The Fas pathway is critical for the maintenance of normal T cell homeostasis. Humans and mice with defects in this pathway exhibit the accumulation of large numbers of peripheral lymphocytes and lupus-like autoimmunity. A major feature of these organisms is the accumulation of non-NK TCRαβ+CD4-CD8- “double negative” (DN) T cells. While regulatory T cells (Tregs) with the DN phenotype have been extensively characterized in Fas-sufficient mice and humans, limited data exist on the role of DN T cells as Tregs in Fas-deficient animals. In fact, most of the literature suggests that the DN T cells accumulating in Fas-deficiency states are pathogenic, contributing to secondary lymph node enlargement and autoimmune disease. In this body of work, data are presented that illustrate that Fas-deficient lymphoproliferative (LPR) DN T cells can act as Tregs in an interferon γ (IFNγ)- and Fas ligand (FasL)-dependent fashion toward Fas-sufficient T cells. LPR DN T cells needed to be able to secrete and respond to IFNγ in order to upregulate surface FasL, in order to ameliorate GVHD mediated by CD4+ T cells in vivo and to suppress the proliferation of and kill activated CD4+ T cells in vitro. FcRγ, a key molecule involved in innate immune responses, can substitute for CD3ζ in the T cell receptor (TCR) of mouse and human T cells in certain circumstances; in doing so, it is essential for the regulatory function of TCR transgenic DN Tregs. FcRγ-deficient LPR mice were found to have exacerbated T cell accumulation and early mortality. We show that while FcRγ expression was required for LPR DN T cells to regulate CD4+ and CD8+ T cells responding to alloantigens in vitro and in vivo, it does not control autologous lymphoproliferation in LPR mice by supporting the function of a regulatory cell, nor does it affect the rate of proliferation of LPR T cells in vivo. Instead, FcRγ-expressing LPR CD4+, CD8+ and DN T cells were found to be undergoing apoptosis at a high rate in vivo, and in contrast to their FcRγ-deficient counterparts, FcRγ+ LPR DN T cells were capable of undergoing TCR restimulation-induced cell death (RICD). The data presented in this thesis therefore show that LPR DN T cells can exhibit IFNγ-, FasL- and FcRγ-dependent regulatory function, and also illustrate a previously unknown function for FcRγ in controlling the expansion of Fas-deficient T cells. The implications of these data for autoimmune lymphoproliferative syndromes, and normal T cell homeostasis, are discussed.

Low affinity Fcg receptor III (FcgRIII, CD16) triggers a variety of cellular events upon binding to the Fc portion of IgG. A real-time flow cytometry method was developed to measure the affinity and kinetics of such low affinity receptor/ligand interactions, which was shown as an easily operated yet powerful tool. Results revealed an unusual temperature dependence of reverse rate of CD16aTM dissociating from IgG. Except for a few studies using mammalian cell CD16s, most kinetics analyses use purified aglycosylated extracellular portion of the molecules, making it impossible to assess the importance of the receptor anchor and glycosylation on ligand binding. We used a micropipette adhesion frequency assay to demonstrate that the anchor length affects the forward rate and affinity of CD16s for IgG in a species specific manner, most likely through conformational changes. Receptor glycosylation dramatically reduced ligand binding by 100 folds. T cell receptor (TCR) is arguably the most important receptor in the adaptive human immune system. Together with coreceptor CD4 or CD8, TCR can discriminate different antigen peptides complexed with major histocompatibility complex (MHC) molecule (pMHC), which differ by as few as only one amino acid, and trigger different T cell responses. When T cell signaling was suppressed, TCR had similar affinity and kinetics for agonist and antagonist pMHC whose binding to CD8 was undetectable. TCR on activated T cell had a higher affinity for pMHCs, suggesting that TCRs organize themselves differently on activated T cells than on naïve T cells. In the absence of inhibitors for signaling, TCR binds agonist pMHC with several orders of magnitude higher affinity than antagonist pMHC. In addition, engagement of TCR by pMHC signals an upregulation of CD8 binding to pMHC, which is much stronger than the TCR-pMHC binding. The transition from weak TCR binding to the strong CD8 binding takes place around 0.75 second after TCR in contact with pMHC and can be reduced by several inhibitors of tyrosine and lipid phosphorylation, membrane rafts, and actin cytoskeleton. These results provide new insights to understanding T cell discrimination.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 6, 2008). Includes bibliographical references.

B lymphocytes play a significant role in autoimmune arthritis, with their function stretching beyond autoantibody production to cytokine secretion and presentation of autoantigen. However, the involvement and activation of different B-cell subset in the autoimmune response is not fully clear. The main focus of this thesis has been to understand the contribution of marginal zone (MZ) B cells in the induction of collagen-induced arthritis (CIA), a mouse model for rheumatoid arthritis (RA). We show that MZ B cells in the spleen of naïve mice display a natural self-reactivity to collagen type II (CII), the autoantigen used for immunization of CIA. The CII-reactive MZ B cells expand rapidly following immunization with CII, and produce IgM and IgG antibodies to CII. They also very efficiently present CII to cognate T cells in vitro and in vivo. Moreover, absence of regulatory receptors such as CR1/2 or FcγRIIb on the MZ B cells increases their proliferation and cytokine production in response to toll-like receptor, but not B-cell receptor, activation. Further, FcγRIIb-deficient MZ B cells present CII to T cells more efficiently than wild-type MZ B cells. We additionally demonstrate for the first time the existence of a small population of nodal MZ B cells in mouse lymph nodes. Similar to splenic MZ B cells, the nodal MZ B cells expand after CIA induction, secrete IgM anti-CII antibodies and can present CII to cognate T cells. Finally, we show that mast cells, associated with ectopic B cell follicles in inflamed RA joints, in coculture with B cells promote their expansion, production of IgM and IgG antibodies as well as upregulation of CD19 and L-selectin. Coculture with mast cells further causes the B cells to upregulate costimulators and class II MHC, important molecules for antigen-presenting function. In summary, my findings suggest that splenic and nodal self-reactive MZ B cells participate in breaking T-cell tolerance to CII in CIA. B-cell intrinsic regulation is needed to keep such autoreactive B cells quiescent. Mast cells can potentiate B-cell responses locally in the arthritic joint, thus feeding the autoimmune reaction.

Submitted by JOHN WILLIAM NEGREIROS TERRONES null (johneter@hotmail.com) on 2018-01-19T17:02:53Z No. of bitstreams: 1 DissertaçãoJohn_JB (1).pdf: 14046795 bytes, checksum: deeec72b971c8015d40780ccc3ee8e37 (MD5) / Approved for entry into archive by Cristina Alexandra de Godoy null (cristina@adm.feis.unesp.br) on 2018-01-19T17:54:25Z (GMT) No. of bitstreams: 1 terrones_jwn_me_ilha.pdf: 14046795 bytes, checksum: deeec72b971c8015d40780ccc3ee8e37 (MD5) / Made available in DSpace on 2018-01-19T17:54:25Z (GMT). No. of bitstreams: 1 terrones_jwn_me_ilha.pdf: 14046795 bytes, checksum: deeec72b971c8015d40780ccc3ee8e37 (MD5) Previous issue date: 2017-09-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho, propõe-se uma metodologia baseada nas incertezas da radiação solar para prever o despacho de demanda e reativos nos sistemas de distribuição radial com geradores fotovoltaicos (GFs). O objetivo dessa metodologia é melhorar o perfil da magnitude de tensão para o dia seguinte, sujeito a certas restrições operativas do sistema elétrico de distribuição. No desenvolvimento dos algoritmos de solução são considerados geradores distribuídos que têm forte dependência das variações da fonte primária de energia (principalmente sistemas fotovoltaicos), ou seja, os limites de injeção de potência dos GFs dependem das incertezas da radiação solar. Este grupo de geradores precisa de considerações que incluam as incertezas na geração de energia. Para isso, elaboram-se cenários de radiação baseado na função de distribuição de probabilidade beta. Assim, são realizados fluxos de potência probabilísticos em múltiplos cenários. Leva-se em consideração, os objetivos a serem otimizados por meio de técnicas multiobjetivo, observando principalmente, os desvios da tensão nos nós e as perdas de potência nas linhas do sistema. A metodologia proposta foi implementada em linguagem de modelagem algébrica com o AMPL para descrever o problema de otimização e resolvido usando o solver comercial CPLEX. Os sistemas testes de 34 e 123 nós foram utilizados para avaliar os modelos matemáticos e a eficiência da técnica de solução proposta para o problema de controle dos sistemas de distribuição radiais com GFs. / This work presents a methodology based on uncertainties of solar radiation to predict demand and reactive dispatch in the radial distribution systems with photovoltaic generators (GFs). The objective is to improve the voltage magnitude profile for a day ahead, subjecting to operation constraints of distribution system. In order to evaluate the optimization criteria for distribution networks, active power balance equations have to be solved. It is considered that distributed generators have a high coherence to variations of the primary energy source (mainly photovoltaic systems), where, the power injection limits of the GFs depend on the uncertainties of the solar radiation. Uncertainties in generation are needed to be considered for this type of generator. Thus, the beta probability distribution function has been employed to include different radiation scenarios in the problem formulation. The aim of this research evolves the development of an optimization tool to predict the reactive power dispatch of GFs considering the uncertainties of solar radiation. For this purpose, the probabilistic power flow is performed under various scenarios. The Multiobjective optimization problem is formulated by including the buses (nodes) voltage deviations and the power losses of distribution lines in two different objective functions. The proposed methodology was implemented in AMPL mathematical model language and solved using the commercial CPLEX solver. The 34 and 123 nodes test systems are applied to show efficiency of presented mathematical models, i.e. control of radial distribution systems with GFs, and the proposed solution method.

Orientador: Antonio Padilha Feltrin / Resumo: Neste trabalho, propõe-se uma metodologia baseada nas incertezas da radiação solar para prever o despacho de demanda e reativos nos sistemas de distribuição radial com geradores fotovoltaicos (GFs). O objetivo dessa metodologia é melhorar o perfil da magnitude de tensão para o dia seguinte, sujeito a certas restrições operativas do sistema elétrico de distribuição. No desenvolvimento dos algoritmos de solução são considerados geradores distribuídos que têm forte dependência das variações da fonte primária de energia (principalmente sistemas fotovoltaicos), ou seja, os limites de injeção de potência dos GFs dependem das incertezas da radiação solar. Este grupo de geradores precisa de considerações que incluam as incertezas na geração de energia. Para isso, elaboram-se cenários de radiação baseado na função de distribuição de probabilidade beta. Assim, são realizados fluxos de potência probabilísticos em múltiplos cenários. Leva-se em consideração, os objetivos a serem otimizados por meio de técnicas multiobjetivo, observando principalmente, os desvios da tensão nos nós e as perdas de potência nas linhas do sistema. A metodologia proposta foi implementada em linguagem de modelagem algébrica com o AMPL para descrever o problema de otimização e resolvido usando o solver comercial CPLEX. Os sistemas testes de 34 e 123 nós foram utilizados para avaliar os modelos matemáticos e a eficiência da técnica de solução proposta para o problema de controle dos sistemas de distribuição radia... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work presents a methodology based on uncertainties of solar radiation to predict demand and reactive dispatch in the radial distribution systems with photovoltaic generators (GFs). The objective is to improve the voltage magnitude profile for a day ahead, subjecting to operation constraints of distribution system. In order to evaluate the optimization criteria for distribution networks, active power balance equations have to be solved. It is considered that distributed generators have a high coherence to variations of the primary energy source (mainly photovoltaic systems), where, the power injection limits of the GFs depend on the uncertainties of the solar radiation. Uncertainties in generation are needed to be considered for this type of generator. Thus, the beta probability distribution function has been employed to include different radiation scenarios in the problem formulation. The aim of this research evolves the development of an optimization tool to predict the reactive power dispatch of GFs considering the uncertainties of solar radiation. For this purpose, the probabilistic power flow is performed under various scenarios. The Multiobjective optimization problem is formulated by including the buses (nodes) voltage deviations and the power losses of distribution lines in two different objective functions. The proposed methodology was implemented in AMPL mathematical model language and solved using the commercial CPLEX solver. The 34 and 123 nodes test systems a... (Complete abstract click electronic access below) / Mestre

Main purpose of this study was to increase protein expression in Chinese hamster ovary (CHO) cells by improving protein secretion of translated proteins. The goal was to find signal peptides from the screening of signal peptide libraries for improvement of protein secretion using a CHO-cell express selection system. Biopharmaceutical products, proteins such as monoclonal antibodies (mAbs), are most commonly produced using mammalian expression systems such as the expression in CHO cells. The posttranslational modifications of the proteins being expressed in CHO cells are similar to the expressional modifications in human cells, why the CHO cells are suitable for production of proteins used for human therapy. The expression of proteins in the cell is a complex mechanism, fundamentally depending on the DNA sequences in the cell nucleus. Secretion of translated proteins has been showed to be a bottleneck when improving expression. Secretion is initiated by the signal peptide, a n-terminal prolongation of the protein that is recognized by a signal recognition particle (SRP) when being translated by the ribosome. The sequence and structure of the signal peptide has been proved to affect secretion and altering the signal peptide could improve secretion even when changing signal peptide between different species. Designing variants of the signal peptides and analyzing protein expression might lead to improvements of the construct design and more protein produced from the cells, which would save time, money and material for the producer. To construct plasmids containing the gene of interest (GOI) and different signal peptides, several gene cloning methods were used. The plasmids were amplified using Escherichia coli (E. coli) transformation. The constructs were expressed by transfection into the CHO cell genome, and expression were analyzed using flow cytometry. When analyzing expression of a Fc-fusion protein with 5 different signal peptides, the signal peptide Azurocidin is the one showing highest expression levels in this study. In addition, IgG kapa and Albumin signal peptides did not show as high protein expression levels, even if they were better than the L1d and H5b signal peptides. Since signal peptides are exchangeable between proteins and species, it might be that Azurocidin is improving secretion and protein expression with other proteins than Fc-fusion proteins which would be an interesting aspect for further studies. When altering signal peptides with library sequences, the experimental challenges were crucial for the protein expression results and due to these issues, no library sequence could be seen to conquer others when it comes to protein expression levels. Transfection and cultivation procedures needs to be studied and improved before being able to draw conclusions about which signal peptide library sequences that might improve secretion and increase the protein expression.

La faisabilité des applications à piles à combustible (PAC) a été largement démontrée à travers le monde. Les efforts de recherche portent actuellement sur l'amélioration de la durée de vie des PAC et la diminution de leur coût. A ce jour, une PAC ne tolère pas les variations rapides de charges qui entraînent très souvent son vieillissement prématuré. Pour pallier cette faiblesse, une hybridation avec un composant électrochimique de stockage (typiquement des supercondensateurs) est généralement proposée via un ou deux convertisseurs statiques, nécessitant l'implantation d'une gestion énergétique. Une partie de ces travaux se situe dans le contexte aéronautique et fait suite au projet européen CELINA piloté par AIRBUS. Le projet européen CELINA (2005-2008) a posé la problématique du remplacement de l'éolienne (RAT) actuelle par une pile à combustible pour le réseau électrique de dernier secours sollicité en cas de perte totale des moteurs ou de la génération électrique. Il alimente les charges essentielles : auxiliaires de puissances presque constantes (calculateurs de bord, …) et les actionneurs de vol (EHA, EMA) qui constituent les principaux consommateurs à caractères très intermittents. Cette étude a permis une classification de trois architectures, dont la validation expérimentale se situant dans le cadre du projet français ISS ayant débuté en 2010 sera exposée. L'hybridation directe entre une PAC et des supercondensateurs présente les avantages de ne pas mettre en jeu de convertisseur statique et d'une autogestion énergétique naturelle. Partant du constat que toutes les applications embarquées utilisant des PAC sont hybridées et qu'un développement d'architecture et de stratégies est effectué pour chaque cas (mise au point de convertisseurs, lois de commande, etc.). Ceci représente un travail considérable et systématique, ce qui freine l'implantation des systèmes PAC dans les applications embarquées. L'objectif est alors d'étudier la faisabilité d'un composant hybride unique jouant le rôle de source de puissance et d'énergie dont la gestion énergétique est transparente pour l'utilisateur et ne nécessitant pas l'ajout d'une hybridation supplémentaire pour ces applications. Cette thématique fait l'objet de ces travaux de thèse en collaboration avec la société française HELION Hydrogen Power. Après une présentation de l'introduction des systèmes PAC en aéronautique centrée autour de l'hybridation directe, la suite des travaux regroupe deux grandes thématiques : la première concerne l'étude des interactions entre PAC et supercondensateurs lors d'une association directe selon trois approches : théorique, expérimentale et par simulation. La seconde concerne la validation expérimentale de trois architectures d'hybridation d'un système PAC retenues pour un contexte aéronautique lors d'études précédentes au laboratoire : une architecture indirecte pour laquelle le stockage possède son convertisseur, une architecture indirecte avec stockage sur le bus DC et une architecture directe. L'objectif de ces travaux étant d'augmenter le niveau de maturité technologique de ces concepts, ainsi que de comparer les différents moyens retenus pour parvenir à l'hybridation d'un système PAC suivant des critères précis. / The feasibility of fuel cell (FC) applications has been demonstrated throughout the world. Research efforts are currently focused on improving the lifetime of the FC and reducing their cost. Until today, a FC does not tolerate rapid variations of load that cause in most cases lifetime reducing. To reduce this defect, hybridization with electrochemical storage component (typically ultracapacitors) is generally suggested via one or two static converters, requiring the implementation of an energy management. Aeronautic applications constitute the framework of these studies. They are the prolongation of studies initiated within the European project CELINA piloted by AIRBUS. The CELINA project (2005-2008) dealt with the replacement of the Ram Air Turbine (RAT) which is currently used for the last emergency electrical network in the case of total losses of engines or electrical generation. This emergency network has to supply the essential loads: the piloting auxiliaries (calculators…) consume a quasi-constant power, and the flight actuators (EHA, EMA) which are the main loads whose consumption is very intermittent. This study resulted in a classification of three architectures for which experimental validation in the framework of the French ISS project started since 2010 will be exposed. The direct hybridization between a FC and ultracapacitors has the advantages of not involving static converter and provide a natural energy management. The statement of facts is that all embedded applications using FC are hybridized, architecture and strategies development is performed for each case (development of converters, control laws, etc.). This represents a significant and systematic work, which limits the implementation of FC in embedded applications. In this work, the objective is to study the feasibility of a single hybrid component acting as a power and energy source for which energy management is transparent to the user and does not require the addition of another hybridization. This work is part of collaboration with the French company HELION Hydrogen Power. After a presentation of the insertion of FC in aeronautics centered on the direct hybridization, two major themes are approached: The first concerns the study of interactions between FC and ultracapacitors in a direct association according to three approaches: theoretical, experimental and simulation. The second concerns the experimental validation of three hybridization architectures for FC considered in previous studies in the laboratory: an architecture for which the indirect storage has its converter, an architecture with indirect storage on the DC bus and a direct hybridization architecture. The objective of this work is to increase the level of technological Readiness level of these concepts, and to compare the different ways considered to achieve the hybridization of a fuel cell system according to specific standards.

Syftet med denna studie är att undersöka och diskutera samtidens porträttering av maskulinitet och avvikelser inom populärkulturen. För att genomföra denna studie analyseras två olika teveserier varav den ena är en dokusåpa och den andra en dramaserie. Båda serierna uppstod vid en och samma tidpunkt på olika delar av världen. Den likhet som fanns mellan dem var att de porträtterade framväxten av en och samma fenomen, nördar som ny manlighet. Analysen genomförs utifrån teoretiska verktyg med grund inom remedering samt teorier om immersivitet och manlighet men även en återkoppling till begreppet freakshow. Uppsatsen är förankrad för att lyfta fram tankar och aspekter kring framväxt av den manliga identiteten men även väcka nya diskussioner. / The purpose of this study is to examine and discuss the current ways of portraying masculinity and deviations within popular culture. For this study, I’ve focused on two different sources, one being a reality-show and the other being a fictional series. Both of these series aired for the first time during 2005, though not in the same country. Despite the distance, both of these series portrayed the appearance of new masculinity, in this case, the fact of being a nerd as a new masculinity. The analysis focuses on examining the series with roots in the remediation theories as well as concepts of the immersive media and masculinity. Also, placing these series in a context where they can be compared to the late freakshow phenomena. This study aims to bring up thoughts around appearances of masculine standards as well as plant seeds for future discussions.

Com o objetivo de estudar a interação do HAdV-41 com células permissivas, primeiramente foi observada a cinética de infecção do HAdV-41 em células HEK-293, durante 7 dias. A seguir, as culturas foram analisadas por MCVL e por MET. O HAdV-41 apresentou um ciclo replicativo lento com liberação da progênie viral por mecanismo não lítico. A seguir, com o intuíto de verificar a participação da proteina FC do HAdV-41 nas etapas de entrada nas células HEK-293 e CaCo2, obteve-se os dodecaedros recombinantes (DR) em células High five (base-Ad3, base-Ad3+FC-Ad41, base-Ad3+FL-Ad41, baseRGEHS-Ad3+FCAd41 e base-Ad3+FAd3). Esses dodecaedros foram inoculados em células HEK-293 e CaCo2. Após a análise por MCVL, observou-se que a proteína FC talvez não desempenhe função na entrada do DR nas células estudadas. A seguir, uma alíquota do DR base-Ad3+FC-Ad41 foi digerida com a enzima pepsina e analisada por WB. Notou-se que a FC sofreu proteólise. Acredita-se que essa proteólise seja necessária para o reconhecimento de receptores no trato gastro-intestinal. Esses resultados fornecerão subsídeos para o desenvolvimento de vetores de terapia gênica direcionada para o epitélio intestinal e vetores vacinais administrados por via oral. / Our objective was study the interaction between HAdV-41 and permissive cells. First, it was observed the kinetic of infection between HAdV-41 and HEK-293 cells, for 7 days. Second, the cultures were analyzed by CLSM and by TEM. The HAdV-41 showed a slower replicative cycle with release of viral progeny by non-lytic mechanisms. In order to verify the participation of SF protein of the HAdV-41 during the phases of entry in HEK-293 and CaCo2 cells, we producted recombinant dodecahedrons (DR) in high five cells (base-Ad3, base-Ad3+SF-Ad41, base-Ad3+SF-Ad41, baseRGEHS-Ad3+SFAd41 and base-Ad3+FAd3). These decahedrons were inoculated in HEK-293 and CaCo2 cells. After analysis with CLSM, observed that SF protein may not have a role in dodecahedron entry in the cells studied. Next, recombinant dodecahedrons base-Ad3+SF-Ad41 and base-Ad3 were digested with pepsin and analyzed by WB. We observed proteolysis of the SF. We believe that this proteolysis may be necessary for the recognition of receptors in intestinal cells. The results obtained will be the base for the development of gene-therapy vectors directed to intestinal epithelium, as well as orally administered vaccine vectors.