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Supervisor:石原 哉 情報科学研究科 博士

Antigen presentation on major histocompatibility complex (MHC) plays an important role in anti-tumor immunity. In addition to epitope presentation on MHC class I that activates cytolytic T cells, antigen presentation on MHC class II and a resulting CD4+ T cell immune response have gained increasing relevance. Preclinical and clinical studies have shown that a successful immunotherapeutic intervention depends on the presentation of neoantigens on MHC class II. However, the exact mechanisms that drive an MHC class II-restricted immune response as well as the relevance of different antigen-presenting cell subsets are not fully understood. Brain tumors such as glioma are characterized by a low mutational load and therefore a reduced abundance of potential neoepitopes. For some glioma associated tumor-driver mutations immunogenicity has been shown; however, the number of targetable epitopes and efficacy of neoantigen specific immunotherapies remains low. In the present thesis several mutations frequently present in low grade gliomas have been screened for immunogenicity. The point mutation CICR215W has been shown to elicit mutation specific immune responses in an MHC-humanized mouse model. This neoepitope was shown to be presented on MHC class II and to lead to a CD4+ T cell-driven immune response. Using single cell T cell receptor sequencing, CICR215W-specific TCRs could be retrieved und functionality was validated in vitro. For in vivo validation, a novel brain tumor model was established using CRISPR-Cas9 mediated genetic manipulations. Locoregionally adoptive transfer of T cell receptor-transgenic T cells led to a reduced tumor growth in CICR215W-mutant brain tumor-bearing mice. Overall, a novel shared therapeutic target in gliomas could be established, that can be targeted via adoptive transfer of T cell receptor transgenic T cells in order to elicit anti-tumor immune responses. Furthermore, the relevance of MHC class II presentation in gliomas has been investigated. Using a novel mouse model allowing for depletion of MHC class II on tumor infiltrating macrophages, it could be shown that prevalence of MHC class II is essential for the response to immunotherapy due to necessary activation of CD4+ T cells. Using antigen-specific tumor models and single-cell transcriptomics it could be shown that the lack of activated CD4+ T cells leads to an exhausted phenotype of tumor-reactive CD8+ T cells that thereupon lose their ability to lyse tumor cells. This effect was only apparent during early stages of T cell activation. A late depletion of MHC class II did not result in any differences in CD8+ T cell activation. In summary, the present thesis describes a novel target for T cell mediated immunotherapy and demonstrates the necessity of MHC class II-restricted antigen presentation on tumor-infiltrating macrophages for successful anti-tumor activity of cytotoxic CD8+ T cells.

Research has highlighted that firms competing in dynamic environments have to balance exploitative (efficiency-directed) activities with explorative (innovation-directed) ones in order to remain internationally competitive. In economically advanced countries, whose competitiveness is innovation-driven, this prerequisite of ambidexterity also holds at the aggregate level of (sea)ports. Ports, being important junctions in international integrated chain systems, however, appear to focus predominantly on exploiting their existing assets and market position, minimizing the costs of freight flows, and enhancing overall efficiency levels. This dissertation contains six different exploratory studies into how efficiency-dominated ports in economically advanced countries can become more ambidextrous and, in turn, strengthen their innovation-driven international competitiveness. These studies variably emphasize how firms, business associations and, in particular, port authorities can play a role in this endeavor. Drawing on case study findings and prior literature, it is shown how new ways of organizing and managing, i.e. management innovation, introduced by these organizations at the intra-, inter- or multi-organizational level may contribute to enhanced resource productivity, greater environmental performance, advancements in technological innovation, improved safety procedures in ports, and to a more innovative business climate in general. Also, it is elaborated how the business model of port authorities and, in this connection, their strategic use of generic policy instruments are related with a port’s level of strategic connectivity and strategic value creation for its country. Several conceptual framework and propositions are developed that provide interesting directions in which future studies on management innovation, multi-organizational collaboration, and port authority strategies may be usefully enriched.

Estudio sobre la Teoría General del Servicio Público y el Derecho a la Educación. Estudio jurídico-administrativo de la Educación en España; se comparan también las Constituciones de Alemania, Francia, Grecia, Italia y Portugal. La enseñanza como derecho humano fundamental, en Constituciones y convenios internacionales, y las posturas de los distintos partidos políticos, conducen a la Teoría General del Servicio Público y finalmente a determinar la naturaleza jurídico-administrativa entre enseñanza estatal o pública y enseñanza no estatal o privada. Declaraciones, convenios internacionales, Constitución y leyes españolas, Constituciones extranjeras, manifiestos de partidos políticos, documentos sobre educación, legislación y bibliografía. Análisis de documentos, legislación y revisión histórica. 1. La cultura implica educación y ésta enseñanza. Con frecuencia se habla de derecho a la cultura y a la educación cuando en realidad se habla de derecho a la enseñanza. 2. El Derecho a la Educación es un derecho fundamental que se debe salvaguardar en toda circunstancia. 3. Todas las fuerzas políticas confluyen en la libertad de enseñanza frente a un monopolio ideológico. Divergencia entre partidos de izquierdas que abogan porque el estado se haga cargo de la enseñanza, y los partidos de derechas que defienden la independencia total de la enseñanza privada. Todos defienden la gratuidad, la obligatoriedad y la descentralización. 4. La enseñanza es una actividad técnica, de interés general, también se da con regularidad. Es por tanto un servicio público, aunque no se da la continuidad. 5. La enseñanza privada no es un servicio público por no ser la administración titular de la misma. Andalucía ESP

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 18-09-2020 Esta tesis tiene embargado el acceso al texto completo hasta el 18-03-2022 Pyruvate Kinase Deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene, and constitutes the main cause of chronic non-spherocytic hemolytic anemia. PKLR gene encodes the erythroid pyruvate kinase protein (RPK) implicated in the last step of the anaerobic glycolysis in red blood cells. The defective enzyme fails to produce normal ATP levels, and erythrocytes from PKD patients suffer hemolysis. The only curative treatment applied so far, is the hematopoietic stem and progenitor cells (HSPC) transplant of a compatible donor, with the associated limitations this technique involves. In this work, we conducted precise gene editing in the endogenous locus. We intended to follow two different approaches based on the homology directed repair pathway (HDR). On one side, we attempted to specifically correct a PKD patient’s mutation (occurring in exon 3 of PKLR gene) by electroporation of a specific ribonucleoprotein (RNP) and a single-stranded oligonucleotide with the corrective sequence in a lymphoblastic cell line derived from the patient. We demonstrated the feasibility of the process although the efficacy needs to be improved to achieve clinical importance. On the other side, we developed a wider strategy, potentially applicable to more patients with a variety of mutations, based in the knockin of a therapeutic donor in the PKLR locus. We tested its introduction in the second intron of the gene, mediated by TALEN nucleases. The donor carried a codon optimized sequence of RPK cDNA and a puromycin selection gene. Although we observed high levels of gene editing in the puromycin resistant cells, the editing efficacy in the engrafted cells was low. To optimize the protocol, we incorporated the use of adeno-associated vectors to mediate the delivery of the therapeutic donor. We were able to efficiently edit HSPCs by using a selection-free system. The ex vivo gene edited cells efficiently reconstituted the human hematopoiesis in primary and secondary recipients, and cells that have undergone HDR were found within the mature and primitive cell subsets. Finally, we mediated gene editing in HSPCs from 3 different PKD patients and edited cells were differentiated in vitro towards the erythroid lineage. At the end of the process, only the cells edited with the therapeutic donor were able to produce normal levels of ATP, demonstrating the restoration of the glycolysis functionality in edited cells and situating this gene editing strategy as a potential therapeutic alternative for the treatment of PKD Para su ejecución, el trabajo de investigación ha sido financiado por proyectos del “Ministerio de Economía, Comercio y Competitividad y Fondo Europeo de Desarrollo Regional (FEDER)” (SAF2014-54885-R y SAF2017-84248-P), “Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III” (Red TERCEL; RD12/0019/0023 y RD16/0011/0011) y Comunidad de Madrid (CellCAM (S2010/BMD-2420 y AvanCell, B2017/BMD-3692)

Il passaggio nell’uomo dalla stazione orizzontale a quella verticale ha richiesto un pavimento pelvico robusto e resistente, atto a sopportare il peso degli organi addominali e pelvici. Gli orifizi per il passaggio attraverso il diaframma pelvico di organi provvisti di muscolatura propria (retto, uretra, e vagina nella femmina), insieme all’invecchiamento fisiologico del connettivo che compone i legamenti ed i muscoli pelvi-perineali, comportano il cedimento delle aree sottoposte a maggior pressione come la regione pelvica. Il prolasso pelvico (definito in letteratura come prolasso urogenitale) è una patologia dell’età adulta del sesso femminile molto frequente, dato che il rischio di subire un intervento chirurgico per il trattamento di un disordine della statica e della dinamica della pelvi è di circa il 10% per una donna con una vita media di 80 anni2,5-6. Tale patologia, oggi più facilmente diagnosticabile grazie allo sviluppo di nuove tecnologie radiologiche dinamiche, si presenta prevalentemente nelle donne di età superiore ai 40 anni e con un’incidenza di circa il 30-40%7,12. La strategia chirurgica adottata è fondata sul principio del sostegno dei 3 compartimenti. Le attuali tecniche prevedono approcci per via trans-vaginale o per via trans-addominale, con o senza l’utilizzo di rinforzi protesici. L’autore riporta nel presente lavoro i risultati preliminari di un nuovo tipo di trattamento chirurgico per la correzione del prolasso tricompartimentale frutto della combinazione di due differenti tecniche, valutandone fattibilità e risultati a breve e medio termine.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 23 de Marzo de 2012

Tese de Doutoramento em Ciências Veterinárias, especialidade de Ciências Biológicas e Biomédicas Germline-encoded autoreactive natural antibodies (NAbs) of the IgM isotype are secreted and circulate as a result of basal immune system stimulation, and constitute an important first line of defense against microorganism invasion, bridging the innate and the adaptive immune responses. NAbs are mostly secreted by positively selected B1a cells, and have been claimed to have a protective role against autoimmunity. Nevertheless, NAbs binding to cell surface self-antigens could have implications in the initiation of autoimmunity. Article I focused on the genetic control of NAbs secretion in healthy mice. Importantly, interferon regulatory factor 4 (Irf4), a transcription factor required for plasma cell differentiation and antibody secretion, was identified as the most probable candidate for the control of homeostatic serum IgM levels in the mouse. Type 1 diabetes (T1D) is a complex autoimmune disease that develops spontaneously in humans and is pathogenically similar in the non-obese-diabetic (NOD) mouse model. B cells are necessary in the NOD diabetogenic process, and the presence of anti-pancreatic beta cell antibodies is the earliest manifestation of T1D. Article II revealed that NOD peritoneal cavity B1a cells are more prone to spontaneously secrete NAbs that recognize pancreatic beta cell autoantigens, which could promote T1D either by enhancing professional antigen presentation of islet antigens, by activating the complement cascade or by directly promoting beta cell damage and self-antigens release. The studies reported in article III have explored these possibilities and have proven that NAbs of NOD B1a cells origin could bind and directly induce oxidative stress on pancreatic beta cells. Moreover, these studies have shown that NOD B1a cells have a lower threshold for innate-like stimulation and have established a link between NOD B1a cells properties, NAbs specificities and impact of IgM binding on beta cells physiology. Finally, article IV provides evidence that early treatment with antibodies that evoke NOD B1a cells proliferation and differentiation into IgM secreting cells correlates with T1D precipitation. In conclusion, this thesis has shown that Irf4 is a critical player in the genetic network that controls IgM secretion in healthy individuals, and that in the NOD mouse model of T1D, a lower threshold for innate like stimulation of peritoneal cavity B1a cells contributes to a naturally increased state of B1a cells activation and autoreactive IgM secretion, determining the initiation and/or contributing to the fueling of beta cells autoimmunity. RESUMO Secreção de IgM natural na saúde e na doença: controlo genético e papel na diabetes tipo 1 - Os autoanticorpos naturais (NAbs) da classe IgM existem no organismo na ausência de imunização e constituem uma primeira linha de defesa fundamental contra infecções. Os NAbs são secretados maioritariamente por células B1a e a sua ligação a autoantigénios na superfície celular pode ter implicações para a iniciação de autoimunidade. O trabalho descrito no artigo I focou-se na compreensão do controlo genético da secreção de NAbs em murganhos saudáveis. Este estudo identificou o interferon regulatory factor 4 (Irf4), um factor de transcrição necessário para a diferenciação de plasmócitos e secreção de anticorpos, como o candidato mais provável para o controlo da homeostasia dos níveis de IgM circulante no murganho. A diabetes tipo 1 (T1D) é uma doença autoimune complexa que se desenvolve espontaneamente nos humanos e que tem uma patogenia semelhante no murganho NOD (non-obese-diabetic). Os linfócitos B são necessários para o processo diabético do NOD, em que a presença de anticorpos anti-células beta pancreáticas é uma das manifestações mais precoces. O artigo II revelou que as células B1a da cavidade peritoneal do NOD têm uma elevada predisposição para secretarem NAbs que reconhecem autoantigénios de células beta pancreáticas e que podem promover o desenvolvimento de T1D quer pelo aumento da apresentação de autoantigénios, quer pela activação da cascata do sistema de complemento, quer pela indução directa de danos nas células beta pancreáticas. A investigação descrita no artigo III provou que os NAbs secretados por células B1a do NOD têm a capacidade de se ligarem e induzirem stress oxidativo nas células beta do pâncreas. Estes estudos revelaram ainda que as células B1a do NOD têm um limiar reduzido para activação inata e estabeleceram uma relação entre as propriedades das células B1a do NOD, as especificidades dos NAbs e o impacto da ligação de IgM na fisiologia das células beta. Finalmente, o artigo IV evidenciou que a indução de proliferação e diferenciação das células B1a em células secretoras de IgM contribui para o início da T1D. Esta tese demonstrou que o Irf4 é um factor de transcrição com um papel fundamental no controlo da secreção de IgM em animais saudáveis e que, no murganho NOD, as células B1a da cavidade peritoneal têm um menor limiar para estimulação inata, que contribui para o seu estado de activação e para a secreção de IgM autoreactiva, determinando a iniciação e/ou contribuindo para a progressão da diabetes tipo 1. Fundação para a Ciência e Tecnologia; Instituto Gulbenkian da Ciência

In the multimodel improvement context, software organizations have the need to incorporate into its processes different practices of several software process improvement models (SPIMs) simultaneously (i.e. CMMI, PSP, ISO 15504 and others). Currently, a clear trend to the specialization of SPIMs to domain specific was identified. Over the last few years, software process architectures have been considered a means to harmonize these technologies. However, it is unclear how to design a software process architecture supporting a multimodel improvement environment. In this research, a method to design a domain-specific software process architecture supporting a multimodel environment (DS-SPAM) with a focus on the capture and organization of the domain knowledge to facilitate its reuse and maintainability is presented. This proposes a structure called the Software Process Architectural Domain Assets Set (SPADAS) as an approach for organizing domain knowledge. This method is described using basic concepts, phases, activities, tasks, roles and artifacts. Thus, our method mostly will assist process stakeholders in the extraction, design and documentation of their software process architecture as part of a multimodel solution