Em nossos dias a crescente busca por portabilidade e desempenho resulta em esforços focados na maximização da duração de bateria dos equipamentos em fabricação, ou seja, busca-se a conflitante solução de circuitos com baixo consumo e ao mesmo tempo com alto desempenho. Neste contexto usualmente na composição de equipamentos portáteis empregam-se SOC´s (Systems On Chip) o que barateia o custo de produção e integração destes circuitos. SOC´s são sistemas completos que executam uma determinada função integrados em uma pastilha de silício única, normalmente possuem memórias SRAM como componente do sistema, que são utilizadas como memórias de alta performance e baixa latência e/ou também como caches. O grande desafio de projeto em memórias SRAMS é a relação de desempenho versus potência consumida a ser otimizada. Basicamente por sua construção estes circuitos apresentam alto consumo de potência, dinâmica e estática, relacionada a primeira diretamente ao aumento de freqüência de operação. Um dos focos desta dissertação é explorar soluções para a redução de consumo de energia tanto dinâmica como estática, sendo a redução de consumo estático de células de memória em standby buscando desempenho, estabilidade e baixo consumo de energia. No desenvolvimento de técnicas para projeto de circuitos analógicos em tecnologias nanométricas, os TST´s (T-Shaped Transistors – Transistor tipo T) surgem como dispositivos com características potenciais para projeto analógico de baixa potência. TSTs / TATs (Trapezoidal Associations of Transistors – Associação Trapezoidal de transistores) são estruturas self-cascode que podem tornar-se uma boa escolha por apresentar redução do leakage, redução na área utilizada e com incremento na regularidade do layout e no casamento entre transistores, propriedade importantíssima para circuitos analógicos. Sendo este o segundo foco deste texto através do estudo e análise das medidas elétricas dos TSTs executadas para comprovação das características destes dispositivos. Também apresenta-se uma análise das possibilidades de utilização dos TSTs em projeto analógico para tecnologias nanométricas. / Nowadays the increasing needs for portability and performance has resulted in efforts to increase battery life, i. e., the conflicting demands for low power consumption and high performance circuits. In this context using SOC´s (System On Chip) in the development for portable equipments composition, an integration of an entire system for a given function in a single silicon die will provide less production costs and less integration costs. SOC´s normally include a SRAM memory as its building block and are used to achieve memories with low latency and short access time or (and) as caches. A performance versus power consumption analysis of SRAM memory building blocks shows a great challenge to be solved. The electrical design aspects of these blocks reveal high power consumption, dynamic and static, and the former is directly proportional to the operating frequency. The design space exploration for dynamic and leakage consumption reduction in these circuits is one of the focus of this work. The main contribution of this topic is the leakage reduction techniques based in performance, stability and low energy consumption for the memory cell stand-by mode. Among the electrical techniques developed for analog circuits at the 20-100 nanometer scale, the TST (T-Shaped Transistors) rises with potential characteristics for analog low power design. TST /TAT (Trapezoidal Associations of Transistors) are selfcascode structures and can be turning into a good alternative for leakage and area reduction. Another point is the increment in mismatch and layout regularity, all these characteristics being very important in analog designs. The TST electrical measurements study and analysis are developed to show the device properties. An analysis of the TST desired properties and extrapolation for nanometer technologies analog design are also presented.

Em nossos dias a crescente busca por portabilidade e desempenho resulta em esforços focados na maximização da duração de bateria dos equipamentos em fabricação, ou seja, busca-se a conflitante solução de circuitos com baixo consumo e ao mesmo tempo com alto desempenho. Neste contexto usualmente na composição de equipamentos portáteis empregam-se SOC´s (Systems On Chip) o que barateia o custo de produção e integração destes circuitos. SOC´s são sistemas completos que executam uma determinada função integrados em uma pastilha de silício única, normalmente possuem memórias SRAM como componente do sistema, que são utilizadas como memórias de alta performance e baixa latência e/ou também como caches. O grande desafio de projeto em memórias SRAMS é a relação de desempenho versus potência consumida a ser otimizada. Basicamente por sua construção estes circuitos apresentam alto consumo de potência, dinâmica e estática, relacionada a primeira diretamente ao aumento de freqüência de operação. Um dos focos desta dissertação é explorar soluções para a redução de consumo de energia tanto dinâmica como estática, sendo a redução de consumo estático de células de memória em standby buscando desempenho, estabilidade e baixo consumo de energia. No desenvolvimento de técnicas para projeto de circuitos analógicos em tecnologias nanométricas, os TST´s (T-Shaped Transistors – Transistor tipo T) surgem como dispositivos com características potenciais para projeto analógico de baixa potência. TSTs / TATs (Trapezoidal Associations of Transistors – Associação Trapezoidal de transistores) são estruturas self-cascode que podem tornar-se uma boa escolha por apresentar redução do leakage, redução na área utilizada e com incremento na regularidade do layout e no casamento entre transistores, propriedade importantíssima para circuitos analógicos. Sendo este o segundo foco deste texto através do estudo e análise das medidas elétricas dos TSTs executadas para comprovação das características destes dispositivos. Também apresenta-se uma análise das possibilidades de utilização dos TSTs em projeto analógico para tecnologias nanométricas. / Nowadays the increasing needs for portability and performance has resulted in efforts to increase battery life, i. e., the conflicting demands for low power consumption and high performance circuits. In this context using SOC´s (System On Chip) in the development for portable equipments composition, an integration of an entire system for a given function in a single silicon die will provide less production costs and less integration costs. SOC´s normally include a SRAM memory as its building block and are used to achieve memories with low latency and short access time or (and) as caches. A performance versus power consumption analysis of SRAM memory building blocks shows a great challenge to be solved. The electrical design aspects of these blocks reveal high power consumption, dynamic and static, and the former is directly proportional to the operating frequency. The design space exploration for dynamic and leakage consumption reduction in these circuits is one of the focus of this work. The main contribution of this topic is the leakage reduction techniques based in performance, stability and low energy consumption for the memory cell stand-by mode. Among the electrical techniques developed for analog circuits at the 20-100 nanometer scale, the TST (T-Shaped Transistors) rises with potential characteristics for analog low power design. TST /TAT (Trapezoidal Associations of Transistors) are selfcascode structures and can be turning into a good alternative for leakage and area reduction. Another point is the increment in mismatch and layout regularity, all these characteristics being very important in analog designs. The TST electrical measurements study and analysis are developed to show the device properties. An analysis of the TST desired properties and extrapolation for nanometer technologies analog design are also presented.

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L’immunothérapie basée sur le transfert de lymphocytes T (LT) spécifiques de la tumeur est une approche prometteuse contre le cancer. Pour activer et amplifier de tels LT, principale étape limitante de cette approche, des cellules présentatrices d’antigène artificielles (CPAA) ont été développées au laboratoire. Ces CPAA ont été construites à partir de fibroblastes murins NIH/3T3 transduits à l’aide de vecteurs gammarétroviraux afin d’exprimer les principaux éléments nécessaires à l’activation de LT humains. Ces CPAA nous permettent d’obtenir des LT mémoires souches (TSCM : CD95+CD45RA+CD62L+CCR7+), LT très peu différenciés récemment identifiés chez l’homme. Ces TSCM ont été décrits comme étant du plus grand intérêt pour l’immunothérapie en raison de leur capacité d’auto-renouvellement et de leur faculté à se différencier en LT effecteursefficaces. Pour optimiser l’amplification de TSCM spécifiques, nous avons notamment étudié les effets sur les LT de l’expression de différentes molécules de costimulation par nos CPAA (CD80, CD70 et 4-1BBL). Les protéines MART-1 et MELOE-1, surexprimées dans les mélanomes, ont été utilisées comme antigènes modèles pour ces travaux. Les CPAA CD80+CD70+ et CD80+CD70+4-1BBL+ sont les plus prometteuses pour maintenir le phénotype des TSCM. Une étude exhaustive des CPAA CD80+CD70+ a montré que nous pouvions obtenir un plus grand nombre de TSCM fonctionnels spécifiques de MART-1 et de MELOE-1 de manière reproductible avec ces CPAA. Dans une seconde étude, nous avons pu montrer que les CPAA CD80+CD70+4-1BBL+ permettaient d’obtenir le plus grand nombre de LT spécifiques fonctionnels et très peu différenciés après purification et restimulation de LT spécifiques stimulés une première fois par les CPAA CD80+CD70+. Ces travaux devraient nous permettre, après le développement d’un modèle murin, de proposer de nouvelles stratégies d’immunothérapie basées sur l’obtention grâce à nos CPAA optimisées de LT spécifiques anti-tumoraux capables d’assurer une protection à long terme aux patients. / Immunotherapy based on the transfer of tumor-specific T lymphocytes (TLs) is a promising approach against cancer. To activate and amplify such TLs, main limiting step of this approach, artificial antigen presenting cells (AAPCs) have been developed in the laboratory. These AAPCs have been constructed from NIH/3T3 murine fibroblasts transduced with gammaretroviral vectors to express the principal elements required to activate human TLs. With these AAPCs, we can obtain anti-tumor stem cell memory TLs (TSCM: CD95+CD45RA+CD62L+CCR7+), which are very limitedly differentiated TLs recently identified in humans. These TLs have been recently described as cells of great interest for immunotherapy because of their self-renewal capacity and their ability to differentiate into effective effector TLs. To improve the amplification of specific TSCM, we notably studied the effects on TLs of the expression of different co-stimulatory molecules by our AAPCs (CD80, CD70 and 4-1BBL). MART-1 and MELOE-1, proteins that are overexpressed in melanoma, were used as model antigens in this work. CD80+CD70+ and CD80+CD70+4-1BBL+ AAPCs appear to be the most promising ones for maintaining a TSCM phenotype. An exhaustive study of CD80+CD70+ AAPCs showed that we could reproducibly get greater numbers of MART-1- and MELOE-1-specific functional TSCM with these AAPCs. In another study, we have shown that CD80+CD70+4-1BBL+ AAPCs enabled us to get the greatest number of functional and very limitedly differentiated specific TLs after purification and restimulation of specific TLs stimulated first with CD80+CD70+ AAPCs. This work should allow us, after the development of a murine model, to propose new immunotherapy strategies based on the possibility of obtaining with our optimized AAPCs anti-tumor specific TLs capable of ensuring patient long term protection.

京都大学 / 0048 / 新制・課程博士 / 博士(情報学) / 甲第20778号 / 情博第658号 / 新制||情報||113(附属図書館) / 京都大学大学院情報学研究科通信情報システム専攻 / (主査)教授 小野寺 秀俊, 教授 佐藤 高史, 教授 黒橋 禎夫 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

The contributions of peptide antigen affinity for TCR in driving T cell memory is unclear. Effector CD4 T cells must recognize cognate antigen again at an effector checkpoint, 5-8 days post-infection, to generate an optimal memory population. In this thesis, we examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We used an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP- peptide variants that bind FluNP TCR with a broad range of avidity. Varying peptide avidity in vivo at the effector checkpoint revealed that higher affinity interactions yielded greater numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN. The major impact of avidity was on memory cell number, not cytokine production, and was already apparent within several days of transfer. These memory cells demonstrated enhanced protection against lethal IAV infection with a robust early day 5 secondary effector response in the lung. We previously showed that autocrine IL-2 production during the effector checkpoint prevented default effector apoptosis and supported memory formation. Here, peptide avidity determined the level of IL-2 produced by effectors while IL-2R expression was unaffected. However, IL-2Ra expression by APC drove more memory cell formation, suggesting that transpresentation of IL-2 by APC at this checkpoint enhanced CD4 memory generation. Secondary memory generation was also avidity-dependent. We propose this pathway selects CD4 effectors of highest affinity to progress to memory and can instruct future vaccine design.

Bakterielle Glykanantigene sind hoch-divers in ihrer Komposition und Verbindung. Antikörper gegen Glykanantigene können vor bakteriellen Infektionen schützen und sind wichtig um die Homöostase zwischen dem Wirt und seinem Mikrobiom aufrecht zu halten. Typischerweise lösen Glykanantigene jedoch Antikörperantworten aus, die sich durch ein vermindertes B Zell-Gedächtnis und niedrig-affine Antikörper mit geringer Spezifität auszeichnen. In dieser Arbeit konnten, mithilfe von biotinylierten Lipopolysaccharide O-Antigenen des opportunistisch-pathogenen Bakteriums Klebsiella pneumoniae (Kp), O-Antigen-spezifische B Zellen innerhalb peripherer Gedächtnis- und intestinaler Effektor-B Zellen identifiziert werden. Durch Einzel-Zell Immunoglobulin-Sequenzierung und Klonierung bzw. rekombinanter Expression von Antikörpern dieser Zellen wird gezeigt, dass, unter natürlichen Umständen, affinitätsgereifte Antikörper gegen definierte Kp Glykanantigene erzeugt werden. Diese Antikörper binden nicht nur Kp O-Serotyp-spezifisch sondern auch spezifisch an strukturell ähnliche Kp O-Antigene und taxonomisch unterschiedliche Mikroorganismen. Die Ergebnisse zeigen, dass Menschen, bei natürlicher Besiedlung, kreuz-spezifische Antikörper gegen Glykanantigene erzeugen und deuten auf einen Mechanismus hin, wie das humorale Immunsystem auf die Glykandiversität des Mikrobioms reagieren und sich anpassen kann. Weiterhin könnten die hier identifizierten Antikörper nützlich für die Behandlung von nosokomiellen Kp Infektionen sein. / Bacterial glycan antigens are highly diverse in composition and linkage. Antibodies against glycan antigens can protect against bacterial infection and are important in maintaining homeostasis between the host and its microbiome. However, glycan antigens typically elicit B cell responses that have impaired long-term memory formation and are comprised of low-affine antibodies with low specificity. In this work, the use of biotinylated Lipopolysaccharide O-antigens of the opportunistic pathogen Klebsiella pneumonia allowed to identify anti-O-antigen B cells in the peripheral memory and intestinal effector B cell pool in healthy humans. Single B cell Immunoglobulin gene sequencing, antibody cloning, and recombinant expression reveal that, under natural circumstances, humans acquire affinity-matured antibodies against defined Kp glycan antigens. Despite their O-serotype-specificity, the antibodies bind to other structurally similar Kp O-serotypes and taxonomically distinct non-Kp microbes. The findings show that humans, under natural exposure, acquire affinity-matured cross-specific anti-glycan antibodies and provide a mechanistic way how the humoral immune system could adapt to the large microbial glycan diversity present in nature. Further, the antibodies identified in this work might be beneficial in treatment of nosocomial Kp infections.

Das Zytokin Interleukin-7 (IL-7) ist für die Entstehung und das Überleben reifer T Zellen von zentraler Bedeutung. Die Gabe von IL-7 führt sowohl in der Maus als auch im Menschen zu erhöhten T Zellzahlen und einem veränderten T Zellphänotyp. Folglich könnte sich die therapeutische Gabe von IL-7 bei Patienten mit geschwächtem Immunsystem positiv auswirken. Diese Hypothese wird derzeit in mehreren klinischen Studien untersucht. Bisher wurde allerdings nur die Wirkung von IL-7 auf T-Zellen studiert. Zu dessen Wirkung auf andere Immun- oder Stromazellen sowie deren IL-7-abhängigen Beitrag zur Regulation der T-Zellhomöostase ist nur wenig bekannt. Daher war es Ziel der Arbeit, den Einfluss einer therapeutischen Gabe von IL-7 auf adoptiv-transferierte T-Zellen in IL-7-Rezeptor (IL-7R)-kompetenten und defizienten lymphopenischen Mäusen zu studieren. Die Untersuchungen bestätigen, dass die Gabe von IL-7 T-Zellantworten unterstützt, zeigen jedoch auch, daß viele dieser Effekte von IL-7R-exprimierenden Wirtszellen abhängig sind. Dies weist darauf hin, dass IL-7R-vermittelte Signale in Wirtszellen indirekt T-Zellantworten beeinflussen. Zudem zeigte sich, dass effiziente anti-Tumor-T Zellantworten von IL 7R-vermittelten Signalen in Wirtszellen abhängen. Vor allem nicht-hämatopoetische Wirtszellen fungieren hier als Regulatoren der IL-7-Therapie-vermittelten T Zelldifferenzierung. Unsere Ergebnisse bestätigen außerdem, dass Stromazellen in verschiedenen Organen il-7 exprimieren und zeigen darüber hinaus, dass diese Zellen durch die Gabe von IL-7 beeinflusst werden. Wir folgern daraus, dass die Effekte der IL-7-Therapie auf T Zellhomöostase teilweise indirekt über il-7-exprimierende Stromazellen vermittelt werden. Um diese Zellen genauer identifizieren und untersuchen zu können, haben wir ein neues transgenes Mausmodell charakterisiert, was es erleichtern wird, die beteiligten molekularen Signalwege zu analysieren und den Erfolg der adoptiven T Zelltherapie zu verbessern. / Interleukin-7 (IL-7) is an essential cytokine required for the development and maintenance of mature T cell. Its availability is limited under normal conditions, but rises during lymphopenia, leading to increased T cell proliferation. The administration of recombinant IL-7 to normal or lymphopenic mice and humans results in increased T cell numbers and altered T cell phenotype. Hence, IL-7 administration could mediate therapeutic benefits in immunocompromised patients and is currently tested in several clinical trials. However, besides its well-studied effects on T cells little is known about the effect of IL-7 on other immune and non-immune cells and their influence on T cell homeostasis. Therefore, we evaluated the effect of IL-7 therapy on adoptively transferred T cells in IL-7 receptor (IL-7R)-competent and IL-7R-deficient lymphopenic mice. We confirm the benefits of IL-7 therapy on T cell responses but additionally show that many of these effects are dependent on IL-7R expression by host cells, indicating that IL-7R signaling in host cells modulates T cell responses. We show that efficient T cell responses against cancer are dependent on host IL-7R signaling. Based on studies in bone-marrow chimeric mice, we identify non-hematopoietic host cells as main regulators of IL-7 therapy-modulated T cell differentiation. We conclude from these data that IL-7 therapy affects non-hematopoietic stromal cells that modulate the success of adoptive T cell therapy. Our results confirm that stromal cells in various organs express il-7 and show that these cells are targeted by IL-7 therapy in vivo. Hence, we propose that il-7-expressing cells regulate IL-7 therapy-modulated T cell homeostasis. To identify and study these il-7 expressing stromal cells in more detail, we characterized a new transgenic mouse model that will facilitate determining the molecular pathways to improve the success of adoptive T cell therapy.

A exposição limitada ao Plasmodium chabaudi induz proeminente imunidade celular, associada à proteção de células T da apoptose. Este estudo tem como objetivo verificar a influência da carga parasitária na geração e manutenção dos linfócitos T e B de memória ao P. chabaudi. Assim, camundongos C57BL/6 foram submetidos à infecção tratada (subpatente) ou não (patente) com cloroquina após a inoculação de 106 eritrócitos parasitados (EP) e analisados nos dias 0, 20, 60, 120 e 200. Com relação à memória de linfócitos T, no dia 20, as freqüências de células CD4+ memória/ativadas e respondedoras aos EP foram significativamente maiores nos animais do grupo subpatente. Os níveis máximos de IgG2a específica foram encontrados no dia 120 em ambos os grupos. O desafio dos animais com 108 EP mostrou que a imunidade protetora declina progressivamente, mas os grupos ainda são capazes de estabelecer resposta secundária eficiente que elimine o parasita. Assim, podemos concluir que a carga parasitária influencia a fase aguda, mas não impede a geração e manutenção das células T e B de memória. / One of the main characteristics of malaria is the intense policlonal activation of splenic T and B lymphocytes induced by the parasite and the consequent elimination, through apoptosis, of part of these cells. However, the limited exposure to the bloodstage malaria seems to induce a prominent cellular immunity, associated with the protection of T lymphocytes from apoptosis. With this in mind, this study aimed to verify the influence of the parasite load in the generation and maintenance of memory T and B cells specific for Plasmodium chabaudi chabaudi AS. In order to evaluate this idea, C57BL/6 mice were infected with 106 parasitized red blood cells (pRBC) and submitted to a patent (untreated) or subpatent infection (controlled with sub-curative doses of chloroquine every time parasitemia reached 1%). Splenocytes from these mice were analyzed at 20, 60, 120 and 200 days after infection, regarding the pRBC-specific T cell proliferation and the expression of surface molecules, as CD4, CD8, CD62L, CD45RB, CD44, CD45R-B220 and IgG. The parasitemia and the splenocyte phenotype were also monitored after the challenge with 108 pRBC. Regarding T cell memory, at day 20 of infection, the frequencies of effector/activated CD4+ T cells (CD62LLOW CD45RBLOW/HIGH) were significantly increased in animals from the patent group, which was strict linked with the highest cellular activation observed in these animals. On the other hand, the total numbers of pRBCproliferating T (CD4+ and CD8+) cells per spleen were approximately 3-fold increased in subpatent animals, indicating that these cells were protected from apoptosis as a result of the limited exposure to the parasite. However, in both groups, these parameters decreased to values similar to those in controls at day 200. The splenocytes from both groups produced Th1 cytokines in response to pRBC in all times of analysis, but at the early phase of infection, Th2 cytokines were also observed, but without differences between the infected groups. Regarding memory B cells, the frequency of sIgG+ cells was increased at day 20 of infection, when 11% and 9% of CD45R+ cells from patent and subpatent animals were positive, respectively. For both groups, specific IgG2a antibodies attained maximum serum levels at day 120, but at day 200, it is possible to observe a significant decrease of these levels only in the serum of patent mice. Moreover, at day 200 of infection, mice of subpatent group showed significantly higher amounts of IgG2a that recognized the intra-erythrocytic forms of the parasite and the surface of infected erythrocytes. Challenge of mice with 108 pRBC showed that protective immunity progressively decline with time and despite the higher levels of specific antibody in subpatent mice, both groups showed similar protection. In experiments of adoptive transference to CD28-/- mice, cells from 200-day infected mice were able to produce specific IgG2a antibodies, in a T CD4+ cell dependent way. In addition, we verified that CD45R+ cells of subpatent mice, when transferred to CD28-/- mice, secreted higher amounts of specific IgG2a and IgG1 antibodies, comparing to cells of patent mice. So, from this work, we can conclude that the parasite load has a great influence in the early immune response to P. chabaudi malaria and it also affects the generation and/or maintenance of memory B cells. Furthermore, according to our data, at least during the analyzed period, the loss of protective immunity against this parasite does not seem to be influenced by the acute-phase parasite load, but it can be a consequence of the progressive decline of T-cell memory response that occurs in patent and subpatent groups with time of infection.