The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-04-05T17:16:52Z No. of bitstreams: 1 COSTA, Jaqueline França - 2009.pdf: 5023458 bytes, checksum: 0aad0e59a4ee55dd3db0b89317f4bd80 (MD5) / Made available in DSpace on 2013-04-05T17:16:52Z (GMT). No. of bitstreams: 1 COSTA, Jaqueline França - 2009.pdf: 5023458 bytes, checksum: 0aad0e59a4ee55dd3db0b89317f4bd80 (MD5) Previous issue date: 2009 / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, Bahia, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A Leishmaniose Cutânea Difusa (LCD) é uma manifestação clínica rara das Leishmanioses, caracterizada pela presença de inúmeros macrófagos intensamente parasitados e baixa reação inflamatoria. No Brasil, a espécie envolvida em casos de LCD é a Leishmania (Leishmania) amazonensis. Tem sido descrito na literatura a exposição e o reconhecimento de fosfatidílserina (PS) na superfície de células apoptóticas fagocitadas por macrófagos como mecanismo de desativação de macrófagos por urna via dependente de TGF-pi e PGE2 (Fadok et al. 1998). Foi demonstrado por Barcinski e colaboradores que formas amastigotas de L. amazonensis expõem PS em sua superfície, em um mecanismo chamado “Mimetismo Apoptótico”. Nesse contexto, nosso objetivo foi investigar a exposição de PS na superfície de isolados de L, amazonensis obtidos de pacientes com LCD e seu papel durante a infecção de macrófagos. Inicialmente, macrófagos peritoneais de camundongos Fl(BALB/c x C57BL/6) estimulados com tioglicolato foram infectados com os diferentes isolados obtidos de pacientes com Leishmaniose Cutânea Localizada (LCL) e LCD. A exposição de PS na superfície das amastigotas purificadas de macrófagos F1 foi determinada por citometria de fluxo. Os isolados obtidos de pacientes com LCD apresentaram maior expressão de PS do que os isolados de pacientes com LCL após 24 horas de infecção. Em seguida, avaliamos se a diferença observada na exposição de PS em amastigotas estaria relacionada à infectividade dos diferentes isolados. Os resultados indicaram que as amastigotas de pacientes com LCD apresentaram maior porcentagem de macrófagos infectados e índice de infecção, quando comparados com amastigotas de pacientes com LCL. Quanto ao mecanismo, 0 grupo infectado com os isolados de pacientes com LCD não apresentou diferença em relação ao grupo LCL quanto a produção de TGF-pl e óxido nítrico, sugerindo que outros mecanismos imunorregulatórios estejam envolvidos. Os resultados em conjunto, sugerem que 0 aumento na exposição de PS nos isolados de L. amazonensis de pacientes com a forma difusa pode representar um mecanismo de adaptação importante para a sobrevivência e estabelecimento da infecção. A elucidação de mecanismos supressores da LCD induzidos por isolados de L. amazonensis poderá ampliar 0 conhecimento sobre a imunorregulação dessa patologia / Diffuse Cutaneous Leishmaniasis (LCD) is a rare clinical manifestation of Leishmaniasis, characterized by a number of macrophages heavily parasitized and low inflammatory reaction. In Brazil, Leishmania (Leishmania) amazonensis is the main specie involved in LCD cases. It has been described that the exposure and recognition of phosphatidylserine (PS) on the surface of apoptotic cells phagocytosed by macrophages is a macrophage deactivation mechanism dependent on TGF-pi and PGE2 (Fadok et al. 1998). Morover, it was demonstrated by Barcinski and colleagues that L. amazonensis amastigotes expose PS on its surface, in a mechanism called ’’Apoptotic Mimicry." In this context, our goal was to investigate the exposure of PS on the surface of L. amazonensis isolates obtained from LCD patients and its role during the infection of macrophages. Initially, peritoneal macrophages from FI mice (BALB/c x C57BL/6) stimulated with thioglycolate were infected with different L. amazonensis strains isolated from patients with Localized Cutaneous Leishmaniasis (LCL) or LCD. The exposure of PS on the surface of amastigotes was determined by flow cytometry using staining to annexin V and propidium iodide. Isolates from LCD patients showed higher PS exposure than the isolates from LCL patients 24 hours after infection. Then, we evaluated whether the differences of PS exposure in amastigotes would correlate with the infectivity of different isolates. Percentage of infected macrophages and infection index were higher in cultures using amastigotes from LCD patients compared to the ones infected with amastigotes from LCL cases. Furthermore, cultures infected with LCD isolates showed no difference to the LCL isolates regarding TGF>pl and nitric oxide production, suggesting that other immuneregulatory mechanisms are involved in this process. These results suggest that the increased exposure of PS in L. amazonensis isolates from patients with diffuse form may represent an important mechanism for the establishment of infection. The elucidation of the LCD suppressing mechanisms induced by L. amazonensis strains may expand the knowledge about the immune regulation of this disease

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22396号 / 薬科博第118号 / 新制||薬科||13(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Phospholipids, such as phosphatidylserine (PS) have been shown to work synergistically with tocopherols to extend the shelf life of oil-in-water emulsions. However, the high cost of PS prevents it from being used as a food additive. This work investigated the potential use of a high PS enzyme-modified lecithin to be used along with α-tocopherol to extend the lag phase of oil-in-water emulsions stabilized using Tween 20. Phospholipase D from Streptomyces sp. and L-serine were used to modify lecithin to increase PS concentration. Enzyme activity was optimized as a function of pH and temperature using a high PC soybean lecithin. The high PS modified lecithin was examined for its ability to enhance the activity of α-tocopherol in Tween 20-stabilized oil-in-water emulsions. The modification was also performed in high PC sunflower lecithin and egg lecithin which were later analyzed for their efficiency in controlling lipid oxidation. α-Tocopherol (3.0 µmol/kg emulsion) alone increased the lag phase of hydroperoxide and hexanal lag phases by 3 and 4 days compared to the control. Authentic PS (15.0 µmol/kg emulsion) increased hydroperoxide and hexanal lag phases by 1 and 3 days, respectively, whereas high PS soy lecithin increased hydroperoxide and hexanal lag phases by 3 and 4 days, respectively. The addition of high PS sunflower and egg lecithin did not have any considerable effects on lag phases compared to the control. Authentic PS (15.0 µmol/kg emulsion) and a-tocopherol (3.0 µmol/kg emulsion) decreased lipid oxidation by increasing the hydroperoxide and hexanal lag phase to 6 and 9 days. The combination of phospholipase D modified high PS lecithins (15.0 µmol/kg emulsion) and a-tocopherol (3.0 µmol/kg emulsion) were able to synergistically increase the antioxidant activity of a-tocopherol increasing the hydroperoxide and hexanal lag phase by 6 and 9 days for soy, 5 days, and 7 days for sunflower and 4 and 6 days for egg lecithin, respectively. This resulted in synergistic antioxidant activity (interaction index > 1.0) except for a-tocopherol and high PS Egg lecithin which showed an additive effect. This research shows that the combination of enzyme-modified high PS lecithin and α-tocopherol could be an effective and commercially viable clean label antioxidant strategy to control lipid oxidation in emulsions.

No description available.

<p>Immunotherapy has significantly improved cancer treatment. However, many tumors are resistant to current immunotherapy due to the highly immunosuppressive tumor microenvironment (TME). Tumor cells can evade immune activation by externalizing phosphatidylserine (PS) on cell surface to trigger anti-inflammatory signals and induce immune tolerance. Recent studies show that PS is upregulated in TME and further increased after chemotherapy. For effective immunotherapy of tumors, the exposed PS needs to be blocked to relieve immunosuppressive TME and sensitize tumors to immune stimulants. </p> <p><br></p> <p>In this study, PS exposure level increased after the chemotherapy Doxil treatment on B16F10 melanoma cells, and the PS exposure reduced the response of antigen-presenting cells (APCs) to immune stimulants such as lipopolysaccharide. Dipicolylamine-Zn (DPA-Zn) shielded the PS exposure resulting from doxorubicin (DOX) treatment and reduced immunosuppressive interaction between tumors and APCs. The PS blockade by DPA-Zn improves the tumor response rate immune stimulants such as GM-CSF, STING agonist cyclic dinucleotides (CDN), anti-PD-L1 antibody. Among the combination at the tested doses, Doxil + DPA-Zn + CDN was the optimal combination that enhanced anti-tumor effect most significantly and prolonged the survival time in immune-cold B16F10 melanoma model. However, the anti-tumor efficacy was limited, which is attributed to poor tumor retention of CDN and DPA-Zn. </p> <p><br></p> <p>To prolong the intratumoral release of DPA-Zn and CDN and maximize the anti-tumor immunity, CDN was formulated as liposomes (CDN@lip), which significantly delayed the release of CDN in vitro and improved anti-tumor efficacy compared with free CDN formulation. Alginate hydrogel showed the potential to sustain release of DPA-Zn. DPA-Zn was loaded in the alginate hydrogel via electrostatic interaction, and the release rate was controlled by additional zinc gluconate. However, zinc caused detrimental effects on skin and can cause mice death at a high dose. To avoid the side effect of subcutaneously administered Zn, the dose of DPA-Zn in alginate hydrogel was readjusted based on the maximum tolerated dose study, and zinc gluconate was replaced with CaSO4.</p>

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第25233号 / 薬博第868号 / 新制||薬||244(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 樋口 ゆり子, 教授 寺田 智祐, 教授 山下 富義 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Promastigotas de Leishmania são encontradas no trato digestivo do inseto vetor e podem ser cultivados em alguns meios de cultura, onde ocorre a diferenciação regulada geneticamente entre procíclicos e metacíclicos infectivos. Amastigotas são intracelulares e irão se estabelecer e se multiplicar dentro dos vacúolos parasitóforos dos macrófagos. Os metacíclicos e os amastigotas possuem capacidade de evasão do sistema de defesa do hospedeiro vertebrado e conseguem estabelecer uma relação parasita/hospedeiro bem-sucedida. A apoptose, em organismos multicelulares, é uma forma controlada geneticamente de suicídio celular. A eliminação das células apoptóticas é feita pelos fagócitos sem que seja induzida uma resposta inflamatória. Em Leishmania, os eventos da apoptose parecem seguir os mesmos dos encontrados em metazoários, tais como: queda do potencial da mitocôndria, ativação de caspases, clivagem de substratos naturais de caspases, além da externalização de fosfatidilserina (PS) e seu papel na modulação da fagocitose. Nossos dados demonstraram que Leishmania é capaz de mimetizar e utilizar um dos fenômenos observados na apoptose em metazoários, a exposição de PS, como um mecanismo adaptativo para se estabelecer como um parasita de mamíferos. / Promastigotes of Leishmania are found in the midgut environment of the vector insect and can be grown in some culture medium. In culture the genetically regulated differentiation from procyclic to infective metacyclic can be observed. Amastigotes are intracellular parasites and which multiply inside phagolysosomes of macrophages. Metacyclics and amastigotes are endowed with the capacity to evade from the innate and adaptive immune system of the vertebrate host and to establish successful infections. Apoptosis, in multicellular organisms, is a form genetically controlled cellular suicide. The elimination of apoptotic cells is made by phagocytes without initiating an inflammatory event. In Leishmania, the events of apoptosis are similar to those observed in multicellular organisms such as: decrease in mitochondrial transmembrane potential, caspase activation, cleavage of caspase substrates besides exposure of phosphatidylserine (PS) and its role in the modulation of phagocytosis. Our data showed that Leishmania is capable of mimicking one of the features of apoptosis, mainly PS exposure, and to use as an adaptive mechanism for survival in mammalian hosts.

A galectina-1 (Gal-1) é uma lectina homodimérica multifuncional capaz de reconhecer e se ligar a beta-galactosídeos por meio de um domínio denominado carbohydrate recognition domain (CRD). A Gal-1 humana (Gal-1h) e a Gal-1 de camundongo (Gal-1c) mantêm 88,15% de homologia e, apesar de não existirem mutações em aminoácidos-chave do CRD, há substituições próximas a esses resíduos. Considerando as implicações dessas diferenças em estrutura e função, e que é comum a utilização de modelos murinos para estudar a função Gal-1, o presente trabalho objetiva analisar comparativamente a Gal-1c e a Gal-1h por meio de ensaios de cristalização e determinação estrutural da Gal-1c, além da avaliação comparativa da atividade lectínica da Gal-1h e da Gal-1c por glycan array e hemaglutinação. Também foi avaliada a capacidade de ambas as Gal-1 em induzir a exposição de fosfatidilserina (FS) em neutrófilos ativados provenientes de medula de camundongos normais ou deficientes de ?-2 integrina (Mac-1), de modo a investigar se a interação Gal-1/Mac-1 estaria envolvida nesse processo. Preparações homogêneas e ativas de Gal-1c e Gal-1h foram utilizadas nos ensaios. Os cristais de Gal-1c foram obtidos em 20% de polietilenoglicol 3350 e 0,2 M de fluoreto de amônio. Os dados de difração de raios X foram coletados e processados, obtendo-se uma estrutura com resolução de 2,4 Å. Observou-se que substituições de aminoácidos entre a Gal-1c e a Gal-1h estão localizadas em regiões expostas ao solvente, próximas do CRD e distantes da interface de dimerização. A análise comparativa entre Gal-1c e Gal-1h mostrou que estas substituições conferem a Gal-1c um caráter mais polar, com consequente aumento da distribuição de volume molecular. Nos ensaios de hemaglutinação, pode-se observar que é necessária uma concentração 2 vezes maior de Gal-1c para aglutinar eritrócitos humanos, de carneiro e de coelho na mesma proporção que a Gal-1h. Por meio do glycan array, pode-se determinar o perfil de ligação a glicanas de ambas as Gal-1. As duas Gal-1 apresentam afinidade por glicanas ramificadas contendo galactose terminal, e a Gal-1h apresentou maior intensidade de ligação às glicanas quando comparada à Gal-1c. Preparações de Gal-1c e Gal-1h induzem níveis semelhantes de exposição de FS na superfície de neutrófilos deficientes ou não de Mac-1, sugerindo que a interação Gal-1/Mac-1 não esteja envolvida no processo de exposição de FS na superfície de neutrófilos ativados. Assim, a diferença sequencial entre a Gal-1c e a Gal-1h é capaz de gerar diferenças estruturais consideráveis que implicam no reconhecimento diferencial de glicanas, o que, entretanto, não se reflete na capacidade de indução de FS na superfície de neutrófilos ativados. Além disso, a interação Gal-1/Mac-1 parece não participar desse processo, o que pode indicar que o papel da Gal-1 no turnover de neutrófilos, via reconhecimento fagocítico, seja um processo complexo e independente dessa interação. / Galectin-1 (Gal-1) is a homodimeric and multifunctional lectin that recognizes and binds to beta-galactoside by a carbohydrate recognition domain (CRD). Human Gal-1 (hGal-1) and mouse Gal-1 (mGal-1) are 88.15% identical, and although there are no mutations in key amino acids within the CRD, there are differences in the amino acids sequence near the CRD. Given the potential of these differences to alter overall structure and function, and the common utilization of murine models to study Gal-1 function, we sought to directly compare key biochemical features of hGal and mGal-1. Thus, we performed crystallization and structure determination assays of mGal-1, and determined the carbohydrate binding specificy of mGal-1 and hGal-1 using a glycan array and using hemagglutination assay. We also evaluated the ability of both Gal-1 to induce exposure of phosphatidylserine (PS) in activated neutrophils from the bone marrow of normal or ?-2 integrin (Mac-1) deficient mice, in order to investigate the involvement of Gal-1/Mac-1 interaction in this process. To accomplish this, homogeneous and active preparations of hGal-1 and mGal-1 were used in the study. mGal-1 crystals were obtained in 20% polyethylene glycol 3350 and 0.2 M ammonium fluoride. Data from X-ray diffraction were collected and processed, yielding a structure with a final resolution of 2.4 Å. The amino acid substitutions found between mGal-1 and hGaI-1 are detected on the solvent-exposed surfaces where the CRDs are located and not on the proteins dimerization surfaces. A comparative structural analysis between mGal-1 and hGal-1 shows that these amino acid substitutions confer to mGal-1 a greater number of ionizable residues, polar character, appearance of the acid regions clustered, and a slight increase of volume distribution. In hemagglutination assays, twice the concentration of mGal-1 was required to cause equivalent agglutination of human, sheep or rabbit erythrocytes as hGal-1. Glycan array analysis demonstrated that both galectins have affinity for branched glycans containing terminal galactose residues. However, hGal-1 appeared to display higher levels of binding that mGal-1. Preparations of mGal-1 and hGal-1 induced similar levels of PS exposure on normal or Mac-1 deficient neutrophils, suggesting that the interaction Gal-1/Mac-1 is not involved in this process. Thus, hGal-1 and mGal-1 appear to possess considerable differences in glycan recognition that likely reflects subtle difference in amino acid sequence. Furthermore, the interaction Gal-1/Mac-1 do not appear to participate in this PS exposure process, which suggest that other Gal-1 receptors are likely important in this process.

Toxoplasma gondii ist ein weit verbreiteter, obligat-intrazellulärer, einzelliger Parasit, der die lebensbedrohliche Krankheit Toxoplasmose in Menschen und Tieren hervorrufen kann. Der schnell replizierende Parasit benötigt erhebliche Mengen an Phospholipiden zur Biogenese intra- und extrazellulärer Membranen. Phosphatidylethanolamin (PtdEtn) ist ein wichtiges und ubiquitäres Phospholipid in Pro- und Eukaryoten und das zweithäufigste Lipid in T. gondii. Dieses kann de novo über den CDP-Ethanolamin Stoffwechselweg oder durch Decarboxylierung von Phosphatidylserin synthetisiert werden. Im Rahmen dieser Arbeit konnte die Expression von zwei distinkten Phosphatidylserin Decarboxylasen (PSDs) in T. gondii nachgewiesen werden: TgPSD1pv ist partiell löslich und wird über Dichte Granula in die Parasitophore Vakuole sekretiert, während sich TgPSD1mt im Mitochondrium von Tachyzoiten befindet. TgPSD1mt ist in der Lage einen Ethanolamin-auxotrophen S. cerevisiae Stamm zu komplementieren. Ein Knock-down von TgPSD1mt in T. gondii verursacht eine verlangsamte Parasitenreplikation, welche zu einem verminderten in vitro Wachstum führt. Der PtdEtn-Gehalt in der Mutante bleibt unverändert, was auf eine stringente Homöostase des zellulären PtdEtn Reservoirs durch alternative Lipidbiogenesewege hindeutet. Tatsächlich verfügt T. gondii zusätzlich über einen aktiven CDP-Ethanolamin Stoffwechselweg im Endoplasmatischen Retikulum, welcher den Verlust von TgPSD1mt partiell kompensieren kann. Das zweite, sekretierte TgPSD1pv-Enzym hingegen scheint für das Parasitenwachstum in vitro entbehrlich zu sein. Infektionsversuche mit radioaktiv markierten Wirtszellen zeigten zudem eine Aufnahme von PtdEtn oder PtdEtn-Derivaten in intrazellulär replizierenden Tachyzoiten. Diese Ergebnisse demonstrieren eine außergewöhnliche Kompartmentalisierung und Plastizität der PtdEtn-Synthese in T. gondii. / Toxoplasma gondii is a remarkably successful and widespread obligate intracellular protozoan parasite, which can cause the potentially life threatening disease Toxoplasmosis in humans and animals. The fast proliferating parasite requires a significant amount of phospholipids for biogenesis of organelles and enclosing vacuolar membranes. Phosphatidylethanolamine (PtdEtn) is one of the most ubiquitous phospholipids and the second most abundant lipid in T. gondii. It can be produced de novo by the CDP-ethanolamine pathway or by decarboxylation of phosphatidylserine. This work revealed the expression of two distinct PtdSer decarboxylase (PSD) enzymes in T. gondii: One of which is Coccidia-specific and partially soluble and secreted into the parasitophorous vacuole via dense granules (TgPSD1pv), and a second enzyme that localizes in the mitochondrion (TgPSD1mt) of tachyzoites. The mitochondrial PSD can complement a S. cerevisiae mutant auxotrophic for ethanolamine. A conditional knockdown of the TgPSD1mt gene impairs the parasite growth in vitro. Surprisingly, the mutant displayed an unaltered total PtdEtn content, which suggests a stringent homeostasis of the cellular PtdEtn pool by alternative routes of lipid biogenesis. Consistently, the parasite encodes an active CDP-ethanolamine pathway in the endoplasmic reticulum. Metabolic labeling of the TgPSD1mt mutant displayed an increased utilization of ethanolamine into PtdEtn, indicating an upregulation of the de novo CDP-ethanolamine pathway. Likewise, exogenous ethanolamine partially restored the growth phenotype of the mutant. In contrast, the TgPSD1pv enzyme is dispensable for the parasite growth. Host cell pre-labeling with radioactive ethanolamine indicated a potential uptake of host-derived PtdEtn or PtdEtn-derivates by intracellular parasites. Taken together, these results demonstrate an exceptional compartmentalization and plasticity of the PtdEtn synthesis in T. gondii.