico foi ensaiada na placa de quimiotaxia com filtro de poro de 8 µm. A fagocitose de neutrófilos apoptóticos por macrófagos da medula óssea foi avaliada por ensaio in vitro. Para verificar os efeitos do ACTH na migração de neutrófilos no processo inflamatório, foi empregado o modelo de bolsa de ar (100 µg/mL; LPS); e o comportamento dos leucócitos circulantes de animais tratados com ACTH foi avaliado pela técnica de microscopia intravital. Os resultados obtidos, que estão apresentados em quatro temáticas, mostraram que: 1) neutrófilos da medula óssea e sangue periférico expressam ANXA1 no citoplasma e membrana, bem como o receptor FPR2, constitutivamente, e a expressão de ambos é regulada pelos GCe. A ANXA1, via receptor FPR2 expresso em células da medula óssea, controlam a maturação neutrofílica e o tráfego destas células da medula óssea para o sangue. A ANXA1, via interação ao FPR2, controla o clearance de neutrófilos do sangue para a medula óssea, modulando o eixo SDF-1α/CXCR4; 2) A administração do ACTH causa neutrofilia e os neutrófilos circulantes são ANXA1+, CD18+, CD49d+, CD62L+, mostrando que injeção do ACTH in vivo altera o fenótipo destas células na circulação. Estas modificações alteram o comportamento dos neutrófilos na circulação, bem como a migração para a bolsa de ar na vigência de inflamação e para os tecidos de clearance. Estes efeitos podem ser dependentes, pelo menos em parte, da inibição de migração orientada, já que quimiotaxia frente ao fMLP ou ao SDF-1α estavam reduzidas. Ainda, o clearance de neutrófilos é reduzido em animais tratados com o ACTH pela menor atividade fagocítica e secretora dos macrófagos medulares; 3) Animais tratados com RU 38486 e ANXA1-/- mobilizam granulócitos da medula óssea para o sangue circulante e, deste compartimento para o foco de inflamação com maior intensidade que o observado em animais controles. O eixo IL-17/IL-23/G-CSF parece estar envolvido na granulopoiese e na mobilização de neutrófilos para o sangue durante a inflamação, mas não é alvo de ação da ANXA1 e o GCe nesta etapa do processo inflamatório. Adicionalmente, foi observado que na vigência de peritonite, as moléculas de adesão, CD49d e CD62L estão envolvidas no processo de migração de neutrófilos da medula óssea para o sangue. Os resultados aqui obtidos permitem concluir que os GCe e a ANXA1 são relevantes para granulopoiese e tráfego dos neutrófilos da medula óssea em condições fisiológicas e na vigência de processo inflamatório. Ainda, em conjunto com os dados da literatura, os nossos resultados podem sugerir a participação da ANXA1 dos GCe na plasticidade fenotípica dos neutrófilos de acordo com os estímulos a que são submetidos, e podem auxiliar na compreensão dos novos conceitos sobre a produção, tempo de vida, localização e funções de neutrófilos. / The traffic leukocytes is a complex process dependent on the action of severals chemical mediators, in addition to perfect cell interaction. Therefore, this study aimed to evaluate the effect of GCe and ANXA1 on SDF-1α/CXCR4 and IL-17/IL-23/G-CSF and on the expression of adhesion molecules CD18, CD49d and CD62L. Balb/C wild type and ANXA1-/- male mice were employed. The analysis were performed at physiological conditions, in the presence of high concentrations of GCe and during of inflammatory process induced by ACTH administration (5 µg/animal, i.p.) or LPS injection (100 µg/kg, i.p.), respectively or in the absence of GCe action, by the action of RU 38486 (RU, 10 mg/kg , i.p.). The involvement of the receptor FPR2 and ANXA1 was assessed by pre-treatment with Ac2-26 (1 mg/kg, i.p.) or BOC2 (10 µg/animal, i.p.) for 4 days, once a day. The quantification of total and differential cell was performed in a Neubauer chamber and stained smears by May-Grunwald and flow cytometry. Quantification of expression of CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L and granulocytic maturation (CD11b/Ly6G) in the bone marrow and circulation were performed by flow cytometry. The expression of ANXA1 on tissues was performed by western blotting and on cells from bone marrow and blood by immunocytochemistry. Quantification of IL-17, IL-23, G-CSF, SDF-1α and corticosterone were performed by ELISA. The chemotaxis of neutrophils from the bone marrow and blood was tested in the chemotaxis chamber with filter pore of 8 microns. The phagocytosis of apoptotic neutrophils by bone marrow macrophages was assessed by in vitro assay. To investigate the effects of ACTH in the migration of neutrophils in the inflammatory process, the model employed was air pouch (100 µg/ ml, LPS), and the behavior of circulating leukocytes from animals treated with ACTH were evaluated by intravital microscopy. The results obtained, which are presented in three sections, showed that: 1) neutrophils from the bone marrow and blood expressed ANXA1 in the cytoplasm and membrane, as well as FPR2, constitutively and the expression of both is regulated by GCe. The ANXA1 via FPR2 receptor expressed in bone marrow cells, controls the neutrophilic maturation and traffic of these cells from the bone marrow into the blood. The ANXA1 via interaction to FPR2 controls the clearance of neutrophils from the blood to the bone marrow by modulating the SDF-1α/CXCR4 axis; 2) the administration of ACTH induces neutrophilia and the circulating neutrophils are ANXA1+, CD18+, CD49d+ and CD62L+, showing that the injection of ACTH in vivo alters the phenotype of these cells in the blood. These modifications alter the behavior of neutrophils in the blood, as well as the migration to the air pouch in the presence of inflammation and to the tissue clearance, and these effects may be dependent, at least in part, on inhibition of migration oriented events, as chemotaxis in response to fMLP or SDF-1α were reduced. Further, the clearance of neutrophils is reduced in animals treated with ACTH due to the lower phagocytic and secretory activity of medullary macrophages; 3) Animals treated with RU 38486 and ANXA1-/- mobilize granulocytes from bone marrow into the blood, and from this compartment to the focus of inflammation with higher intensity than that observed in the control group. The axis IL-17/IL-23/G-CSF seems to be involved in granulopoiesis and mobilization of neutrophils into the blood during inflammation, but it is not the target of action of ANXA1 and GCe at this step of inflammatory process. Additionally, it was observed that in the presence of peritonitis, the adhesion molecules, CD49d and CD62L are involved in the migration of neutrophils from the bone marrow into the blood. The results obtained allow concluding that the GCe and ANXA1 are relevant to the granulopoiesis and the traffic of neutrophils from bone marrow under physiological conditions and in the presence of inflammation. Furthermore, together with literature data, the data presented here may suggest the involvement of ANXA1 the GCe in phenotypic plasticity of neutrophils according to the stimuli that are submitted, and may support to understand the new concepts of production, half-life, location and function of neutrophils.

Introduction Adipocytes are part of hematopoietic microenvironment, even though they are generally believed to play no active role during hematopoiesis. We have shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. Material and Methode To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. Results Morphological and immunophenotypic analysis of FLFCs in co-culture with CD34+ cells revealed that FLFCs constitutively produced M-CSF and induced CD34+ differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with G-CSF production. Both functions were also recovered when FLFCs cultured in contact with CD34+ cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. Conclusion Our data provide the first evidence that adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes.

O tráfego de leucócitos é um processo complexo, dependente da ação de inúmeras substâncias químicas, além da perfeita interação celular. Desta forma, este estudo teve como objetivo avaliar a ação dos GCe e da ANXA1 sobre o eixo SDF-1α/CXCR4 e IL-17/IL-23/G-CSF e sobre a expressão de moléculas de adesão CD18, CD49d e CD62L. Foram utilizados camundongos machos Balb/C selvagens (WT) ou ANXA1-/-. As avaliações foram realizadas em condições basais, na presença de altas concentrações de GCe e na vigência de processo inflamatório, induzidos pela administração de ACTH (5 µg/animal, i.p.) ou pela injeção de LPS (100 µg/kg, i.p.), respectivamente, ou na ausência da ação dos GCe, pela ação do RU 38486 (RU, 10 mg/kg, i.p.). A participação da ANXA1 e do receptor FPR2 foi avaliada pelo pré-tratamento com Ac2-26 (1 mg/Kg, i.p.) ou com BOC2 (10 µg/animal, i.p.) durante 4 dias, 1 vez ao dia. A quantificação total e diferencial das células foi realizada em câmara de Neubauer e em esfregaços corados por May-Grunwald ou citometria de fluxo. As quantificações de CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L e maturação granulocítica (CD11b/Ly6G) em células da medula e da circulação foram realizadas por citometria de fluxo. A expressão de ANXA1 nos tecidos do estomago e do baço foi realizada por western blotting e nas células da medula óssea e sangue circulante foi realizada por imunofluorescência. As quantificações de IL-17, IL-23, G-CSF, SDF-1α e corticosterona foram realizadas por ELISA. A quimiotaxia de neutrófilos da medula óssea e sangue periférico foi ensaiada na placa de quimiotaxia com filtro de poro de 8 µm. A fagocitose de neutrófilos apoptóticos por macrófagos da medula óssea foi avaliada por ensaio in vitro. Para verificar os efeitos do ACTH na migração de neutrófilos no processo inflamatório, foi empregado o modelo de bolsa de ar (100 µg/mL; LPS); e o comportamento dos leucócitos circulantes de animais tratados com ACTH foi avaliado pela técnica de microscopia intravital. Os resultados obtidos, que estão apresentados em quatro temáticas, mostraram que: 1) neutrófilos da medula óssea e sangue periférico expressam ANXA1 no citoplasma e membrana, bem como o receptor FPR2, constitutivamente, e a expressão de ambos é regulada pelos GCe. A ANXA1, via receptor FPR2 expresso em células da medula óssea, controlam a maturação neutrofílica e o tráfego destas células da medula óssea para o sangue. A ANXA1, via interação ao FPR2, controla o clearance de neutrófilos do sangue para a medula óssea, modulando o eixo SDF-1α/CXCR4; 2) A administração do ACTH causa neutrofilia e os neutrófilos circulantes são ANXA1+, CD18+, CD49d+, CD62L+, mostrando que injeção do ACTH in vivo altera o fenótipo destas células na circulação. Estas modificações alteram o comportamento dos neutrófilos na circulação, bem como a migração para a bolsa de ar na vigência de inflamação e para os tecidos de clearance. Estes efeitos podem ser dependentes, pelo menos em parte, da inibição de migração orientada, já que quimiotaxia frente ao fMLP ou ao SDF-1α estavam reduzidas. Ainda, o clearance de neutrófilos é reduzido em animais tratados com o ACTH pela menor atividade fagocítica e secretora dos macrófagos medulares; 3) Animais tratados com RU 38486 e ANXA1-/- mobilizam granulócitos da medula óssea para o sangue circulante e, deste compartimento para o foco de inflamação com maior intensidade que o observado em animais controles. O eixo IL-17/IL-23/G-CSF parece estar envolvido na granulopoiese e na mobilização de neutrófilos para o sangue durante a inflamação, mas não é alvo de ação da ANXA1 e o GCe nesta etapa do processo inflamatório. Adicionalmente, foi observado que na vigência de peritonite, as moléculas de adesão, CD49d e CD62L estão envolvidas no processo de migração de neutrófilos da medula óssea para o sangue. Os resultados aqui obtidos permitem concluir que os GCe e a ANXA1 são relevantes para granulopoiese e tráfego dos neutrófilos da medula óssea em condições fisiológicas e na vigência de processo inflamatório. Ainda, em conjunto com os dados da literatura, os nossos resultados podem sugerir a participação da ANXA1 dos GCe na plasticidade fenotípica dos neutrófilos de acordo com os estímulos a que são submetidos, e podem auxiliar na compreensão dos novos conceitos sobre a produção, tempo de vida, localização e funções de neutrófilos. / The traffic leukocytes is a complex process dependent on the action of severals chemical mediators, in addition to perfect cell interaction. Therefore, this study aimed to evaluate the effect of GCe and ANXA1 on SDF-1α/CXCR4 and IL-17/IL-23/G-CSF and on the expression of adhesion molecules CD18, CD49d and CD62L. Balb/C wild type and ANXA1-/- male mice were employed. The analysis were performed at physiological conditions, in the presence of high concentrations of GCe and during of inflammatory process induced by ACTH administration (5 µg/animal, i.p.) or LPS injection (100 µg/kg, i.p.), respectively or in the absence of GCe action, by the action of RU 38486 (RU, 10 mg/kg , i.p.). The involvement of the receptor FPR2 and ANXA1 was assessed by pre-treatment with Ac2-26 (1 mg/kg, i.p.) or BOC2 (10 µg/animal, i.p.) for 4 days, once a day. The quantification of total and differential cell was performed in a Neubauer chamber and stained smears by May-Grunwald and flow cytometry. Quantification of expression of CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L and granulocytic maturation (CD11b/Ly6G) in the bone marrow and circulation were performed by flow cytometry. The expression of ANXA1 on tissues was performed by western blotting and on cells from bone marrow and blood by immunocytochemistry. Quantification of IL-17, IL-23, G-CSF, SDF-1α and corticosterone were performed by ELISA. The chemotaxis of neutrophils from the bone marrow and blood was tested in the chemotaxis chamber with filter pore of 8 microns. The phagocytosis of apoptotic neutrophils by bone marrow macrophages was assessed by in vitro assay. To investigate the effects of ACTH in the migration of neutrophils in the inflammatory process, the model employed was air pouch (100 µg/ ml, LPS), and the behavior of circulating leukocytes from animals treated with ACTH were evaluated by intravital microscopy. The results obtained, which are presented in three sections, showed that: 1) neutrophils from the bone marrow and blood expressed ANXA1 in the cytoplasm and membrane, as well as FPR2, constitutively and the expression of both is regulated by GCe. The ANXA1 via FPR2 receptor expressed in bone marrow cells, controls the neutrophilic maturation and traffic of these cells from the bone marrow into the blood. The ANXA1 via interaction to FPR2 controls the clearance of neutrophils from the blood to the bone marrow by modulating the SDF-1α/CXCR4 axis; 2) the administration of ACTH induces neutrophilia and the circulating neutrophils are ANXA1+, CD18+, CD49d+ and CD62L+, showing that the injection of ACTH in vivo alters the phenotype of these cells in the blood. These modifications alter the behavior of neutrophils in the blood, as well as the migration to the air pouch in the presence of inflammation and to the tissue clearance, and these effects may be dependent, at least in part, on inhibition of migration oriented events, as chemotaxis in response to fMLP or SDF-1α were reduced. Further, the clearance of neutrophils is reduced in animals treated with ACTH due to the lower phagocytic and secretory activity of medullary macrophages; 3) Animals treated with RU 38486 and ANXA1-/- mobilize granulocytes from bone marrow into the blood, and from this compartment to the focus of inflammation with higher intensity than that observed in the control group. The axis IL-17/IL-23/G-CSF seems to be involved in granulopoiesis and mobilization of neutrophils into the blood during inflammation, but it is not the target of action of ANXA1 and GCe at this step of inflammatory process. Additionally, it was observed that in the presence of peritonitis, the adhesion molecules, CD49d and CD62L are involved in the migration of neutrophils from the bone marrow into the blood. The results obtained allow concluding that the GCe and ANXA1 are relevant to the granulopoiesis and the traffic of neutrophils from bone marrow under physiological conditions and in the presence of inflammation. Furthermore, together with literature data, the data presented here may suggest the involvement of ANXA1 the GCe in phenotypic plasticity of neutrophils according to the stimuli that are submitted, and may support to understand the new concepts of production, half-life, location and function of neutrophils.

Abstract The filtration of pulp suspension is one of the key papermaking processes. It is measured using many kinds of analyzers and modeled using both physical and empirical mathematical expressions. The main target of this thesis was to develop an empirical mathematical model to describe the filtration of a drainage analyzer. In this work the diameter of the screen (10 cm), as well as the amount (1000 cm3), consistency (0.3%) and temperature (20 ˚C) of the sample were (about) the same as those of the Canadian Standard Freeness analyzer. The analyzer was equipped with the measurements of filtrated volume, driving pressure and filtrate consistency. The pressures from the underpressure of 80 kPa to the overpressure of 80 kPa could be used. Pulp types of SGW, PGW, TMP and chemical pulp with freeness levels from 30 to 600 ml were analyzed. A conditioner of the sample volume, temperature and consistency measurements was constructed. The error of freeness value caused by the errors of the conditioner was evaluated to be less than 1%. The equations for the calculation of the possible initial values of the controlled quantities were derived. The optically measured consistencies of the filtrates were different for different pulp types and decreased close to zero before the filtrated volume of 150 cm3. The filtration seemed to change the portion of optically active fines. In the model of this work the filtration time (t) is proportional to an experimental power of the filtrated volume (V): Ve = kt. An auxiliary constant (Ve = kt + V0e) was used during the regression analyses to overcome the disturbances and non-validity of the model in the beginning of the filtration. The correlation coefficients of the fit were higher than 0.999. The exponent 2 suggested by Darcy’s law is a special case while the exponent has also been close to 1 or 3. The curves of specific filtration resistances, based on the model and pressure measurements, were shown to change during the course of drainage and as a function of pressure additionally dependent on the pulp type. The model was successfully used to calculate freeness values and to filter noise from the measurements. / Tiivistelmä Paperimassasulpun suotauttaminen on paperinvalmistuksen avainprosesseja. Sitä on mitattu monenlaisilla analysaattoreilla ja kuvattu sekä fysikaalisilla että kokeellisilla matemaattisilla malleilla. Tämän tutkimuksen päätavoite on kehittää suotautuvuusanalysaattorin kokeellista matemaattista mallia. Tutkimuksessa viiran halkaisija (10 cm) sekä näytteen määrä (1000 cm3), sakeus (0.3 %) ja lämpötila (20 ˚C) olivat suunnilleen samat kuin Canadian standard freeness –analysaattorissa. Järjestelmä mittasi suotautettua tilavuutta, suotauttavaa painetta ja suodoksen sakeutta. Suotautuspaineet olivat 80 kPa:n ali- ja ylipaineen väliltä. Testattavana oli hioketta, painehioketta ja termohierrettä sekä kemiallista massaa, joiden freeness oli 30 ml:sta 600 ml:aan. Analysaattoriin rakennettu vakiointijärjestelmä sääti näytteen sakeuden, tilavuuden ja lämpötilan niin tarkasti halutuiksi, että näiden vaihtelu ei olisi muuttanut freeness-arvoa edes prosenttia. Suureiden kehittymiselle johdettiin kaavat. Niiden avulla voidaan laskea ne näytteen arvoalueet, joilta halutut tavoitearvot voidaan saavuttaa. Optisesti mitattu suodoksen sakeus riippui massatyypistä ja hiipui lähes nollaksi ennen kuin 150 ml oli suotautettu. Suotautus muutti optisesti aktiivisen hienoaineen osuutta. Tämän työn suodoskäyrän mallissa aika (t) on verrannollinen suotautetun tilavuuden (V) kokeellisen potenssiin: Ve = kt. Mallinnuksen ajaksi lisätään apuparametri (Ve = kt+V0e), jotta suotautuksen alku ei huononna mallia. Sovituksen korrelaatiokerroin oli yli 0.999. Eksponentin arvo vaihteli vähän yli yhdestä melkein kolmeen, joten Darcyn lain mukainen eksponentin arvo 2 osoittautui erikoistapaukseksi. Mallin ja painemittauksen avulla lasketut ominaisresistanssit muuttuivat suotautuksen kuluessa ja riippuivat myös massatyypistä. Mallin avulla voitiin laskea näytteen freeness sekä suodattaa mittauskohinaa.

O Fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) produzido em Escherichia coli é uma proteína não glicosilada com 175 aminoácidos, de grande importância clínica para o tratamento de neutropenias. O presente trabalho propõe a construção de dois sistemas de expressão em E. coli, um sistema para obtenção do rhG-CSF no citoplasma e outro para secreção da proteína recombinante no meio de cultura utilizando a sequência sinal da L-asparaginase II. Os dois sistemas de expressão foram testados e comparados. A partir desses dados, passou-se para as etapas de obtenção do rhG-CSF com o sistema de expressão sem a sequência sinal. As etapas de renaturação e purificação foram eficientes obtendo-se uma proteína com adequado grau de pureza, integridade estrutural e atividade biológica. Essa proteína também foi utilizada com sucesso para a produção de anticorpos policlonais em camundongos. Com os resultados obtidos, a proteína rhG-CSF mostrou-se viável para estudos posteriores em bioreatores e produção em escala-piloto. / The recombinant human granulocyte colony stimulating factor (rhG-CSF) is a non-glycosylated protein with 175 amino acids. This factor plays an important role in hematopoietic cell proliferation and has been widely used for treating neutropenia. The purpose of this work is to construct two expression systems in E. coli; a system for obtaining rhG-CSF in the cytoplasm and the other for secretion of recombinant protein in the culture medium using the signal sequence of L-asparaginase II. The two expression systems were tested and compared. From these data, the next steps for obtaining the rhG-CSF were done with the expression system without the signal sequence. The refolding and purification steps were efficient, resulting in a protein with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of polyclonal antibodies in mice. With these results, the protein rhG-CSF was feasible for further studies in bioreactors and pilot scale production.

Offentliga företag har fått ett större intresse av att införa Business Intelligence-lösningar. Graden av framgång varierar mellan olika typer av industrier på grund av komplexa processer som är relaterade till höga kostnader. Syftet med studien var att identifiera de kritiska framgångsfaktorerna för att framgångsrikt införa en BI-lösning ur ett CFO-perspektiv. BI har en bred kategori av olika applikationer och teknologier som används för att samla in, lagra och analysera data för att sedan extrahera den. Denna data görs sedan tillgänglig för anställda i företaget för att hjälpa beslutsfattare att skapa professionella och informerade affärsbeslut. Studien genomfördes med hjälp av en kvalitativ innehållsanalys tillsammans med semi-strukturerade intervjuer. Intervjuer genomfördes med sju olika CFO:er för att få sina perspektiv angående vilka som är de kritiska framgångsfaktorer för att införa en BI-lösning. Innehållsanalysen genererade nio kategorier med flera underkategorier. Varje kategori och underkategori identifierar CSF ur ett CFO-perspektiv. De kategorier som identifierades var: Företagsledare med genomtänkta arbetslag, hållbar datakvalitet, slutanvändaren i fokus, extern hjälp bidrar med ett bredare perspektiv och kapacitet, mål och visioner, stöd från ledningen, iterativt tillvägagångssätt, affärsdriven expansion och agila tekniska ramar, och att ha rätt förutsättningar under hela implementeringen. Både studielitteraturen, CFO:erna och CFO:ernas rankning av CSF drar slutsatsen att alla framgångsfaktorer verkligen behövs för en framgångsrik BI-lösning. Alla CSF prioriteras däremot olika inom alla tre datakällor som fanns tillgängliga för studien. / Public companies have gotten a bigger interest in implementing Business Intelligence solutions. The level of success rate varies between various types of industries because of complex processes that are related to high costs. The aim of the study was to identify the critical success factors for implementing a BI-solution successfully from a Chief Financial Officers perspective. BI has a broad category of various applications and technologies that are used to collect, store and analyze data and then extract it and make the data available to employees in the company in order to help decision makers to create professional and informed business decisions. The study was conducted using a qualitative content analysis and semi-structured interviews making use of seven different CFO’s to follow up on their perspective on organizational critical successfactors for implementing a BI-solution. The content analysis generated nine categories with several sub-categories. Each category and sub-category defining CSF from a CFO’s point of view. The categories identified were: Business leaders with well-thought-out work teams, sustainable data quality, the end user in focus, external help contributes witha broader perspective and capacity, goals and visions, management support, iterative approach, business-driven expansion and agile technical framework, and having the right conditions through out the implementation. Both the study literature, respondents and the respondents’ ranking of the CSF conclude that all success factors are indeed CSF needed for a successful BI-implementation. However, the CSF were prioritized differently within all three data sources of material that were available for the study.