Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in Human Embryonic Kidney cells (HEK/TLR2) and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1 and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1 or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. TLR2 may utilize both TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins contributing to inflammatory disease. Two critical factors in chronic HCV infection are inflammatory disease and immune evasion. We have demonstrated that TLR2 and its co-receptors play a role in inflammatory-mediated induction via HCV NS3 and core administration. It has recently been shown that HCV targets the TLR3 pathway to aid in immune evasion. TLR3 is only one of four viral recognition receptors located within the endosome and it is plausible that HCV may target others. We hypothesized that HCV infection may interfere with the expression and function of TLR7, a sensor of single stranded RNA. Investigating any effect on TLR7 by HCV may reveal a new mechanism for HCV immune evasion. Low levels of both TLR7 mRNA and protein were measured in HCV replicating cells compared to control cells while reducing HCV infection with either IFNα or restrictive culture conditions restored the decreased TLR7 expression. Downstream of the TLR7 pathway, an increased baseline IRF7 nuclear translocation was observed in HCV replicating cells compared to controls. Stimulation with a TLR7 ligand, R837, resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV replicating cells showed impaired IRF7 activation. Use of RNA polymerase inhibitors on hepatoma cells, control and HCV replicating, revealed a shorter TLR7 half life in HCV replicating cells compared to control cells which was not seen in TLR5 mRNA. These data suggest that reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and results in impaired TLR7-induced IRF7-mediated cell activation. In conclusion, Hepatitis C Virus manipulates specific Toll-like receptors’ expression and their signaling pathways to induce cytokine production. HCV utilizes surface receptors TLR2 and its co-receptors which once activated could contribute to inflammatory disease by production of inflammatory cytokines and possibly immune evasion. HCV down-regulates TLR7, a viral recognition receptor, by decreasing mRNA stability which could facilitate evasion of host immune surveillance.

Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.

Tolerance-based stem cell transplantation using sub-lethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. In order to study these two issues, we first established that mouse bone marrow recipients treated with sub-lethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. Our conditioning regimen of 6 Gy irradiation, a short course of anti-CD154 mAb and 25 million fully allogeneic BALB/c bone marrow cells consistently produced long-term, stable, and multilineage chimerism in C57BL/6 recipients. Furthermore, chimeric mice displayed donor-specific transplantation tolerance, as BALB/c skin allografts were permanently accepted while third-party CBA/JCr skin allografts were promptly rejected. We next determined both the safety and efficacy of this protocol by infecting chimeric mice with lymphocytic choriomeningitis virus (LCMV) either at the time of transplantation or at several time points afterwards. Infection with LCMV at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Surprisingly, infected allograft recipients also failed to clear the virus and died. Post-mortem study revealed hypoplastic bone marrow and spleens. Hypoplasia and death in these mice required the combination of 6 Gy irradiation, LCMV infection on the day of transplantation, and an allogeneic bone marrow transplant but did not require the presence of anti-CDl54 mAb. Allochimeric mice infected with LCMV 15 days after transplantation were able to survive and maintain their bone marrow graft, indicating that the deleterious effects of LCMV infection on host and graft survival are confined to a narrow window of time during the tolerization and transplantation process. The final section of this thesis studied the mechanisms of graft rejection and death in sublethally irradiated recipients of allogeneic bone marrow and infection with LCMV at the time of bone marrow transplantation. Infection of interferon-α/β receptor knockout mice at the time of transplantation prevented the engraftment of allogeneic bone marrow, but the mice survived. Therefore, IFN-αβ is involved in the development of marrow hypoplasia and death, whereas a second mechanism is involved in blocking the development of chimerism in these mice. Through the use of depleting mAb's and knockout mice we demonstrate that three types of recipients survived and became chimeric after being given sublethal irradiation, anti-CD154 mAb, an allogeneic bone marrow transplant and a day 0 LCMV infection: mice depleted of CD8+ T cells, CD8 knockout mice, and TCR-αβ knockout mice. Our data indicate that the mediator of bone marrow allograft destruction in LCMV-infected mice treated with costimulatory blockade is a radioresistant CD8+ NK1.1- TCRαβ+ T cell. We conclude that a non-cytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sub-lethally irradiated mice treated with costimulation blockade. The abrogation of allogeneic bone marrow engraftment is mediated by a population of CD8+ NK1.1- TCRαβ+ T cells and the mediator of hypoplasia and death is viral induction of IFN-αβ.

Submitted by FERNANDO PARRA DOS ANJOS LIMA null (engfernandoparra@gmail.com) on 2016-10-31T11:47:54Z No. of bitstreams: 1 Fernando Parra A. Lima.pdf: 3667307 bytes, checksum: 0d206b9c09566cdb11de101b84976228 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-11-07T16:42:09Z (GMT) No. of bitstreams: 1 lima_fpa_dr_ilha.pdf: 3363973 bytes, checksum: d8849cdd159a11920c497d025a8ae16a (MD5) / Made available in DSpace on 2016-11-07T16:42:09Z (GMT). No. of bitstreams: 1 lima_fpa_dr_ilha.pdf: 3363973 bytes, checksum: d8849cdd159a11920c497d025a8ae16a (MD5) Previous issue date: 2016-09-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Esta pesquisa é dedicada ao desenvolvimento de uma metodologia para a realização do diagnóstico de distúrbios de tensão de sistemas de distribuição de energia elétrica, baseada no uso de sistemas imunológicos artificiais (SIA). Trata-se da proposição de um novo paradigma no ambiente dos SIA que confere o aprendizado de modo contínuo (plasticidade). Esta concepção permite compor um sistema de diagnóstico apto a aprender continuamente, contemplando novos tipos de distúrbios advindos da constante evolução do setor elétrico, sem a necessidade de reiniciar o processo de aprendizado. Neste contexto, empregam-se dois algoritmos imunológicos artificiais, sendo o algoritmo de seleção negativa, responsável pelo processo de reconhecimento de padrões, e o algoritmo de seleção clonal responsável pelo processo de aprendizado. A principal aplicação deste novo método é auxiliar na operação do sistema durante distúrbios, bem como, supervisionar o sistema de proteção, e estar apto a acompanhar a evolução dos sistemas elétricos adquirindo conhecimento continuamente. Para avaliar a eficácia e o desempenho deste novo método foram realizadas simulações de distúrbios de tensão em sistemas de distribuições de energia elétrica com 5, 33, 84 e 134 barras, no software ATP/EMTP. Os resultados obtidos com esta abordagem mostram robustez e eficiência quando comparados à literatura. / This work develops a methodology to realize voltage disturbance diagnosis in electrical distribution systems, based on Artificial Immune Systems (AIS). It is a proposition of a new paradigm in AIS environment, which provides a continuous learning (plasticity). This conception allows composing a diagnosis system able to continuous learn, when new disturbances appear due to the constant evolution of the power systems, without needing to reinitialize the learning. This way, two artificial immune algorithms are used, such as the negative selection algorithm executing the pattern recognition process, and the clonal selection algorithm, executing the learning process. The main application of this new method is to aid the system operation during disturbances, as well as, supervise the system protection and be able to carry on the evolution of the electrical systems acquiring knowledge continuously. To evaluate the efficiency and the performance of this new method, voltage disturbance simulations were executed in electrical distributions systems with 5, 33, 84 and 134-bus in ATP/EMTP software. Results show robustness and efficiency when compared with those in the literature.

Orientador: Carlos Roberto Minussi / Resumo: Esta pesquisa é dedicada ao desenvolvimento de uma metodologia para a realização do diagnóstico de distúrbios de tensão de sistemas de distribuição de energia elétrica, baseada no uso de sistemas imunológicos artificiais (SIA). Trata-se da proposição de um novo paradigma no ambiente dos SIA que confere o aprendizado de modo contínuo (plasticidade). Esta concepção permite compor um sistema de diagnóstico apto a aprender continuamente, contemplando novos tipos de distúrbios advindos da constante evolução do setor elétrico, sem a necessidade de reiniciar o processo de aprendizado. Neste contexto, empregam-se dois algoritmos imunológicos artificiais, sendo o algoritmo de seleção negativa, responsável pelo processo de reconhecimento de padrões, e o algoritmo de seleção clonal responsável pelo processo de aprendizado. A principal aplicação deste novo método é auxiliar na operação do sistema durante distúrbios, bem como, supervisionar o sistema de proteção, e estar apto a acompanhar a evolução dos sistemas elétricos adquirindo conhecimento continuamente. Para avaliar a eficácia e o desempenho deste novo método foram realizadas simulações de distúrbios de tensão em sistemas de distribuições de energia elétrica com 5, 33, 84 e 134 barras, no software ATP/EMTP. Os resultados obtidos com esta abordagem mostram robustez e eficiência quando comparados à literatura. / Doutor

Orientadores: Fernando Jose Von Zuben, Jonatas Manzolli / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-06T18:10:14Z (GMT). No. of bitstreams: 1 Caetano_MarceloFreitas_M.pdf: 11758987 bytes, checksum: 2521dc99ef68d0b0a4e06b9ea4751fc5 (MD5) Previous issue date: 2006 / Resumo: Não há limitações teóricas para o uso do computador como fonte de sons musicais. O computador digital permite a produção de qualquer som concebivel dada a seqüência correta de números (amostras digitais). No entanto, produzir uma dada seqüência de números que corresponda a um som musical que possua determinadas características perceptivas desejadas é uma tarefa de difícil resolução. Grande parte dos métodos e sistemas de síntese sonora digital utiliza modelos e/ou incorpora técnicas que não levam em conta a natureza dinâmica dos sons musicais ou que não foram originalmente desenvolvidas para manipulação musical. Neste trabalho, é apresentada uma abordagem populacional para síntese sonora no domínio temporal. Foi estudado um espaço sonoro e um conjunto de atratores, isto é, um conjunto de formas de onda com qualidades sonoras desejadas e definidas a priori, e foi possível obter sons que possuem características associadas a um ou mais atratores, representando variantes dos mesmos. Este método de síntese de sons musicais pode ser interpretado como um processo de busca no espaço vetorial que contém todas as possibilidades sonoras decorrentes da representação adotada, e tem por objetivo a criação de formas de onda digítalizadas com características emergentes e potencial para serem utilizadas em diversas aplicações musicais. Os resultados representam variantes e/ou possuem íntersecções das características próprias dos atratores, responsáveis por indicar as regiões de interesse do espaço de busca. A proposta de pesquisa envolveu a utilização de algoritmos bioinspirados - os quais expressam propriedades de sistemas auto-organizados e adaptativos - como definidores de processos de geração e estruturação dos elementos sonoros, entendidos aqui como problemas de otimização. A auto-organização e os mecanismos de manutenção de diversidade e de adaptação, intrínsecos aos sistemas bio-inspirados, fundamentam a proposta no sentido de viabilizarem a emergência temporal de estruturas estáveis sem um elemento organizador externo / Abstract: There are no theoretical limitations to the use of the computer as a source of musical sounds. The digital computer allows for the production of any conceivable sound given the carrect sequence af numbers (digital samples). Nevertheless, producing the correct sequence of numbers that correspond to a musical sound expressing predefined perceptual characteristics is a very difficult task. Most sound synthesis methods and systems utilize models and/or incorporate techniques which do not take into account the dynamic nature of musical sounds or were not originally developed for the manipulation of musical tones. In this work we are proposing a populational sound synthesis approach in the time domain. A soundspace and a set of attractors, i.e. waveforms containing a priari desired features or qualities, and a population of agents communicating by means of local interaction were studied, and it was possible to attain sounds which share some qualities from more than one of the attractors, resulting exclusively from low-Ievel rules followed by these agents. This sound synthesis method can be regarded as a search in the vector space that contains ali the possible sounds resulting from the adopted representation, and its objective is to synthesize digital waveforms that possess emergent properties and the potential to be used in musical applications. The resulting sounds are variants or hybrids that share some of the intrinsic features of the attractors, which are responsible for indicating the regions of interest in the search space. This proposal involved the use of bio-inspired algorithms, which express features of adaptive, self-organizing systems, as definers of generating and structuring processes of sound elements, regarded herein as optimization processes. Self-organization and diversity maintenance and adaptation mechanisms, intrinsic to bio-inspired systems, lay the foundations of this proposal so as to make viable the temporal emergence of stable structures without an externa I organizing element / Mestrado / Engenharia de Computação / Mestre em Engenharia Elétrica

Orientadores: Fernando Jose Von Zuben, Mauricio Fernandes Figueiredo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-06T19:35:31Z (GMT). No. of bitstreams: 1 Michelan_Roberto_M.pdf: 4495515 bytes, checksum: aed72feefc89070579190e862ea0f740 (MD5) Previous issue date: 2006 / Resumo: A concepção de sistemas autônomos de navegação para robôs móveis, havendo múltiplos objetivos simultâneos a serem atendidos, como a coleta de lixo com manutenção da integridade, requer a adoção de técnicas refinadas de coordenação de módulos de comportamento elementar. Modelos de redes imunológicas artificiais podem então ser empregados na proposição de um controlador concebido com base em um processo de mapeamento dinâmico. Os anticorpos da rede são responsáveis pelos módulos de comportamento elementar, na forma de regras do tipo <condição>-<ação>, e as conexões são responsáveis pelos mecanismos de estímulo e supressão entre os anticorpos. A rede iniciará uma resposta imunológica sempre que lhe forem apresentados os antígenos. Estes antígenos representam a situação atual capturada pelos sensores do robô. A dinâmica da rede é baseada no nível de concentração dos anticorpos, definida com base na interação dos anticorpos e dos anticorpos com os antígenos. De acordo com o nível de concentração, um anticorpo é escolhido para definir a ação do robô. Um processo evolutivo é então responsável por definir um padrão de conexões para a rede imunológica, a partir de uma população de redes candidatas, capaz de maximizar o atendimento dos objetivos durante a navegação. Resulta então um sistema híbrido que tem a rede imunológica como responsável por introduzir um processo dinâmico de tomada de decisão e tem agora a computação evolutiva como responsável por definir a estrutura da rede. Para que fosse possível avaliar os controladores (redes imunológicas) a cada geração do processo evolutivo, um ambiente virtual foi desenvolvido para simulação computacional, com base nas características do problema de navegação. As redes imunológicas obtidas através do processo evolutivo foram analisadas e testadas em novas situações, apresentando capacidade de coordenação em tarefas simples e complexas. Os experimentos preliminares com um robô real do tipo Khepera II indicaram a eficácia da ferramenta de navegação / Abstract: The design of an autonomous navigation system for mobile robots, with simultaneous objectives to be satisfied, as garbage collection with maintenance of integrity, requires refined coordination mechanisms to deal with modules of elementary behavior. Models of artificial immune networks can then be applied to produce a controller based on dynamic mapping. The antibodies of the immune network are responsible for the modules of elementary behavior, in the form of <condition>-<action> rules, and the connections are responsible for the mechanisms of stimulation and suppression of antibodies. The network will always start an immune response when antigens are presented. These antigens represent the current output of the robot sensors. The network dynamics is based on the levels of antibody concentration, provided by interaction among antibodies, and among antibodies and antigens. Based on its concentration level, an antibody is chosen to define the robot action. An evolutionary process is then used to define the connection pattern of the immune network, from a population of candidate networks, capable of maximizing the objectives during navigation. As a consequence, a hybrid system is conceived, with an immune network implementing a dynamic process of decision-making, and an evolutionary algorithm defining the network structure. To be able to evaluate the controllers (immune networks) at each iteration of the evolutionary process, a virtual environment was developed for computer simulation, based on the characteristics of the navigation problem. The immune networks obtained by evolution were analyzed and tested in new situations and presented coordination capability in simple and complex tasks. The preliminary experiments on a real Khepera II robot indicated the efficacy of the navigation tool / Mestrado / Engenharia de Computação / Mestre em Engenharia Elétrica

Jak3, a member of the Janus family of tyrosine kinases, is essential for signaling via the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These Jak3-dependent cytokines primarily activate STAT5 and are critical for lymphoid generation and differentiation. Using naïve CD4+ T cells from Jak3-deficient mice and wild type CD4+ T cells treated with a pharmacological inhibitor of Jak3, we report that Jak3-dependent cytokine signals are not required for the proliferation of naïve CD4+ T cells. This is illustrated by the similar percentage of divided cells, comparable cell divisions, intact cell cycle progression and unaffected regulation of cell cycle proteins in the absence of Jak3. In contrast to proliferation, differentiation of naïve CD4+ T cells into Th1 effector cells requires Jak3-dependent cytokine signals. In the absence of Jak3, naïve CD4+ T cells proliferate robustly, but produce little IFN-γ after Th1 polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4, nor to impaired up-regulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that while Jak3-dependent signals are dispensable for naïve CD4+ T cell proliferation, Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 differentiation.

The innate immune system is the first line of defense against infectious agents. It is essential for protection against pathogens and stimulation of long-term adaptive immune responses. Therefore, deciphering the mechanisms of the innate immune system is crucial for understanding the integrated systems of host defense against microbial infections, which is conserved from insects to humans. Despite lacking a conventional adaptive immune system, insects can mount a robust immune response against a wide array of microbial pathogens. These innate immune mechanisms have been widely studied in Drosophila melanogaster, because of the model system’s powerful genetic, genomic and molecular tools. The Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophilahumoral immune response is the rapid induction of antimicrobial peptide genes in the fat body, the homolog of the mammalian liver. Expression of these antimicrobial peptide genes is controlled by two distinct immune signaling pathways, the Toll pathway and the IMD (immune deficiency) pathway. The Toll pathway is activated by fungal and Gram-positive bacterial infections, whereas the IMD pathway responds to Gram-negative bacteria. Both pathways culminate in activation of the Rel/NF-кB transcription factors DIF (Dorsal-related immunity factor), Dorsal and Relish, which in turn translocate to the nucleus to induce the antimicrobial peptide genes. DIF and Dorsal are activated by the Toll pathway and control induction of antimicrobial peptide genes such as Drosomycin. The NF-кB precursor Relish, which is composed of an N-terminal Rel homology domain and a C-terminal IкB-like domain, is activated by the IMD pathway and initiates transcription of antimicrobial peptide genes such as Diptericin. Although many components of the Drosophila immune signaling pathways have been identified, the detailed mechanisms of signal trans

T lymphocytes are distinguished by the expression of αβ TCR or γδ TCR on their cell surface. The kinetic differences in the effector functions classifies γδ T cells as innate-like lymphocytes and αβ T cells as adaptive lymphocytes. Although distinct, αβ and γδ T cell lineages produce a common array of cytokines to mount an effective immune response against a pathogen. The production of cytokine IL-17 is a shared characteristic between the γδ T (Tγδ17) cells and the CD4 T (Th17) cells. γδ T cells develop into Tγδ17 cells in the thymus whereas CD4 T cells differentiate into Th17 cells in response to antigens in the peripheral lymphoid tissues. γδ T cells exported from the thymus, as pre-made effectors, are the early IL-17 producers compared with the late IL-17 producing Th17 cells. In this thesis we describe how TGFβ-SMAD2 dependent pathway selectively regulates Th17 cell differentiation but not Tγδ17 cells generation. We further illustrate the requirement of WNT-HMG box transcription factor (TF) signaling for the thymic programming of Tγδ17 cells. Cytokine TGFβ in co-operation with IL-6 induces the differentiation of Th17 cells. Conversely, TGFβ signaling also regulates the differentiation and maintenance of CD4+FOXP3+ regulatory T cells. The mechanism by which TGFβ signals synergize with IL-6 to generate inflammatory versus immunosuppressive T cell subsets is unclear. TGFβ signaling activates receptor SMADs, SMAD2 and SMAD3, which associate with a variety of nuclear factors to regulate gene transcription. Defining relative contributions of distinct SMAD molecules for CD4 T cell differentiation is critical for mapping the versatile intracellular TGFβ signaling pathways that tailor TGFβ activities to the state of host interaction with pathogens. We show here that SMAD2 is essential for Th17 cell differentiation and that it acts in part by modulating the expression of IL-6R on T cells. While mice lacking SMAD2 specifically in T cells do not develop spontaneous lymphoproliferative autoimmunity, Smad2-/- T cells are impaired in their response to TGFβ in vitro and in vivo and they are more pathogenic than controls when transferred into lymphopenic mice. These results demonstrate that SMAD2 is essential for TGFβ signaling in CD4+ T effector cell differentiation and that it possesses functional capabilities distinct from SMAD3. Although SMAD2 is essential for the differentiation of Th17 cells, TGFβ signaling via SMAD2 is not required for the thymic programming of innate Tγδ17 cells. Among different γδ T cells, Vγ2+ (V2) γδ T cells are the major IL-17 producing subsets. We demonstrate that Sry-high mobility group (HMG) box TFs regulate the development of V2 Tγδ17 cells. We show that the HMG box TF, SOX13 functions in a positive loop for the intrathymic generation of V2 Tγδ17 cells. SOX13 regulates the programming of Tγδ17 cells by controlling the expression of B-lymphoid kinase (BLK) in developing immature V2 γδ T cells. BLK is an Src-family kinase expressed by all Tγδ17 cells. Furthermore, we show another HMG box TF, TCF1, the nuclear effector of canonical WNT signaling, is the primary negative regulator of IL-17 production by all γδ T cells. We propose that the antagonism of SOX13 and TCF1 determines the generation of IL-17 producing γδ T cells. We also show that extrinsic cues from αβ T cells do not affect the generation of IL-17 producing γδ T cells. Using OP9-DL1 culture system, we demonstrate that the progenitors of V2 Tγδ17 cells are the c-Kit+ early thymic precursors.