Orientador: Ricardo de Lima Zollner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T22:53:38Z (GMT). No. of bitstreams: 1 Brito_RhubiaBethaniaSocorroLemosde_M.pdf: 1643193 bytes, checksum: 3883ffa4fdb888928db0d59dc9e0a674 (MD5) Previous issue date: 2006 / Resumo: A imunoterapia é a estratégia empregada no tratamento das doenças alérgicas que, quando bem indicada, é considerada eficaz com redução dos sintomas. Apesar disso, uma das desvantagens é a possibilidade de surgirem reações sistêmicas adversas durante o tratamento. Dentro do contexto de inovação tecnológica, o surgimento da nanotecnologia oferece a perspectiva de novas formas de elaboração de vacinas e imunoterápicos, com estruturas com propriedades de encapsulação da droga/alérgeno permitindo a construção de sistemas de liberação controlada. Dentre os diversos sistemas em estudo, as microesferas de PLGA apresentam-se como promissores, uma vez que têm a propriedade de, juntamente com o antígeno/alérgeno, modular a resposta imune por meio das células apresentadoras de antígenos que os fagocitam, propiciando apresentação antigênica lenta e gradual. Com objetivo de estudarmos a resposta imune de camundongos BALB/c previamente sensibilizados com ovalbumina (OVA, antígeno Th2), injetamos dose única de microesferas de PLGA encapsuladas com OVA e microesferas vazias como controle. Os animais eram sacrificados 30, 60, 90 e 120 dias pós-injeção das microesferas. Após os 120 dias de observação, nossos resultados sugerem que as microesferas encapsuladas com OVA, promovem modulação de resposta com perfil misto Th2/Th1, com elevação predominante da citocina IFN-? e tendência de elevação de IL-12. Além disso, pode-se observar diminuição significativa dos níveis séricos da subclasse IgG1 representativa da resposta Th2, embora a IgE permaneça inalterada / Abstract: The immunotherapy is a strategy used in the treatment of allergic diseases when properly indicated is considered efficient with reduction of the symptoms. In spite of that, one of its disadvantages is the possibility to adverse systemic reactions appear during the treatment. In the context of technological innovation, the sprouting of the nanotechnology offers the perspective of new forms of immunotherapies using structures with properties of encapsulation allergen/drug allowing the construction of controlled delivery systems. Amongst the different systems in study, the PLGA microspheres seem to be promising. Microspheres with the encapsulated allergen/antigen, modulate the immune response through the antigens presenters cells that phagocyte them, propitiating slow and gradual antigenic presentation. Aiming to study the immune response of BALB/c mice previously sensitized with ovalbumin (OVA, Th2 antigen), we inject single dose of PLGA microspheres encapsulated with OVA and empty microspheres as control. The animals were sacrificed 30, 60, 90 and 120 days after-injection of the microspheres (ME-PLGA). Followed the 120 days of observation, our results suggest that microspheres encapsulated with OVA promote modulation of immune response with mixing profile Th2/Th1, however with statistical significant increase of cytokines IFN-_?and tendency of IL-12 increase. Moreover, it can be observed statistical significant reduction of the serum levels of IgG1 subclass representative of the Th2 profile, although the IgE remains unchanged. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Orientador: Francisco Benedito Teixeira Pessine / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-18T17:10:14Z (GMT). No. of bitstreams: 1 Baldim_Victor_M.pdf: 1808226 bytes, checksum: 7d81445a4d4297b7466e764b52804247 (MD5) Previous issue date: 2011 / Resumo: Neste trabalho preparamos e utilizamos nanopartículas de PLGA-DOTAP encapsulando o peptídeo P17, que bloqueia a ação da citocina imunossupressora TGF-b, no tratamento de camundongos BALB/c fêmea, inoculados via subcutânea com células de hepatocarcinoma (CHC) murino BNL ou de câncer colo-retal (CCR) murino CT26. No modelo de CHC, nanopartículas foram administradas em conjunto com vírus adenoassociados recombinantes, cujo DNA viral codificava a citocina imunoestimuladora IL-12. No modelo de CCR, nanopartículas foram testadas como potencializadoras de uma vacina contra câncer colo-retal. Além disso, a biodistribuição de nanopartículas de PLGA-DOTAP, funcionalizadas ou não com a glicoproteína assialofetuína, foi verificada pelos métodos de bioluminescência em câmara escura com detector CCD e por PCR quantitativa em tempo real. Como resultado, observou-se que nanopartículas de PLGA-DOTAP encapsulando o fármaco P17 são capazes de potencializar a vacina contra CCR e que a glicoproteína assialofetuína é capaz de direcioná-las ao fígado / Abstract: In this research we prepared and used PLGA-DOTAP nanoparticles loaded with the peptide P17, which blocks the action of the imunossupressive cytokine TGF-b, in the treatment of female BALB/c mice inoculated subcutaneously with BNL (murine hepatocellular carcinoma (HCC) cells) or CT26 (murine colorectal cancer (CRC) cells). In the HCC model, nanoparticles were administered together with recombinant adeno-associated virus whose viral DNA encodes the immunostimulatory cytokine IL-12. In the CRC model, nanoparticles were tested as vaccine booster against colorectal cancer. In addition, the biodistribution of PLGA-DOTAP nanoparticles functionalized or not with the asialofetuin glycoprotein was verified by bioluminescence and real-time quantitative PCR. As a result, it was observed that P17 containing DOTAP-PLGA nanoparticles are able to increase vaccine efficiency and that asialofetuin glycoprotein is able to direct them to the liver / Mestrado / Físico-Química / Mestre em Química

Prostate cancer is the most common cancer in men and the third leading cause of cancer-related deaths in Canadian men. Despite hormone and radiation therapies, most patients progress to late-stage metastatic castrate-resistant prostate cancer (mCRPC). 177Lu-PSMA617 radioligand therapy (rLT) is a radioactive biochemical substance that targets the human prostate-specific membrane antigen (hPSMA). This rLT has been used in compassionate trials in mCRPC patients and has been demonstrated significant clinical efficacy. However, recent findings suggest that this efficacy is short-lived, and most patients exhibit tumor recurrence [96]. Here we establish a murine model to study the anti-tumor effects and the corresponding immune response of 177Lu-PSMA617 rLT on prostate cancer. We generated a doxycycline-inducible hPSMA-expressing murine prostate cancer (hPSMA TRAMP-C2) cell line with high binding responses to PSMA617. Using this system, we evaluated the in vitro and in vivo binding of 177Lu-PSMA617 to the hPSMA TRAMP-C2 cell line. Here, we show that the hPSMA TRAMP-C2 cell line expresses hPSMA upon doxycycline induction and that 177Lu-PSMA617 can bind to its target in vitro and in vivo. Together, these results show that the developed hPSMA TRAMP-C2 cell line can be used to investigate therapeutic and immunological responses targeted against PSMA in prostate cancer. / Graduate

Les bactéries entéropathogènes du genre Salmonella et Shigella sont transmises par les aliments ou l’eau et sont responsables de nombreuses infections entériques chez les animaux et les humains. Ces maladies infectieuses restent une cause importante de morbidité et de mortalité dans les pays en voie de développement. L’existence de multiples sérotypes de Salmonella et de Shigella ainsi que l’émergence de souches résistantes aux antibiotiques, nécessite le développement de vaccins efficaces et large spectre. Ces bactéries utilisent un système d’injection de leurs protéines effectrices, appelé injectisome ou encore Système de Sécrétion de Type III (SST3), nécessaire à leur pathogénicité. Alors que les protéines effectrices injectées au moyen de cet injectisome sont variées et dépendent essentiellement du type cellulaire cible et donc de la spécificité du pathogène, certaines des protéines structurales composant l’injectisome sont relativement bien conservées parmi les différentes bactéries pathogènes, notamment les protéines de l’aiguille : PrgI et MxiH, et celles de la coiffe de l’aiguille : SipD et IpaD, respectivement de Salmonella et Shigella. Ces protéines, fortement impliquées dans la virulence des bactéries, semblent donc être des cibles de choix pour lutter contre des infections opportunistes impliquant ces bactéries pathogènes.Le premier objectif de cette thèse était d’évaluer l’immunogénicité et l’effet protecteur des protéines structurales de l’injectisome citées précédemment contre les infections à Salmonella et Shigella. Les protéines recombinantes préparées et produites au laboratoire ont été utilisées de façon séparée ou en combinaison pour immuniser des souris par différentes routes. Ensuite, les réponses immunitaires des souris ainsi immunisées ont été analysées par des tests immunométriques. Enfin, le potentiel immunogène et vaccinant de ces protéines structurales a été évalué en infectant les souris immunisées avec 100 DL50 de Salmonella par voie orale ou de Shigella par voie intranasale. Le meilleur résultat a été obtenu en utilisant la voie intra-gastrique pour les immunisations avec environ 70% de protection. Cette stratégie a permis également d’évaluer la pertinence de cette approche vaccinale dans un modèle murin de protection croisée (entre 25 et 60%). Le deuxième objectif de cette thèse était d’évaluer le pouvoir protecteur d’anticorps monoclonaux murins reconnaissant les régions conservées des protéines SipD et IpaD. Les anticorps obtenus ont été caractérisés et leur pouvoir neutralisant a été évalué in vivo dans un modèle murin d’infection avec Salmonella ou Shigella (jusqu’à 60% de protection).L’ensemble de ces travaux montre que l’utilisation de certaines protéines structurales conservées de l’injectisome de bactéries entéropathogènes présente un intérêt vaccinal et immunothérapeutique pour aider au traitement de certaines salmonelloses et shigelloses. / Salmonella and Shigella species are food and water borne pathogens that are responsible for enteric infections in both humans and animals. These infectious diseases are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of antibio- resistant strains, require the development of protective and broad-spectrum vaccines. All these bacteria utilize a system for injection of their effectors, called injectisome or Type III Secretion System (T3SS), necessary for their pathogenicity. While effector proteins are varied and depend essentially on the cellular target and thus on the specificity of the pathogen, the structural proteins that form the injectisome are common to all virulent Salmonella and Shigella spp., particularly the needle proteins PrgI and MxiH and the needle-tip proteins SipD and IpaD of Salmonella and Shigella respectively. These proteins, strongly involved in the virulence of the bacteria, appear to be ideal candidate antigens for a subunit-based, broad spectrum vaccine.The first aim of my PhD was to evaluate the immunogenicity and protective efficacy of structural proteins of the above-mentioned injectisome against Salmonella and Shigella infections. The recombinant proteins were prepared and produced in the laboratory and were used alone or in combination to immunize mice using different routes. The immune responses of immunized mice were then analyzed by immunometric assays. Finally, the protective efficacy was evaluated in a mouse model of intestinal (Salmonella) or pulmonary (Shigella) challenge. The best result was obtained by orogastric immunization with 70% of protection. This strategy also allowed to estimate the relevance of this approach in a mouse model of crossed protection (from 25 to 60%). The second objective of my PhD was to evaluate the protective efficacy of murine monoclonal antibodies recognizing conserved regions of SipD and IpaD proteins. The obtained antibodies were characterized and their therapeutic effect was evaluated in vivo with a Salmonella and Shigella infection murine model (up to 60% of protection).To conclude, this work showed that some conserved structural proteins composing the injectisome of enteropathogenic bacteria is of interest for treatment of enteric diseases caused by Salmonella and Shigella.

Les cellules NK sont des lymphocytes de l’immunité innée qui ont un rôle majeur dans le contrôle précoce des infections virales et dans l’immunosurveillance des tumeurs. Cependant, en cas de stimulation chronique, un état d’anergie, où les cellules NK n’exercent plus leur fonction a été mis en évidence. Les mécanismes conduisant à cette perte de fonction demeurent mal caractérisés et s’il s’agit d’une cause ou d’une conséquence de la chronicité n’est pas non plus déterminé. Cibler les cellules NK constitue une perspective thérapeutique de choix mais nécessite une meilleure compréhension de ces aspects. L’objectif de ce travail de thèse s’articule autour de trois points. Premièrement, nous avons généré un modèle tumoral murin sensible aux cellules NK permettant l’étude de la réponse anti-tumorale et l’établissement d’une stimulation chronique. Deuxièmement, l’utilisation de ce modèle a permis d’investiguer les mécanismes conduisant à la perte de fonctionnalité des cellules NK et de tester des stratégies de restauration. Enfin, nous avons mené une étude parallèle, chez l’homme, par analyse d’une cohorte de patients chroniquement infectés par le virus de l’Hépatite B dans le but de déterminer le phénotype des cellules NK et d’identifier les causes conduisant à leur perte de fonction dans ce contexte / NK cells are innate lymphocytes which play a crucial role in the early control of viral infection and in tumor immunosurveillance. However, a state of tolerance, where NK cells are poorly functional, occurs in the context of chronic stimulation. The mechanisms leading to this process remain poorly understood and whether this is a cause, or a consequence of chronicity is unknown. Targeting NK cells appears to be a potent therapeutic strategy but requires further investigation. With the lack of clarity in the field this work had three main objectives. First, we engineered a tumoral mouse model that was strongly immunogenic for NK cells and thus allowed us to study the anti-tumoral response of NK cells and to trigger chronic stimulation. Then, we used this model to investigate the mechanisms driving NK cell loss of function and to test potential therapeutic strategies to reverse this state. Finally, in the context of human chronic infection we analyzed samples from HBV infected patients in order to determine the phenotype, function and signaling capacity of NK cells to identify the drivers of NK cell dysfunction

Les modèles animaux actuellement disponibles pour l’étude des leucémies humaines ne sont pas adaptés pour le développement optimal de nouvelles thérapies ciblées. Au cours de ce projet, qui s’inscrit dans une double perspective fondamentale et industrielle, nous avons cherché à générer un modèle versatile de leucémogénèse humaine chez la souris humanisée BRGS (BALB/c Rag2-/- IL-2Rγc-/- SIRPα.NOD). Les animaux sont greffés avec des progéniteurs hématopoïétiques transduits par des vecteurs lentiviraux surexprimant les oncogènes MYC et BCL2 et placés sous le contrôle d’un promoteur ubiquitaire (EF1α ou SFFV). Le suivi longitudinal des animaux sur une période de 5 mois montre que seule la construction SFFV/Myc-T2A-Bcl2 entraîne la transformation des progéniteurs hématopoïétiques. Entre 12 à 14 semaines post-greffe, >90% des animaux développent des lympho-proliférations de type pro-B (CD19+CD10+CD9+CD20-cytIgM-) infiltrant principalement la rate et la moelle osseuse, et circulant en abondance dans le sang. Le caractère transmissible des tumeurs est validé par des greffes secondaires de tumeurs spléniques. Les cultures in vitro de progéniteurs hématopoïétiques suggèrent que l’émergence des blastes est liée à la réactivation d’un programme B latent dans les précurseurs T, dont le développement est bloqué. Nous avons développé en parallèle un modèle de tumeur autologue. L’ensemble de ces résultats valide le modèle de leucémogénèse humaine développé chez la souris humanisée BRGS et ouvre des perspectives pour la caractérisation fonctionnelle des mécanismes de leucémogénèse, et la validation préclinique de nouvelles stratégies anti-tumorales. / Existing animal models for the study of human leukemia are not accurate for the proper development of innovative, targeted therapies. The aim of this project, which contains both a fundamental and an industrial perspective, therefore was to develop a new, versatile model of human leukemogenesis in the BRGS (BALB/c Rag2-/- IL-2Rγc-/- SIRPα.NOD) humanized mouse. Animals are grafted with hematopoietic progenitors transduced with lentivirals vectors to allow overexpression of MYC and BCL2 proteins under the control of an ubiquitous promotor (EF1α or SFFV). Longitudinal monitoring of the animals over five months shows that only the SFFV/Myc-T2A-Bcl2 construction induces the transformation of humans hematopoietics progenitors. Between 12 and 14 weeks post-transplantation, more than 90% of the animals develop pro-B lympho-proliferations (CD19+CD10+CD9+CD20-cytIgM-), with tumor cells being mainly found in the spleen, the bone marrow and in blood. Tumor transferability is achievable through secondary transplantation in immunodeficient mouse recipients. In vitro culture of bone marrow T cell progenitors suggest that the blasts arise from these cells after reactivation of a latent B cell program with blockade of their T cell development. In parallel, we have also developed an autologous tumor model. Altogether, these results validate the human leukemogenesis model constructed here in humanized BRGS mice and provide attractive prospects regarding the functional characterization of leukemogenesis and a preclinical validation of new anti-tumor strategies.

abstract: The success of genetically-modified T-cells in treating hematological malignancies has accelerated the research timeline for Chimeric Antigen Receptor-T (CAR-T) cell therapy. Since there are only two approved products (Kymriah and Yescarta), the process knowledge is limited. This leads to a low efficiency at manufacturing stage with serious challenges corresponding to high cost and scalability. In addition, the individualized nature of the therapy limits inventory and creates a high risk of product loss due to supply chain failure. The sector needs a new manufacturing paradigm capable of quickly responding to individualized demands while considering complex system dynamics. The research formulates the problem of Chimeric Antigen Receptor-T (CAR-T) manufacturing design, understanding the performance for large scale production of personalized therapies. The solution looks to develop a simulation environment for bio-manufacturing systems with single-use equipment. The result is BioMan: a discrete-event simulation model that considers the role of therapy's individualized nature, type of processing and quality-management policies on process yield and time, while dealing with the available resource constraints simultaneously. The tool will be useful to understand the impact of varying factor inputs on Chimeric Antigen Receptor-T (CAR-T) cell manufacturing and will eventually facilitate the decision-maker to finalize the right strategies achieving better processing, high resource utilization, and less failure rates. / Dissertation/Thesis / Masters Thesis Industrial Engineering 2020

abstract: Immunotherapy has received great attention recently, as it has become a powerful tool in fighting certain types of cancer. Immunotherapeutic drugs strengthen the immune system's natural ability to identify and eradicate cancer cells. This work focuses on immune checkpoint inhibitor and oncolytic virus therapies. Immune checkpoint inhibitors act as blocking mechanisms against the binding partner proteins, enabling T-cell activation and stimulation of the immune response. Oncolytic virus therapy utilizes genetically engineered viruses that kill cancer cells upon lysing. To elucidate the interactions between a growing tumor and the employed drugs, mathematical modeling has proven instrumental. This dissertation introduces and analyzes three different ordinary differential equation models to investigate tumor immunotherapy dynamics. The first model considers a monotherapy employing the immune checkpoint inhibitor anti-PD-1. The dynamics both with and without anti-PD-1 are studied, and mathematical analysis is performed in the case when no anti-PD-1 is administrated. Simulations are carried out to explore the effects of continuous treatment versus intermittent treatment. The outcome of the simulations does not demonstrate elimination of the tumor, suggesting the need for a combination type of treatment. An extension of the aforementioned model is deployed to investigate the pairing of an immune checkpoint inhibitor anti-PD-L1 with an immunostimulant NHS-muIL12. Additionally, a generic drug-free model is developed to explore the dynamics of both exponential and logistic tumor growth functions. Experimental data are used for model fitting and parameter estimation in the monotherapy cases. The model is utilized to predict the outcome of combination therapy, and reveals a synergistic effect: Compared to the monotherapy case, only one-third of the dosage can successfully control the tumor in the combination case. Finally, the treatment impact of oncolytic virus therapy in a previously developed and fit model is explored. To determine if one can trust the predictive abilities of the model, a practical identifiability analysis is performed. Particularly, the profile likelihood curves demonstrate practical unidentifiability, when all parameters are simultaneously fit. This observation poses concerns about the predictive abilities of the model. Further investigation showed that if half of the model parameters can be measured through biological experimentation, practical identifiability is achieved. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics 2020

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22035号 / 医博第4520号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 佐藤 俊哉, 教授 福原 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22643号 / 医博第4626号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 斎藤 通紀, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM