Plants are sessile organisms and constantly encounter a myriad of pathogens; therefore, they rely on highly effective defense system for their survival. Our understanding of how plant immunity is triggered and regulated has seen tremendous progress over the last two decades, with many important players identified in the model systems, Arabidopsis thaliana. Mitogen activated protein kinases play a central role in signal transduction in biotic and abiotic stresses. MAPK pathways are regulated by three-interlinked protein kinases (MAPKKK, MAPKK, MAPK), which are sequentially activated by phosphorylation. The activation of the three MAPKs MPK3, MPK4 and MPK6 is one of the earliest cellular responses following pathogen attack leading to the phosphorylation of appropriate cytosolic or nuclear targets to regulate cellular processes. However, only few targets of MPK3, MPK4 and MPK6 have been identified and validated so far and many MAPK substrates remain to be discovered. We performed largescale phosphoproteomics on mock treated and flg22 treated WT and the three loss-of-function mutants mpk3, mpk4 and mpk6 to identify novel MAPKs substrates and their cellular functions in response to pathogen attack. We identify and validated some of the differentially phosphorylated cytosolic and chromatin targets of MPK3, MPK4 and MPK6. DEK2, a nuclear protein involved in multiple chromatin-related processes, was identified in the phosphoproteomics screen as an in vivo target of MPK6 and it interacts in planta and is phosphorylated in vitro by the three immune MAPKs. dek2 loss-of-function mutants were susceptible to bacterial as well as fungal pathogens. Additionally, transcriptome data of the dek2-1 mutant show that DEK2 is a transcriptional repressor inclusive of defense related genes and hormone synthesis and signaling genes. We determined that DEK2 is a reader of the histone mark, H3K9me1, by Microscale thermophoresis. From ChIP-Seq analysis, DEK2 was found to be enriched at class I TCP binding motif regions. We further need to determine whether DEK2 binds to TCP transcription factors directly or indirectly. Finally, based on our data we postulate a hypothetical working model for the function of DEK2 as a transcriptional repressor and a reader of H3K9me1 mark.

The adaptive immune system is a complex network of cells that protect the body from invasion by foreign pathogens. Crucial to the function of the adaptive immune system is the activation, proliferation and differentiation of T cells in response to foreign pathogen presentation by antigen presenting cells. T cell activation is driven through different signaling pathways that are dependent on phosphorylation of substrates by kinases. In the PLC pathway that activates the il2 gene program, Protein Kinase C-q (PKCq) and Notch1 localize to the immunological synapse and help drive the signaling cascade that leads to robust T cell activation. It has been previously shown that PKCq and Notch1, both interact with the CBM complex at the immunological synapse. Additionally, PKCq and Notch1 both have specific cytoplasmic and nuclear functions that help drive the il2 gene program. Here, we demonstrate the localization of PKCq and Notch1 constructs transfected into HEK 293 cells. The use of deletion constructs of Notch1 was intended to inform us of what functional domain of Notch1 was responsible for the interaction with PKCq, however no direct interaction was demonstrated with the PKCq and Notch1 constructs used in these experiments. We hypothesize that this is likely due to the inactive form of PKCq found in our construct, or a result of the cell type used in these experiments.

The onset of immune response against microbial stimuli activates induction of many anti- inflammatory genes and ISGs for effective clearance of the pathogen. This response includes transcriptional activation of several non-coding transcripts such as miRNAs and long non-coding RNAs (lncRNAs). LncRNAs constitutes the largest class of non-coding genome and are arbitrarily described as transcripts greater than 200 base pairs. Similar to protein coding mRNAs, lncRNAs are RNA polymerase II transcripts and undergo mRNA processing such as capping, splicing and polyadenylation. In recent years, high throughput sequencing has enabled an in-depth exploration of the human genome and subsequent discovery of lncRNAs. Several studies have highlighted the crucial role of lncRNAs in many biological processes including as regulators of gene expression as well as molecular effectors of host-pathogen driven immune responses. To date, majority of lncRNAs have been studied in murine models with limited understanding in human cells. In order to elucidate the role of lncRNAs in human immune cell regulation, the goal of this thesis is to identify and characterize novel lncRNAs critical to host-pathogen innate immune responses. RNA sequencing in LPS, IAV and HSV stimulated cells revealed lncRNA LUCAT1 as most differentially regulated lncRNA. CRISPR-cas9 and shRNA mediated depletion of LUCAT1 showed enhanced IFN-I genes signature, which was suppressed upon overexpression of LUCAT1. Additionally, LPS stimulated hDCs showed enrichment of LUCAT1 in the nucleus and its association with the chromatin markers. Further, LUCAT1 depletion contributed to enhanced occupancy of transcriptional coactivators at the promoters of IFN-I genes. Global identification of RNA associated proteins revealed LUCAT1 association with STAT1 in the nucleus thus emphasizing its role in transcriptional regulation of Type I IFN genes in inflammatory responses. This thesis furthers the understanding about the molecular factors affecting immune regulation and describes the novel role of LUCAT1 as an attenuator of immune cell response to pathogens.

No description available.

Les anticorps bloquant les points de contrôle immunitaires modifient profondément la gestion des patients atteints de cancer. À la pointe de cette nouvelle classe d'agents anticancéreux, les anticorps anti-PD-1 / PD-L1 peuvent ainsi restaurer une réponse efficace des cellules T antitumorales. En conséquence, ces agents sont maintenant approuvés dans divers types de tumeurs, tels que le mélanome, le cancer bronchique non à petites cellules, le cancer du rein, les tumeurs ORL ou le cancer de la vessie. Ces nouvelles immunothérapies entraînent également de nouveaux profils de réponse tumorale tels que des réponses tumorales retardées ou des pseudoprogressions. Au fil de l’expérience acquise avec ces traitements, il a été observé chez certains patients un état de progression rapide de la maladie, ce qui pourrait suggérer que le blocage de points de contrôle immunitaire pourrait avoir un effet délétère en accélérant la maladie chez un sous-groupe de patients. Ce travail de thèse a permis de caractériser sur le plan clinique et biologique ce phénomène d’accélération de la croissance tumorale sous immunothérapie anti-checkpoint que nous avons définit “maladie hyperprogressive” (HPD). L’analyse transcriptomique d’échantillons tumoraux de ces patients a permis d’orienter vers un rôle spécifique de l’environnement myeloide. / Immune checkpoint blocking antibodies are profoundly changing the management of patients with cancer. At the forefront of this novel anticancer agent class, anti-PD-1/PD-L1 antibodies can exhibit a significant activity by restoring an efficient antitumor T-cell response. As a result, these agents are now approved in various tumor types such as melanoma, squamous, and nonsquamous non–small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC) or bladder cancer. Interestingly, these new immunotherapies also result in novel tumor response patterns such as delayed tumor responses or pseudoprogressions. As experience grows with these therapeutics, anecdotal reports are relating rapid disease progressions, which could suggest that immune checkpoint blockade may have a deleterious effect by accelerating the disease in a subset of patients. This thesis work has made it possible to characterize clinically and biologically this phenomenon of accelerated tumor growth under anti-checkpoint immunotherapy, which we have defined as “hyperprogressive disease” (HPD). Transcriptomic analysis of tumour samples from these patients suggested a specific role for the myeloid environment.

In the version of this article initially published, author Raphael Duivenvoorden’s last name was spelt incorrectly as Duivenwoorden. The error has been corrected in the HTML and PDF versions of the article.

The immune system rapidly mounts an innate immune response to invading pathogens that is accompanied by antigen-presentation, to promote the development of the adaptive immune response. These responses orchestrate through signal transduction by PRRs that recognize PAMPs, which results in the expression of various cytokines and mediators to promote pathogen control. Herein, we investigated the role of the type I interferon (IFN)- and the p38MAPK- pathways in response to infection with Salmonella Typhimurium (ST). We delved into the mechanisms through which IFNAR1-signaling results in host susceptibility against ST and show that while STAT2 and IRF9 promote susceptibility against ST, this is antagonized by STAT1. Our results indicate that IFNAR1-signaling induces IL-10 production through the ISGF3 complex, which indeed inhibits the production of IL-1β (via NLRP3 and caspase-1) resulting in a state of resistance against ST. Furthermore, our work elucidates that MK2, which is a p38MAPK substrate promotes host resistance, which is contradictory to type I IFNs despite the fact that MK2 regulates cytokine expression in a similar pattern to IRF9. We demonstrate that MK2 inhibits inflammasome signaling via NLRP3, caspase-1 and caspase11. We also reveal a role for MK2 in regulating IL-1β production via distinct signaling pathways including inhibition of MSK1/2 besides activation of the autophagic machinery; which also contribute to the enhanced inflammasome activation seen in Mk2- deficient cells. Thus, our observations illuminate the fact that the type I IFN pathway and the p38MAPK pathway are only dependent on each other to a certain extent in modulating the innate immune response to Salmonella infection, thereby bringing about varied outcomes in the infected host.

archives@tulane.edu / International conventions do not set down rules on state immunity and leave it to national courts to decide the nature and scope of state immunity. The inevitable result of this state-centrist approach is the evolution of divergent views among states on the reach of state immunity. In the early years of international relations, the accepted view was that states enjoyed absolute sovereign immunity and that as a result no state, without its consent, was subject to the national jurisdiction of another state. Gradually many states, mainly through judicial decisions, moved towards a qualified doctrine of immunity enabling a degree of submission by one state to the jurisdiction of another. This restricted view of sovereign immunity was prompted by the changing nature of socio-economic and political circumstances, with states taking an increasingly felt presence in trading and commercial activities. It is generally the developed countries that were eager to embrace the restrictive view of state immunity, which enabled their nationals to press claims against errant foreign states. Naturally, the developing countries tended to favor the absolute doctrine of state immunity, in order to resist claims, however well founded, made against them. Today, most developing countries still insist on absolute state immunity. While not yet codified in an international convention, the doctrine of state immunity has found its way into customary international law. In identifying and interpreting international customary law of state immunity, national courts frequently refer to and follow judicial decisions of foreign jurisdictions. This practice enables states to learn from different legal techniques and criteria that are used in other jurisdictions to demarcate the scope of the doctrine of state immunity. An area where states have reached some common understanding is the enforcement of arbitral awards—imposing measures of constraint against state assets. While judicial enforcement of arbitral awards is the much preferred and most prevalent means of subjecting state assets to seizure or attachment, there are some notable non-judicial remedial measures which may aid the aggrieved investors in satisfying their claims against state parties to a dispute. These non-judicial means of relief rely on the willingness of the investors’ parent state to pursue their cause with the recalcitrant state. The parent state’s willingness is dictated by political considerations in contrast to non political nature of judicial proceedings. It is commonly agreed that an independent judicial process is much preferable to politically motivated non-judicial avenues of relief. As such, attention of judges, scholars and lawmakers must focus on refining judicial processes and building effective enforcement mechanisms. This calls for widely agreed principles of state immunity and a commonly shared enforcement mechanism. Having identified problems arising from a lack of universal agreement on state immunity and the diversity and, more dishearteningly, the inadequacy of forms of enforcement available to an aggrieved claimant, this thesis proposes that the international community must work towards the setting up of a central enforcement agency, a functional model of enforcement. This thesis suggests that the central mechanism of enforcement could be set up through reaching an international treaty or convention or modifying the existing mechanisms. / 1 / Zixin Meng

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21413号 / 医博第4403号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 伊達 洋至, 教授 中川 一路 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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