Investigation of glycosyltransferases from oat

Plants produce a diversity of secondary metabolites crucial for their survival into specific ecological niches. Many of these compounds are glycosides generated by the action of family one UDP-dependant glycosyltransferases (UGTs). Glycosylated products of UGTs are known to be essential for reproductive fitness, defence against pathogens, and signalling; UGTs also have a role in the detoxification of xenobiotics. To date, little is known about monocot UGTs compare to their dicot counterparts, despite their potential role in defence and modification of health-promoting component of cereals essential to human diet. This thesis focuses on identification and functional investigation of UGTs expressed in in the diploid oat species Avena strigosa. Chapters 1 and 2 consist of the General Introduction and Material and Methods, respectively. In chapter 3, a systematic analysis of root-expressed UGTs was carried out using transcriptomic and proteomic approaches. A subset of UGTs was then selected for biochemical analysis. Of particular interest were candidates for glycosylation of avenacin, an antimicrobial triterpenoid glycoside that protects oat against fungi infection. In chapter 4, the sugar donor specificity of the recombinant UGTs and their activity towards different triterpenoid acceptors was investigated. In chapter 5, a transient expression system was established in Nicotiana benthamiana in order to investigate UGT activity. Heterologous co-expression of UGTs with early avenacin biosynthetic pathway enzymes leads to biosynthesis of new-to-nature triterpenoid glycosides, so providing a powerful system for functional analysis of terpene glycosylation in planta. In chapter 6, the catalytic properties of the UGT collection towards different plant natural products was investigated, leading to the production of glycosides of interest. The contribution of this study to the understanding of the evolution and function of monocot UGTs and to their potential commercial exploitation is discussed in the chapter 7

Analysis of Two New Arabinosyltransferases Belonging to the Carbohydrate-Active Enzyme (CAZY) Glycosyl Transferase Family1 Provides Insights into Disease Resistance and Sugar Donor Specificity

Glycosylation of small molecules is critical for numerous biological processes in plants, including hormone homeostasis, neutralization of xenobiotics, and synthesis and storage of specialized metabolites. Glycosylation of plant natural products is usually carried out by uridine diphosphate-dependent glycosyltransferases (UGTs). Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits such as disease resistance and flavor and have numerous pharmaceutical applications. Most characterised plant natural product UGTs are glucosyltransferases, and little is known about enzymes that add other sugars. Here we report the discovery and characterization of AsAAT1 (UGT99D1), which is required for biosynthesis of the antifungal saponin avenacin A-1 in oat. This enzyme adds L-arabinose to the triterpene scaffold at the C-3 position, a modification critical for disease resistance. The only previously reported plant natural product arabinosyltransferase is a flavonoid arabinosyltransferase from Arabidopsis. We show that AsAAT1 has high specificity for UDP-β-L-arabinopyranose, identify two amino acids required for sugar donor specificity, and through targeted mutagenesis convert AsAAT1 into a glucosyltransferase. We further identify a second arabinosyltransferase potentially implicated in the biosynthesis of saponins that determine bitterness in soybean. Our investigations suggest independent evolution of UDP-arabinose sugar donor specificity in arabinosyltransferases in monocots and eudicots

Immune checkpoint blockade – how does it work in brain metastases?

Immune checkpoints restrain the immune system following its activation and their inhibition unleashes anti-tumor immune responses. Immune checkpoint inhibitors revolutionized the treatment of several cancer types, including melanoma, and immune checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies is becoming a frontline therapy in metastatic melanoma. Notably, up to 60% of metastatic melanoma patients develop metastases in the brain. Brain metastases (BrM) are also very common in patients with lung and breast cancer, and occur in ∼20–40% of patients across different cancer types. Metastases in the brain are associated with poor prognosis due to the lack of efficient therapies. In the past, patients with BrM used to be excluded from immune-based clinical trials due to the assumption that such therapies may not work in the context of “immune-specialized” environment in the brain, or may cause harm. However, recent trials in patients with BrM demonstrated safety and intracranial activity of anti-PD-1 and anti-CTLA-4 therapy. We here discuss how immune checkpoint therapy works in BrM, with focus on T cells and the cross-talk between BrM, the immune system, and tumors growing outside the brain. We discuss major open questions in our understanding of what is required for an effective immune checkpoint inhibitor therapy in BrM

Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation

Human cytomegalovirus (HCMV) induces a uniquely high frequency of virus-specific effector/memory CD8+ T-cells, a phenomenon termed “memory inflation”. Thus, HCMV-based vaccines are particularly interesting in order to stimulate a sustained and strong cellular immune response against cancer. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with high lethality and inevitable relapse. The current standard treatment does not significantly improve the desperate situation underlining the urgent need to develop novel approaches. Although HCMV is highly fastidious with regard to species and cell type, GBM cell lines are susceptible to HCMV. In order to generate HCMV-based therapeutic vaccine candidates, we deleted all HCMV-encoded proteins (immunoevasins) that interfere with MHC class I presentation. The aim being to use the viral vector as an adjuvant for presentation of endogenous tumor antigens, the presentation of high levels of vector-encoded neoantigens and finally the repurposing of bystander HCMV-specific CD8+ T cells to fight the tumor. As neoantigen, we exemplarily used the E6 and E7 proteins of human papillomavirus type 16 (HPV-16) as a non-transforming fusion protein (E6/E7) that covers all relevant antigenic peptides. Surprisingly, GBM cells infected with E6/E7-expressing HCMV-vectors failed to stimulate E6-specific T cells despite high level expression of E6/E7 protein. Further experiments revealed that MHC class I presentation of E6/E7 is impaired by the HCMV-vector although it lacks all known immunoevasins. We also generated HCMV-based vectors that express E6-derived peptide fused to HCMV proteins. GBM cells infected with these vectors efficiently stimulated E6-specific T cells. Thus, fusion of antigenic sequences to HCMV proteins is required for efficient presentation via MHC class I molecules during infection. Taken together, these results provide the preclinical basis for development of HCMV-based vaccines and also reveal a novel HCMV-encoded block of MHC class I presentation

Central nervous system lymphatic unit, immunity, and epilepsy : Is there a link?

Summary The recent definition of a network of lymphatic vessels in the meninges surrounding the brain and the spinal cord has advanced our knowledge on the functional anatomy of fluid movement within the central nervous system (CNS). Meningeal lymphatic vessels along dural sinuses and main nerves contribute to cerebrospinal fluid (CSF) drainage, integrating the cerebrovascular and periventricular routes, and forming a circuit that we here define as the CNS-lymphatic unit. The latter unit is important for parenchymal waste clearance, brain homeostasis, and the regulation of immune or inflammatory processes within the brain. Disruption of fluid drain mechanisms may promote or sustain CNS disease, conceivably applicable to epilepsy where extracellular accumulation of macromolecules and metabolic by-products occur in the interstitial and perivascular spaces. Herein we address an emerging concept and propose a theoretical framework on: (a) how a defect of brain clearance of macromolecules could favor neuronal hyperexcitability and seizures, and (b) whether meningeal lymphatic vessel dysfunction contributes to the neuroimmune cross-talk in epileptic pathophysiology. We propose possible molecular interventions targeting meningeal lymphatic dysfunctions, a potential target for immune-mediated epilepsy.Peer reviewe

A noncanonical vacuolar sugar transferase required for biosynthesis of antimicrobial defense compounds in oat

Plants produce an array of natural products with important ecological functions. These compounds are often decorated with oligosaccharide groups that influence bioactivity, but the biosynthesis of such sugar chains is not well understood. Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits, as exemplified by avenacins, antimicrobial defense compounds produced by oats. Avenacins have a branched trisaccharide moiety consisting of L-arabinose linked to 2 D-glucose molecules that is critical for antifungal activity. Plant natural product glycosylation is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). We previously characterized the arabinosyltransferase that initiates the avenacin sugar chain; however, the enzymes that add the 2 remaining D-glucose molecules have remained elusive. Here we characterize the enzymes that catalyze these last 2 glucosylation steps. AsUGT91G16 is a classical cytosolic UGT that adds a 1,2-linked D-glucose molecule to L-arabinose. Unexpectedly, the enzyme that adds the final 1,4-linked D-glucose (AsTG1) is not a UGT, but rather a sugar transferase belonging to Glycosyl Hydrolase family 1 (GH1). Unlike classical UGTs, AsTG1 is vacuolar. Analysis of oat mutants reveals that AsTG1 corresponds to Sad3, a previously uncharacterized locus shown by mutation to be required for avenacin biosynthesis. AsTG1 and AsUGT91G16 form part of the avenacin biosynthetic gene cluster. Our demonstration that a vacuolar transglucosidase family member plays a critical role in triterpene biosynthesis highlights the importance of considering other classes of carbohydrate-active enzymes in addition to UGTs as candidates when elucidating pathways for the biosynthesis of glycosylated natural products in plants

Subtelomeric assembly of a multi-gene pathway for antimicrobial defense compounds in cereals

Non-random gene organization in eukaryotes plays a significant role in genome evolution. Here, we investigate the origin of a biosynthetic gene cluster for production of defence compounds in oat—the avenacin cluster. We elucidate the structure and organisation of this 12-gene cluster, characterise the last two missing pathway steps, and reconstitute the entire pathway in tobacco by transient expression. We show that the cluster has formed de novo since the divergence of oats in a subtelomeric region of the genome that lacks homology with other grasses, and that gene order is approximately colinear with the biosynthetic pathway. We speculate that the positioning of the late pathway genes furthest away from the telomere may mitigate against a ‘self-poisoning’ scenario in which toxic intermediates accumulate as a result of telomeric gene deletions. Our investigations reveal a striking example of adaptive evolution underpinned by remarkable genome plasticity

Highly integrated workflows for exploring cardiovascular conditions: Exemplars of precision medicine in Alzheimer's disease and aortic dissection

For precision medicine to be implemented through the lens of in silico technology, it is imperative that biophysical research workflows offer insight into treatments that are specific to a particular illness and to a particular subject. The boundaries of precision medicine can be extended using multiscale, biophysics-centred workflows that consider the fundamental underpinnings of the constituents of cells and tissues and their dynamic environments. Utilising numerical techniques that can capture the broad spectrum of biological flows within complex, deformable and permeable organs and tissues is of paramount importance when considering the core prerequisites of any state-of-the-art precision medicine pipeline. In this work, a succinct breakdown of two precision medicine pipelines developed within two Virtual Physiological Human (VPH) projects are given. The first workflow is targeted on the trajectory of Alzheimer's Disease, and caters for novel hypothesis testing through a multicompartmental poroelastic model which is integrated with a high throughput imaging workflow and subject-specific blood flow variability model. The second workflow gives rise to the patient specific exploration of Aortic Dissections via a multi-scale and compliant model, harnessing imaging, computational fluid-dynamics (CFD) and dynamic boundary conditions. Results relating to the first workflow include some core outputs of the multiporoelastic modelling framework, and the representation of peri-arterial swelling and peri-venous drainage solution fields. The latter solution fields were statistically analysed for a cohort of thirty-five subjects (stratified with respect to disease status, gender and activity level). The second workflow allowed for a better understanding of complex aortic dissection cases utilising both a rigid-wall model informed by minimal and clinically common datasets as well as a moving-wall model informed by rich datasets

Etre une fille et s'engager dans une filière scolaire de garçons (la place des activités physiques et sportives dans la construction de l'<atypicité> scolaire)

Ce travail propose une analyse de la construction du genre, et des socialisations qui contribuent à reproduire et/ou atténuer, voire questionner les rapports sociaux de sexe imbriqués à ceux de classe, en étudiant le cas de filles engagées dans des filières scolaires majoritairement masculines, et plus précisément à saisir les relations entre les choix scolaires des interrogées et leurs rapports au corps et aux pratiques physiques et sportives. L'enquête a consisté en un recueil de questionnaires (269) auprès d' "atypiques" et de "typiques" scolaires, de 21 entretiens auprès d'atypiques engagées dans des voies professionnelles, technologiques, ou générales, et des séquences d'observation en lycée. Ces différents outils ont permis de réaliser une analyse comparative entre typiques et atypiques, et au sein du groupe des atypiques (configuration familiale, modes d'engagements corporel et sportif ... ). La première partie des résultats présente des portraits des familles des atypiques enquêtées: répartition sexuée du travail domestique relativement traditionnelle, mais souvent plus favorisées et plus sportives que celles des "typiques" de même niveau. La seconde partie concerne les goûts enfantins des interrogées, remarquant déjà des différences, entre typiques et atypiques et au sein du groupe des atypiques (selon classes sociales). La dernière partie approfondit enfin la question des relations, à partir de l'adolescence, entre orientation scolaire atypique et engagement dans le monde sportif, confirmant alors l'importance de la socialisation sportive dans la construction d'un rapport spécifique aux études, et plus largement au monde, différencié selon la classe.ln order to analyze socializations that contributes to reproduce and/or to weaken, or even to question gender and class social relations, this research work deals with the case of young women who decide to follow a generally male school programme ; the analysis focuses particularly on understanding the relations between school choices and bodily hexis. The survey consisted in a collection of questionnaires (269) filled by girls with atypical or typical school choices, of 21 interviews with "atypical" girls engaged in various scholar sectors, and of observation's periods in high school. These various analytical tools allowed us to carry out a comparative analysis between "typical" and "atypical" girls, and within the group of atypical girls (family configuration, physical and sports commitments, etc.). The first results presented introduce the portraits of the families of atypical girls investigated: families are frequently wealthier and "sportier" than those of the "typical" girls of the same school level, despite a traditional gendered domestic work distribution. The second part concerns the childish tastes of the questioned girls : in the childhood we can already notice differences, between typical and atypical girls and within the group of atypical (according to social classes). The last part, finally, goes further into the question of the relations, starting from adolescence, between atypical school choices and commitment toward the sports world; thus confirming the importance of sports socialization in the construction of a specific relationship to scholar education, and more widely to the world, differentiated according to social classes.ORSAY-PARIS11-Bib. STAPS (914712103) / SudocSudocFranceF