Salicylic acid binding protein 2 (SABP2) is known to play a key role in Salicylic acid mediated defense pathway. SBIP-470 is SABP2 interacting protein that might be putatively involved in transfer of lipids. SBIP-470 was cloned without the signal peptide and expressed in E. coli. In vitro lipid binding assay using recombinant SBIP-470 failed to detect lipid binding. In vitro lipid transfer assay showed recombinant SBIP-470 does not transfer phospholipid. Study has shown that SBIP-470 is highly inducible upon infection with viral as well as bacterial pathogens. Induction of SBIP-470 expression upon the TMV infection most likely depends upon the SABP2 while its expression upon non-host bacterial pathogens is most probably inhibited by the SABP2. A study of Arabidopsis knockout mutants (ltp12 mutant and ltp2 mutant) lacking the SBIP-470 homolog genes showed defects in growth phenotype, and they were found susceptible to bacterial pathogens.

Fungicide use with different mechanisms of action as well the pulverization moment are the factors to be considered on the asian soybean rust control. Since many products have shown lost of efficiency, emerges the need of new alternatives to the control of soybean rust. The study aimed to quantify the effect of the systemic resistance inducer Acibenzolar-S-Methyl and the combination of fungicides molecules of the triazole + strobilurin + carboxamide applied in different stages in relation to the soybean rust control and yield. Two field experiments were conducted in greenhouse on the harvest year of 2010/2011. The experiment 1, with the pathogen inoculation at the phenological stage R5.1, was performed on factorial scheme (2x5x6), and the factors consisted of: Factor A: two soybeans cultivars (BMX Potência RR and Coodetec 219 RR); Factor D: treatments with Acibenzolar-S-Methyl (12,5 g a.i.ha-1), Azoxystrobin + Cyproconazole (60 + 24 g a.i.ha-1), Acibenzolar-SMethyl + Azoxystrobin + Cyproconazole (12,5 + 60 + 24 g a.i.ha-1), Pyraclostrobin + Epoxiconazole + Fluxapyroxad (64,8 + 40 + 40 g a.i.ha-1) and a testimony trait in each cultivar; factor E: Application of the treatments at the stages V6, R1, R5.1, 10, 17 and 24 days after the artificial inoculation (DAI) of Phakopsora pachyrhizi. The experiment 2, with the pathogen inoculation at the stage R1 was performed in factorial scheme (2x5x5) differing from the experiment 1 only on the factor E: application of the treatments in R1, 5, 10, 17 and 24 days after the inoculation. Was evaluated the area under the disease progress curve and the number of days to the appearance of the first pustule in leaflets of three distinct ages, also the average weight of seeds per experimental unit. The treatments presented differentiated behavior when exposed to different combinations between the cultivars, application period and leaflets age. Generally the combinations of Acibenzolar-S-Methyl + Azoxystrobin + Cyproconazole and Pyraclostrobin + Epoxiconazole + Fluxapyroxad resulted in better disease control and higher soybeans yield at the earlier and late applications in relation to the pathogen inoculation moment, without differing of Azoxystrobin + Cyproconazole in the others application moments. / O uso de fungicidas com diferentes mecanismos de ação, assim como a época de aplicação dos mesmos, são fatores que devem ser levados em consideração para o controle da ferrugem asiática na soja. Uma vez que muitos produtos têm apresentado perda de eficiência, surge a necessidade de novas alternativas de controle dessa doença. Com este trabalho buscou-se quantificar o efeito do uso do indutor de resistência Acibenzolar-S-Metil e da mistura de moléculas fungicidas dos grupos triazol + estrobilurina + carboxamida aplicados em diferentes épocas do ciclo da cultura em relação ao controle da ferrugem asiática e produtividade da soja. Para isso dois experimentos foram executados em estufa plástica na safra agrícola 2010/2011. O experimento 1, com a inoculação do patógeno no estádio fenológico R5.1, foi realizado em arranjo fatorial (2x5x6), cujos fatores foram compostos por: fator A: duas cultivares de soja (BMX Potência RR e Coodetec 219 RR); fator D: tratamentos com Acibenzolar-S-Metil (12,5 g i.a.ha-1), Azoxistrobina + Ciproconazol (60 + 24 g i.a.ha-1), Acibenzolar-S-Metil + Azoxistrobina + Ciproconazol (12,5 + 60 + 24 g i.a.ha-1), Piraclostrobina + Epoxiconazol + Fluxapyroxad (64,8 + 40 + 40 g i.a.ha-1) e um tratamento testemunha em cada cultivar; fator E: aplicação dos tratamentos em V6, R1, R5.1, 10 , 17 e 24 dias após a inoculação (DAI) artificial de Phakopsora pachyrhizi. O experimento 2, com a inoculação do patógeno no estádio fenológico R1, foi realizado em arranjo fatorial (2x5x5) diferindo-se do experimento 1 apenas para o fator E: aplicação dos tratamentos em R1, 5, 10 , 17 e 24 dias após a inoculação (DAI). Avaliou-se a área abaixo da curva de progresso da doença (AACPD) e o número de dias para aparecimento da primeira pústula em trifólios de três idades distintas, além do peso de grãos por parcela. Os tratamentos apresentaram comportamento diferenciado quando expostos a diferentes combinações entre cultivares, épocas de aplicação e idade de trifólios. De modo geral as misturas de Acibenzolar-S-Metil + Azoxistrobina + Ciproconazol e Piraclostrobina + Epoxiconazol + Fluxapyroxad resultaram em melhor controle da doença e maior produtividade de soja nas aplicações mais precoces e tardias em relação ao momento da inoculação do patógeno, não diferindo de Azoxistrobina + Ciproconazol nas demais épocas de aplicação dos tratamentos.

The paucity of well validated preclinical models is frequently cited as a contributing factor to the high attrition rates seen in clinical oncological trials. There remains a critical need to develop models which are accurately able to recapitulate the features of human disease. The aims of this study were to use genetically engineered mouse models (GEMMs) to explore the efficacy of novel treatment strategies in HER2 positive breast cancer and to further develop the model to facilitate the study of mechanisms underpinning drug resistance. Using the BLG--HER2KI-PTEN+/- model, we demonstrated that Src plays an important role in the early stages of tumour development. Chemopreventative treatment with dasatinib delayed tumour inititation (p= 0.046, Wilcoxon signed rank test) and prolonged overall survival (OS) (p=0.06, Wilcoxon signed rank test). Dasatinib treatment also induced squamous metaplasia in 66% of drug treated tumours. We used 2 cell lines derived from this model to further explore dasatinib’s mechanism of action and demonstrated reduced proliferation, migration and invasion following in vitro treatment. Due to the prolonged tumour latency and the low metastatic rate seen in this model, further studies were undertaken with the MMTV-NIC model. This model also allowed us to study the impact of PTEN loss on therapeutic response. We validated this model by treating a cohort of MMTV-NIC PTEN+/- mice with paclitaxel and demonstrated prolonged OS (p=0.035, Gehan Breslow Wilcoxon test). AZD8931 is an equipotent signalling inhibitor of HER2, HER3 and EGFR. We observed heterogeneity in tumour response but overall AZD8931 treatment prolonged OS in both MMTV-NIC PTEN FL/+ and MMTV-NIC PTEN+/- models. PTEN loss was associated with reduced sensitivity to AZD8931 and failure to suppress Src activity, suggesting these may be suitable predictive biomarkers of AZD8931 response. To facilitate further studies exploring resistance, we transplanted MMTV-NIC PTEN+/- fragments into syngeneic mice and generated 3 tumours with acquired resistance to AZD8931. These tumours displayed differing resistance strategies; 1 tumour continued to express HER2 whilst the remaining 2 underwent EMT and lost HER2 expression reflecting to a very limited degree some of the heterogeneity of resistance strategies seen in human disease. To further explore resistance to HER2 targeting tyrosine kinase inhibitors, we generated a panel of human cell lines with acquired resistance to AZD8931 and lapatinib. Western blotting demonstrated loss of HER2, HER3 and PTEN in all resistant lines. Acquisition of resistance was associated with a marked change in phenotype and western blotting confirmed all lines had undergone EMT. We used a combination of RPPA and mass spectrometry to further characterise the AZD8931 resistant lines and identified multiple potential novel proteins involved in the resistant phenotype, including several implicated in EMT. In conclusion, when coupled with appropriate in vitro techniques, the MMTV-NIC model is a valuable tool for selection of emerging drugs to carry forward into clinical trials of HER2 positive breast cancer.

En raison du manque d'efficacité et de la toxicité des thérapies conventionnelles contre le cancer, la recherche s'est concentrée sur le développement de nouvelles stratégies. Ces efforts ont été à l'origine de l'essor de l'immunothérapie, dont les acteurs les plus récents sont les anticorps monoclonaux ciblant les points de contrôles immunitaires (PCI). Parmi ces inhibiteurs des PCI, on retrouve les anticorps ciblant la protéine de surface « Programmed Cell Death 1 », les anti-PD1, et ceux ciblant son ligand, « Programmed Cell Death Ligand 1 », les anti-PDL-1. Ces anticorps ont démontré une efficacité spectaculaire dans plusieurs types de cancers, et sont aujourd'hui couramment utilisés en clinique comme thérapies dans le mélanome, le cancer du poumon, de la vessie et du rein. Cependant, ces traitements ne profitent pas à tous les patients atteints de cancer, avec en moyenne 60% de résistance innée, et 25% de résistance acquise après une réponse primaire aux anticorps, variable selon le type de tumeur. Les phénomènes impliqués dans la résistance sont à l'heure actuelle peu connus. Ainsi, l'objectif de mon projet de recherche consistait à établir des modèles in vivo de résistance acquise aux anti-PD1 et anti-PDL 1. Pour ce faire, nous avons utilisé des tumeurs syngéniques de rein (RENCA), de vessie (MB49 et MBT-2) et de colon (MC38) et des souris immunocompétentes, que nous avons rendues résistantes aux traitements en les soumettant à des séries de réimplantation de tumeurs et de traitements, induisant une pression de sélection jusqu'à l'obtention d'un phénotype résistant. Le succès du blocage de l'axe PD1/ PDL-1 étant fortement lié à l'état du microenvironnement tumoral, nous avons mis en place un protocole d'immunophénotypage. Nous avons ainsi pu observer les cellules au profil « anti-tumoral », telles que les cellules T, les Natural Killer, et les macrophages M1, mais également les cellules ayant une fonction immunosuppressive, telles que les macrophages M2, les MDSC, les Treg. Enfin, certaines études ayant identifié une sur-régulation des PCI inhibiteurs alternatifs dans les cas de résistance acquise à l'anti-PD1, nous avons également observé l'expression de LAG3, TIM3 et TIGIT en plus de l'expression de PD1 et PDL-1. Nous avons ainsi pu déterminer que la résistance semble très fortement dépendante du modèle tumoral, même si nous avons pu identifier une diminution des macrophages M1 anti-tumoraux dans l'ensemble des modèles résistants à l'anti- PD1, et une augmentation des Treg dans les modèles résistants à l'anti-PDL-1, suggérant un mécanisme commun de résistance propre respectivement à l'anti-PD1 et à l'anti-PDL-1. Suite à l'identification par Zaretsky et al. de gènes impliqués dans la voie interféron dans des cas de résistance acquise de mélanome traité à l'anti- PD1, nous avons également décidé d'étudier le profil moléculaire des tumeurs résistantes. Cela nous a permis d'identifier 5 gènes communs entre les modèles anti- PD1 et anti-PDL-1 résistants, dont SERPINF1 et FCNA qui semblent prometteurs comme cibles à valider. Enfin, en parallèle de l'établissement et de la caractérisation des modèles de résistance acquise, nous avons testé de nouvelles approches thérapeutiques de potentialisation des anticorps anti-PD1 et anti-PDL-1 en combinaison avec des chimiothérapies de référence pour le cancer étudié. Nous avons ainsi démontré une potentialisation dans les modèles sauvages de cancer du côlon MC38 et de la vessie MB49, aucun effet de la combinaison dans le modèle de cancer du sein métastatique 4T1, et une inhibition de l'effet de l'anti-PDL-1 avec la combinaison dans le modèle de vessie MBT-2. L'immunophénotypage nous a permis de constater ici aussi des différences très importantes entre les modèles tumoraux, au niveau basal et après traitement [etc...] / Because of the limited efficacy and the toxicity of conventional therapies to fight cancer, researchers focused on the new trategies. These efforts lead to the emergence of immunotherapies, whose msot recent actors are the monoclonal antibodies targeting immune checkpoint (ICP). Among those ICP inhibitors, we found antibodies targeting the surface protein « Programmed Cell Death 1 », called anti- PD1, and those targeting its ligand, « Programmed Cell Death Ligand 1 », called anti- PDL-1. Those antibodies shown a great efficacy in a wide diveristy of cancers, and are currently used for clinical practice in the case of melanoma, lung cancer, bladder cancer and renal cell carcinoma. However, those treatments don’t benefit to all tumor bearing patients, with a mean of 60% of innate resistance, and 25% of acquired resistance following a primary response, variable according to tumor type. Phenomena involved in resistance are currently poorly described. In this context, the aim of my project was to establish in vivo preclinical models of acquired resistance to anti-PD1 and anti-PDL-1. To do that, we used syngeneic renal cancer (RENCA), bladder cancer (MB49 and MBT-2), and colorectal cancer (MC38), and immunocompetent mice, that we have made resistant by serial reimplantations of tumors pieces and serial treatments, inducing a selection pressure until we obtained a resistant phenotype. The efficiency of PD1/PDL-1 axis blocking is strongly linked to the microenvironment composition, as a result we realized an immunophenotyping protocol. We observed anti-tumor cells as T cells, Natural Killer cells, and M1 macrophages, but also cells harboring immunosuppressive functions, as M2 macrophages, MDSC, and Treg. Moreover, some studies have identified an upregulation of alternatives ICP in the context of acquired resistance to anti-PD1, so we also observed the expression of LAG3, TIM3 and TIGIT besides PD1 and PDL-1 expression. We shown that resistance is strongly dependant to the tumor model, even if we identified a decrease of anti-tumor M1 macrophages is models resistant to anti-PD1, and an increase of Treg in models resistant to anti-PDL-1, suggesting a common mechanism of resistance specific to respectively anti PD1 and anti-PDL-1. Following Zaretsky and al. identification of genes involved in interferon pathway in the case of acquired resistance to anti-PD1 in melanoma, we decided to study the molecular profile of resistant tumors. We identified 5 common genes differently modulated between anti-PD1 and anti-PDL-1 resistant models, including SERPINF1 and FCNA which seems to be promising as targets to validate. Lastly, in parallel to establishment and characterization of preclinical models of acquired resistance, we tested new therapeutical approches of anti-PD1 and anti- PDL-1 potentiation in combination with reference chemotherapies. We shown a synergy in wild-type colorectal and bladder cancers (MC38 and MB49), no effect of the combination in metastatic breast cancer 4T1, and an inhibition of anti-PDL 1 effect in bladder cancer MBT-2. Immunphenotyping of tumors allowed us to observe here also high differences between tumor models, both at baseline and after treatments initiation. To conclude, even if our results need a validation with patients samples, we demonstrated that different cellular and molecular modifications could be involved in resistance to anti-PD1 and anti-PDL-1, and that resistance could be bypass with chemotherapy combination, according to tumor type

Tobacco SABP2, a 29. kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar-. S-methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2-silenced tobacco plants when treated with ASM, fail to express PR-1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2-silenced plants. These results show that functional SABP2 is required for ASM-mediated induction of resistance.

Soybean cyst nematode (SCN), a major pathogen of soybean worldwide, causes huge losses in soybean production. Various approaches including cloning of genes to combat this devastating disease help to better understand the cellular function and immune responses of plants. Membrane fusion genes are the important regulatory parts of vesicular transport system, which works through packaging of intracellular compounds and delivering them to apoplast or nematode feeding sites to induce an incompatible reaction. The incompatible nature of membrane fusion proteins such as SNAP25, Munc18, Syntaxin, Synaptobrevin, NSF, Synaptotagmin and alpha-SNAP are conserved in eukaryotes and regulate the intracellular function to combat abiotic and biotic stress in plants. Overexpression of these genes in G. max [Williams 82(PI518671)] which is a susceptible cultivar of soybean to nematodes resulted in a reduction of the SCN population providing further insights of molecular and genetic approaches to solve the SCN problems in agriculture.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24781号 / 医博第4973号 / 新制||医||1066(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 中島 貴子, 教授 溝脇 尚志, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Tobacco SABP2 has been shown to display high affinity for salicylic acid (SA) and methylsalicylate (MeSA) and plays an important role in SAR signal development. Using biochemical approach, SABP2 has been shown to demonstrate strong esterase activity in converting MeSA to SA. Recent study shows that tetra fluoroacetophenone, a synthetic analog of SA, competitively inhibits SABP2 esterase activity as well as suppresses SAR signal development in tobacco mosaic virus (TMV)-infected tobacco plants. Not much has been studied on the effect of pesticides on plant defenses. Because both AChE and SABP2 are esterase-like proteins belonging to α/β hydroxylase superfamily, we hypothesize that pesticides may inhibit the MeSA esterase activity of SABP2 and block SAR development. Biochemical and molecular biology techniques were used to test this hypothesis. SAR in tobacco-TMV plant-pathogen system is measured by significant decrease in TMV-induced lesion sizes in secondarily inoculated distal leaves.

Most rice protection methods have currently used toxic chemicals to control pathogens and pests, which leads to environmental pollution. Systemic acquired resistance (SAR) taking advantage of natural defence reaction of plants could be proposed as an alternative, ecologically friendly approach for plant protection. Its application into rice production could minimize the chemicals quantity used and could contribute to the decrease of environmental pollution and the development of sustainable agriculture. The research was conducted to select the most effective chemical and suitable method to improve the health of rice plants infected by grassy stunt disease in net-house of Can Tho University. SAR chemicals were used at very low concentrations (in mM). Results showed that the height of rice plants treated with SAR chemicals was higher than that of plants untreated. Besides, the number of diseased plants was reduced and the ratio of firm grain and yield increased when plants were applied by SAR. Among the used substances, oxalic acid provided the best systemic acquired resistance. With oxalic acid, seed soaking was better than seed coating in systemic acquired resistance against rice grassy stunt disease. / Hầu hết các phương pháp sản xuất lúa hiện nay đều sử dụng các hóa chất độc hại trong việc phòng trừ bệnh và côn trùng gây hại, nên dẫn đến ô nhiễm môi trường. Kích thích tính kháng lưu dẫn giúp kích hoạt cơ chế tự nhiên kháng bệnh của cây có thể là giải pháp bảo vệ thực vật thay thế an toàn với môi trường. Việc ứng dụng tiến bộ này vào trong sản xuất lúa có thể làm giảm lượng hóa chất sử dụng, đóng góp vào việc giảm thiểu ô nhiễm môi trường và sự phát triển của một nền nông nghiệp bền vững. Nghiên cứu đã được thực hiện tại nhà lưới trường Đại học Cần Thơ để tuyển chọn hóa chất và phương pháp sử dụng hóa chất để tăng cường sức khỏe giúp cây lúa vượt qua bệnh vàng lùn. Hóa chất kích kháng được sử dụng ở một nồng độ rất thấp (đơn vị là mM). Kết quả cho thấy chiều cao cây lúa khi xử lý chất kích kháng tốt hơn so đối chứng không xử lý. Bên cạnh đó, số cây lúa nhiễm bệnh giảm, tỉ lệ hạt chắc và năng suất tăng khi cây lúa được xử lý với chất kích kháng. Trong số các chất kích kháng đã sử dụng, acid oxalic cho hiệu quả vượt trội. Với chất acid oxalic, phương pháp ngâm hạt cho hiệu quả kích kháng tốt hơn phương pháp áo hạt.

Cultivars of Agrostis stolonifera showed weak and strong responsiveness to the systemic acquired resistance (SAR) activator, benzothiadiazole (BTH), or the induced systemic resistance (ISR) activator, 2R, 3R-butanediol (BD). Next Generation RNA sequencing was used to identify 2163 putative transcripts with increased expression in BTH versus water-treated A. stolonifera. Among three BTH-induced genes, AsASP-2 and AsHIR-1 were induced faster, while AsLOX-1 had stronger transient induction, in one out of two strongly BTH-responsive cultivars. Three ISR-responsive genes, AsGNS-5, AsOPR-4 and AsAOS-1, showed no greater induction or priming in the strongly versus weakly BD-responsive cultivars. Cultivars of A. stolonifera vary significantly in their response to defense activators, however this is not consistently related to defense gene expression. To quantify disease severity, chlorophyll fluorescence imaging of the maximum quantum efficiency of photosystem II (Fv/Fm) was tested on Nicotiana benthamiana infected with Colletotrichum orbiculare. Leaf areas of healthy, non-necrotic affected and necrotic tissue could be individually quantified, which demonstrated that BD delayed symptom development by approx. 24-hour and reduced non-necrotic affected tissue compared to controls. Chlorophyll fluorescence imaging can quantify and reveal novel features about induced disease resistance.