abstract: Cancer is a heterogeneous disease with discrete oncogenic mechanisms. P53 mutation is the most common oncogenic mutation in many cancers including breast cancer. This dissertation focuses on fundamental genetic alterations enforced by p53 mutation as an indirect target. p53 mutation upregulates the mevalonate pathway genes altering cholesterol biosynthesis and prenylation. Prenylation, a lipid modification, is required for small GTPases signaling cascades. Project 1 demonstrates that prenylation inhibition can specifically target cells harboring p53 mutation resulting in reduced tumor proliferation and migration. Mutating p53 is associated with Ras and RhoA activation and statin prevents this activity by inhibiting prenylation. Ras-related pathway genes were selected from the transcriptomic analysis for evaluating correlation to statin sensitivity. A gene signature of seventeen genes and TP53 genotype (referred to as MPR signature) is generated to predict response to statins. MPR signature is validated through two datasets of drug screening in cell lines. As advancements in targeted gene modification are rising, the CRISPR-Cas9 technology has emerged as a new cancer therapeutic strategy. One of the important risk factors in gene therapy is the immune recognition of the exogenous therapeutic tool, resulting in obstruction of treatment and possibly serious health consequences. Project 2 describes a method development that can potentially improve the safety and efficacy of gene-targeting proteins. A cohort of 155 healthy individuals was screened for pre-existing B cell and T cell immune response to the S. pyogenes Cas9 protein. We detected antibodies against Cas9 in more than 10% of the healthy population and identified two immunodominant T cell epitopes of this protein. A de-immunized Cas9 that maintains the wild-type functionality was engineered by mutating the identified T cell epitopes. The gene signature and method described here have the potential to improve strategies for genome-driven tumor targeting. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2017

No description available.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25008号 / 医科博第155号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 伊藤 貴浩, 教授 藤田 恭之, 教授 伊藤 能永 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The development of a malaria vaccine is necessary for disease eradication. Successful vaccine candidates to date have targeted the asymptomatic, pre-erythrocytic stage of the disease, however even the most efficacious vaccines are only partially protective. Research undertaken in our laboratory has demonstrated that one such regimen, using an 8 week prime-boost viral vector approach of ChAd63 ME-TRAP and MVA ME-TRAP, induces sterile efficacy in 21% of vaccinees, with a key role identified for TRAP-specific CD8⁺ T cells. The work described in this thesis explores the most immunogenic regimen by which to administer these two pre-erythrocytic malaria vaccines. A shortening of the prime-boost interval from 8 to 4 weeks, and the addition of an extra ChAd63 ME-TRAP priming vaccination, both demonstrated improved T cell immunogenicity over the standard 8 week regimen. Further to this, novel assays were developed to aid the evaluation of vaccine-induced immune responses. Adaptations of the existing methodology for ELISpot analysis and to whole blood flow cytometry techniques, enabled more detailed analyses of paediatric vaccine-induced T cell responses in The Gambia. This work also permitted the comparison of vaccine immunogenicity in this paediatric population, with malaria-naïve and malaria-exposed adult vaccinees. The results suggest that vaccine-induced T cell responses in infants of 8 weeks and older are comparable to that of adults. A second approach involved the development of a novel functional assay. This assay quantitatively measured the in vitro inhibition of intrahepatic Plasmodium parasite development using T cells from ChAd63.MVA ME-TRAP vaccinated volunteers. The assay demonstrated the ability of CD8⁺ T cells to inhibit parasite development in a TRAP-specific manner, and provides a platform with which to further explore pre-erythrocytic immune responses.

<p>Malaria is one of the major infectious diseases in the world with regard to mortality and morbidity, and the development of a vaccine against the malaria parasite <i>Plasmodium falciparum</i> is considered of high priority. The aim of the work presented in this thesis was to develop and characterize recombinant vaccine constructs based on the <i>P. falciparum</i> asexual blood-stage antigen Pf332. We have studied the humoral responses in mice elicited by various types of constructs, including naked DNA plasmids, naked mRNA, alphavirus, and peptides. Immunological memory was successfully induced against the repetitive EB200 fragment of Pf332, although the antibody titers were generally low and the highest titers were unexpectedly obtained with a conventional DNA plasmid. In another study, we also demonstrated the ability to circumvent genetically restricted immune responses in mice against two malaria epitopes, one of them derived from Pf332, by inclusion of universal T-cell epitopes into multiple antigen peptide constructs. However, the overall variability of the responses stressed the importance of including several epitopes in a future malaria vaccine. Further, the recent completion of sequencing of Pf332 enabled us to identify and characterize the immunogenic properties of a non-repeat fragment of the Pf332, termed C231. Our analyses of C231 showed that antibodies raised against the recombinant protein possess an <i>in vitro</i> parasite inhibitory capacity similar to that of antibodies against recombinant EB200. Furthermore, the recognition of C231 by antibodies in sera from individuals naturally primed to <i>P. falciparum</i>, correlated well with that previously observed for the corresponding sera and EB200. When analyzing the IgG subclass distribution of anti-C231 antibodies, we noted a bias towards IgG2 and IgG3 relative to IgG1, differing from the subclass profiles of IgG binding crude <i>P. falciparum</i> antigen, which were dominated by IgG1. Taken together, the work presented herein is likely to facilitate further studies on Pf332 as a potential target for protective immune responses, and amounts to a small step towards the realization of a malaria vaccine.</p>

A papaína é uma enzima proteolítica empregada no debridamento e cicatrização de feridas. Contudo, problemas de estabilidade na forma farmacêutica, bem como reações alérgicas reportadas por pacientes submetidos à tratamentos com a enzima, culminaram na restrição aos produtos contendo papaína para uso tópico por órgãos regulatórios internacionais. Este trabalho objetivou desenvolver hidrogel nanoestruturado contendo complexo de papaína e ciclodextrina visando obter forma farmacêutica estável e eficaz como curativo dérmico, com redução da resposta imunológica. A síntese do hidrogel foi realizada combinando fenômenos de cristalização e/ou reticulação e esterilização simultânea induzida por radiação gama, de modo a promover nanoestruturação adequada da membrana para veiculação da papaína nativa e do complexo. O complexo e o produto final tiveram suas propriedades biológicas e físico-químicas avaliadas. O hidrogel a base de PVA contendo complexo de papaína-ciclodextrina apresentou características adequadas para aplicação como curativo, além de apresentar indícios de redução na resposta imunológica e melhora na citocompatibilidade quando comparado à papaína nativa, isso devido ao encapsulamento molecular com a ciclodextrina e à alta retenção do complexo por parte da matriz. Por outro lado, a irradiação, não alterou o perfil citotóxico da enzima, mas acarretou leve diminuição em seu potencial imunogênico. O hidrogel se mostrou promissor para uso como curativo e demonstrou potencial redução nas reações adversas desencadeadas pelo uso da papaína. / Papain is a proteolytic enzyme applied for wound healing and debridement. However, stability issues as well as allergenic reactions reported by patients submitted to papain pharmaceutics led to restriction of papain containing products for topical use by international regulatory agencies. This work aimed the development of nanostructured hydrogel containing papain and cyclodextrin complex or papain in order to obtain stable and suitable pharmaceutical form as a wound dressing with reduced allergenic properties. The hydrogel synthesis was performed by combining freezing cycles and the simultaneous crosslinking and sterilization process promoted by gamma irradiation to achieve a nanostructured hydrogel for the loading of the papain and cyclodextrin complex. The biological and physical-chemical properties of the complex and the final product were assayed. The PVA based hydrogel containing cyclodextrin-papain complex presented desirable characteristics for wound dressing purposes. In addition, an in vitro shift in the immunological response and an increase in the cytocompatibility if compared to native papain were observed as a function of the molecular encapsulation with cyclodextrin. The process of irradiation was not capable of altering papain cytotoxicity, but conferred a slight decrease in the immunogenic properties. In conclusion, the developed hydrogel was promising as a novel papain containing dressing with potential reduced adverse reactions.

A leishmaniose é uma doença negligenciada causada por protozoários unicelulares do gênero Leishmania. De acordo com a Organização Mundial de Saúde, a doença é classificada em duas formas clínicas: leishmaniose tegumentar (LT) e leishmaniose visceral (LV). A LV, também conhecida como calazar, é a forma mais grave, sendo potencialmente fatal em indivíduos não tratados. Atualmente, um dos grandes desafios encontrados nos estudos acerca da crescente urbanização da leishmaniose visceral (LV), é o desenvolvimento de vacinas com elevada eficácia para induzir proteção contra infecção por Leishmania. Neste contexto, o presente estudo foi elaborado de modo a se realizar uma análise preliminar de segurança e imunogenicidade de um possível candidato vacinal contra LV, constituído por um peptídeo sintético da glicoproteína gp63 de Leishmania major com predição para MHC de classe I, utilizando o hamster (Mesocricetus auratus) como modelo experimental. Um total de nove animais foram distribuídos em três grupos experimentais, entre os quais: (i) grupo controle (C, n=3), que recebeu 100 μL de solução salina estéril a 0,85%; (ii) grupo inoculado com adjuvante Montanide ISA-61VG (ISA, n=3) que recebeu 30 μL de Montanide, diluída em 70 μL de solução salina estéril a 0,85% e (iii) grupo imunizado com peptídeo MCH-I de Leishmania major e o adjuvante Montanide ISA-61VG (Lm-ISA, n=3) que recebeu 30 μL do antígeno/dose, diluído em 40 μL de solução salina estéril a 0,85% e emulsionados com 30 μL do adjuvante oleoso Montanide ISA-61VG. Os inóculos foram administrados por via subcutânea em três doses com intervalos de 14 dias. Amostras de sangue, soro e fragmentos do baço foram coletados para realização de diferentes análises laboratoriais, em tempos distintos. Perfil bioquímico da função renal e hepática, quadro leucocitário, titulação de anticorpos e linfoproliferação de esplenócitos foram alvos deste trabalho. Nossos resultados revelaram que a formulação foi inócua e não tóxica para os animais, uma vez que os níveis séricos de ureia, creatinina e das enzimas hepáticas: alanina aminostranferase (ALT), aspartato aminotransferase (AST) e fosfatase alcalina (FA), se mantiveram dentro dos padrões de normalidade descrito da literatura. Além disso, a formulação se mostrou capaz de induzir uma resposta humoral específica anti-Leishmania, através das dosagens de IgG total. Em resposta à série branca e à linfoproliferação das células do baço, foi verificado a existência de memória imunológica através do aumento significativo da população de leucócitos, bem como pela alta atividade linfoproliferativa obtida no grupo vacinal. / Leishmaniasis is a neglected disease caused by unicellular protozoa of the genus Leishmania. According to the World Health Organization, the disease is classified into two clinical forms: tegumentary leishmaniasis (LT) and visceral leishmaniasis (LV). LV, also known as calazar, is the most severe form, potentially fatal in untreated individuals. Actually, one of the major challenges encountered in studies of the increasing urbanization of visceral leishmaniasis (LV) is the development of highly effective vaccines to induce protection against Leishmania infection. In this context, the present study was elaborated in order to carry out a preliminary analysis of the safety and immunogenicity of a possible candidate vaccine against LV, constituted of a synthetic peptide of the glycoprotein gp63 of Leishmania major with prediction for MHC of class I, using the hamster (Mesocricetus auratus) as an experimental model. A total of nine animals were distributed in three experimental groups, among them: (i) control group (C, n = 3), that received 100 μL 0.85% sterile saline solution; (Ii) Montanide ISA-61VG (ISA, n = 3) adjuvant group that received 30 μL of Montanide, diluted in 70 μL of 0.85% sterile saline and (iii) group immunized with MCH-I peptide Leishmania Major and Montanide ISA-61VG adjuvant (Lm-ISA, n = 3) that received 30 μL of the antigen/dose, diluted in 40 μL of 0.85% sterile saline and emulsified with 30 μL of the Montanide ISA-61VG oily adjuvant. The inocula were administered subcutaneously in three doses at 14 day intervals. Samples of blood, serum and spleen fragments were collected for different laboratory tests at different times. Biochemical profile of renal and hepatic function, leukocyte framework, antibody titration and lymphoproliferation of splenocytes were the targets of this study. Our results revealed that the formulation was innocuous and non-toxic to the animals, since serum levels of urea, creatinine and hepatic enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AF) were maintained within the patterns of normality described in the literature. In addition, the formulation was capable to induce a humoral anti-Leishmania specific response by total IgG dosages. In response to the white series and lymphoproliferation of spleen cells, the existence of immunological memory was verified through a significant increase in the leukocyte population, as well as the high lymphoproliferative activity obtained in the vaccine group.

A papaína é uma enzima proteolítica empregada no debridamento e cicatrização de feridas. Contudo, problemas de estabilidade na forma farmacêutica, bem como reações alérgicas reportadas por pacientes submetidos à tratamentos com a enzima, culminaram na restrição aos produtos contendo papaína para uso tópico por órgãos regulatórios internacionais. Este trabalho objetivou desenvolver hidrogel nanoestruturado contendo complexo de papaína e ciclodextrina visando obter forma farmacêutica estável e eficaz como curativo dérmico, com redução da resposta imunológica. A síntese do hidrogel foi realizada combinando fenômenos de cristalização e/ou reticulação e esterilização simultânea induzida por radiação gama, de modo a promover nanoestruturação adequada da membrana para veiculação da papaína nativa e do complexo. O complexo e o produto final tiveram suas propriedades biológicas e físico-químicas avaliadas. O hidrogel a base de PVA contendo complexo de papaína-ciclodextrina apresentou características adequadas para aplicação como curativo, além de apresentar indícios de redução na resposta imunológica e melhora na citocompatibilidade quando comparado à papaína nativa, isso devido ao encapsulamento molecular com a ciclodextrina e à alta retenção do complexo por parte da matriz. Por outro lado, a irradiação, não alterou o perfil citotóxico da enzima, mas acarretou leve diminuição em seu potencial imunogênico. O hidrogel se mostrou promissor para uso como curativo e demonstrou potencial redução nas reações adversas desencadeadas pelo uso da papaína. / Papain is a proteolytic enzyme applied for wound healing and debridement. However, stability issues as well as allergenic reactions reported by patients submitted to papain pharmaceutics led to restriction of papain containing products for topical use by international regulatory agencies. This work aimed the development of nanostructured hydrogel containing papain and cyclodextrin complex or papain in order to obtain stable and suitable pharmaceutical form as a wound dressing with reduced allergenic properties. The hydrogel synthesis was performed by combining freezing cycles and the simultaneous crosslinking and sterilization process promoted by gamma irradiation to achieve a nanostructured hydrogel for the loading of the papain and cyclodextrin complex. The biological and physical-chemical properties of the complex and the final product were assayed. The PVA based hydrogel containing cyclodextrin-papain complex presented desirable characteristics for wound dressing purposes. In addition, an in vitro shift in the immunological response and an increase in the cytocompatibility if compared to native papain were observed as a function of the molecular encapsulation with cyclodextrin. The process of irradiation was not capable of altering papain cytotoxicity, but conferred a slight decrease in the immunogenic properties. In conclusion, the developed hydrogel was promising as a novel papain containing dressing with potential reduced adverse reactions.

A cromoblastomicose é uma micose subcutânea, com alto índice de morbidade, causada por fungos demácios, sendo o mais recorrente agente etiológico o Fonsecaea pedrosoi. Já foi descrito em todos continentes e é mais recorrente em países de clima quente, sua incidência é de grande importância no Brasil. Raramente os indivíduos se curam, pois, não há terapia padrão por conta da baixa eficácia das terapêuticas antifúngicas atuais contra a cromoblastomicose. Pouco se sabe sobre a relação parasita hospedeiro desta infeção. Os métodos de diagnóstico laboratorial se limitam a visualização das estruturas fúngicas presentes nos tecidos do hospedeiro. Decorrente da falta de informação sobre a cromoblastomicose uma doença de tamanha morbidade, surge à necessidade de estudar melhor a resposta imunológica e seus antígenos. Nosso trabalho mostrou que algumas proteínas secretadas em meio nutricionalmente pobre (exoantígenos) se mostraram imunorreativas, contra os soros de camundongos BALB/c com diferentes tempos de infecção, assim como proteínas derivadas da ruptura do fungo total através da sonicação. A proteína que se mostrou majoritariamente imunorreativa foi a de aproximadamente 50KDa, outros trabalhos descrevem proteínas de alta relevância com peso molecular próximo deste, sugerindo uma possível relação entre os achados. O sequenciamento destas bandas mostrou homologia significante com enoyl redutase, uma enzima envolvida na síntese de ácidos graxos. / The chromoblastomycosis is a subcutaneous mycosis with high morbidity, caused mostly by Fonsecaea pedrosoi, a dematious fungus. The chromoblastomycosis has been described in all continents and is more recurrent in hot climate countries, with a greatest incidence in Brazil. The host rarely heal because the therapy is low efficacy of current antifungal therapies against chromoblastomycosis. Little is known about the relationship of Fonsecaea pedrosoi with the host. The diagnostic methods is limited to visualize fungal structures, present in the host tissues. Arising from lack of information about the chromoblastomycosis, is necessary to characterize new antigens. Our work has shown that some secreted proteins in medium (exoantigens) were immunoreactive against sera from infected mice, with different times of infection, as well as proteins derived from the fungus rupture by sonication. The antigen has approximately 50 kDa. Other studies describe proteins with high relevance close this molecular weight, suggesting a possible relationship between the findings. The sequencing of these bands showed significant homology with enoyl reductase, an enzyme involved in fatty acid synthesis.