Étude de la variante d’histone H2A.Z et du cycle de phosphorylation de l’ARN polymérase II chez Saccharomyces cerevisiae

La chromatine est plus qu’un système d’empaquetage de l’ADN ; elle est le support de toutes les réactions liées à l’ADN dans le noyau des cellules eucaryotes et participe au contrôle de l’accès de l’ARN polymérase II (ARNPolII) à l’ADN. Responsable de la transcription de tous les ARNm des cellules eucaryotes, l’ARNPolII doit, suivant son recrutement aux promoteurs des gènes, transcrire l’ADN en traversant la matrice chromatinienne. Grâce au domaine C-terminal (CTD) de sa sous-unité Rpb1, elle coordonne la maturation de l’ARNm en cours de synthèse ainsi que les modifications de la chromatine, concomitantes à la transcription. Cette thèse s’intéresse à deux aspects de la transcription : la matrice, avec la localisation de la variante d’histone H2A.Z, et la machinerie de transcription avec le cycle de phosphorylation du CTD de l’ARNPolII. Suivant l’introduction, le chapitre 2 de cette thèse constitue un protocole détaillé et annoté de la technique de ChIP-chip, chez la levure Saccharomyces cerevisiae. Cette technique phare dans l’étude in vivo des phénomènes liés à l’ADN a grandement facilité l’étude du rôle de la chromatine dans les phénomènes nucléaires, en permettant de localiser sur le génome les marques et les variantes d’histones. Ce chapitre souligne l’importance de contrôles adéquats, spécifiques à l’étude de la chromatine. Au chapitre 3, grâce à la méthode de ChIP-chip, la variante d’histone H2A.Z est cartographiée au génome de la levure Saccharomyces cerevisiae avec une résolution d’environ 300 paires de bases. Nos résultats montrent que H2A.Z orne un à deux nucléosomes au promoteur de la majorité des gènes. L’enrichissement de H2A.Z est anticorrélé à la transcription et nos résultats suggèrent qu’elle prépare la chromatine pour l’activation des gènes. De plus H2A.Z semble réguler la localisation des nucléosomes. Le chapitre suivant s’intéresse à la transcription sous l’angle de la machinerie de transcription en se focalisant sur le cycle de phosphorylation de l’ARN polymérase II. Le domaine C-terminal de sa plus large sous-unité est formé de répétitions d’un heptapeptide YSPTSPS dont les résidus peuvent être modifiés au cours de la transcription. Cette étude localise les marques de phosphorylation des trois résidus sérine de manière systématique dans des souches mutantes des kinases et phosphatases. Nos travaux confirment le profil universel des marques de phosphorylations aux gènes transcrits. Appuyés par des essais in vitro, ils révèlent l’interaction complexe des enzymes impliqués dans la phosphorylation, et identifient Ssu72 comme la phosphatase de la sérine 7. Cet article appuie également la notion de « variantes » des marques de phosphorylation bien que leur étude spécifique s’avère encore difficile. La discussion fait le point sur les travaux qui ont suivi ces articles, et sur les expériences excitantes en cours dans notre laboratoire.Chromatin is more than just the eucaryotic DNA packaging system; it is the substrate of all reactions involving DNA in eukaryotic cells and actively regulates RNA Polymerase II (RNAPolII) access to DNA. Responsible for all mRNA transcription in eucaryotes, the RNAPolII must, following its recruitment to the pre-initiation complex, overcome the chromatin barrier in order to transcribe genes. The RNAPolII CTD allows for the co-transcriptional coordination of mRNA maturation and chromatin modifications. The work covered in this thesis addresses two aspects of transcription: the chromatin substrate, with the localization of H2A variant, H2A.Z, and the transcription complex with the phosphorylation cycle of the RNAPolII CTD. Following the introduction, chapter 2 constitutes a detailed and annotated Saccharomyces cerevisiae ChIP-chip protocol, from the culture to the hybridization of the array, with an emphasis on the proper controls required for chromatin study. This technique, extremely powerful for the in vivo study of all DNA transactions, leads to a better understanding of chromatin function in nuclear phenomena, thanks to the localization of histone variants and modifications. The third chapter maps the H2A.Z variant across the yeast genome at ~300 base pairs resolution using ChIP-chip. Our data shows that H2A.Z is incorporated into one or two promoter-bound nucleosomes at the majority of genes. H2A.Z enrichment is anticorrelated with transcription, and the results suggest that it configures chromatin structure to poise genes for transcriptional activation. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning. The next chapter focuses on the transcription machinery and, more precisely, on the phosphorylation cycle of RNAPolII. The CTD contains repetitions of a heptapeptide (YSPTSPS) on which all serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Here, we systematically profiled the location of the RNAPII phospho-isoforms in wild-type cells and mutants for most CTD modifying enzymes. The results provide evidence for a uniform CTD cycle across genes. Together with results from in vitro assays, these data reveal a complex interplay between the modifying enzymes, identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes. Moreover, it reinforces the notion of variants of the phosphorylation marks, even though the exact nature of the variant is still difficult to identify. The discussion introduces the studies that followed this work, including new projects conceived in our lab

Synthesis, structural determination and antimicrobial evaluation of two novel CoII and ZnII halogenometallates as efficient catalysts for the acetalization reaction of aldehydes

Background: Complexes of imidazole derivatives with transition metal ions have attracted much attention because of their biological and pharmacological activities, such as antimicrobial, antifungal, antiallergic, antitumoural and antimetastatic properties. In addition, imidazoles occupy an important place owing to their meaningful catalytic activity in several processes, such as in hydroamination, hydrosilylation, Heck reaction and Henry reaction. In this work, we describe the crystallization of two halogenometallate based on 2-methylimidazole. Their IR, thermal analysis, catalytic properties and antibacterial activities have also been investigated. Results: Two new isostructural organic-inorganic hybrid materials, based on 2-methyl-1H-imidazole, 1 and 2, were synthesized and fully structurally characterized. The analysis of their crystal packing reveals non-covalent interactions, including C/N–HCl hydrogen bonds and stacking interactions, to be the main factor governing the supramolecular assembly of the crystalline complexes. The thermal decomposition of the complexes is a mono-stage process, confirmed by the three-dimensional representation of the powder diffraction patterns (TDXD). The catalytic structure exhibited promising activity using MeOH as solvent and as the unique source of acetalization. Moreover, the antimicrobial results suggested that metal-complexes exhibit significant antimicrobial activity. Conclusion: This study highlights again the structural and the biological diversities within the field of inorganic–organic hybrids. [Figure not available: see fulltext.]

A humanised anti-IGF-1R monoclonal antibody (AVE1642) enhances Bortezomib-induced apoptosis in myeloma cells lacking CD45

The humanised form of an antagonistic anti-IGF-1R mAb (AVE1642) selectively inhibits the growth of CD45neg myeloma cells. AVE1642 strongly increased bortezomib-induced apoptosis, correlated with an increase of Noxa expression. These results support the therapeutic use of anti-IGF-1R/bortezomib in CD45neg Myeloma patients, particularly those with the most aggressive form, t(4,14)

Screening the Medicines for Malaria Venture (MMV) Pandemic Response Box chemical library on Caenorhabditis elegans identifies re-profiled candidate anthelmintic drug leads

The 3 major classes of soil transmitted helminths (whipworm, hookworm and Ascaris) affect 1.5 billion people worldwide mostly in poor countries, where they have adverse effects on child development, nutrition, and the work capacity of adults. Although there are drugs effective on Ascaris, notably the benzimidazoles, those same drugs show poor efficacy particularly against whipworm (Trichuris trichiura) and to a certain extent hookworm. Parasitic nematodes also infect farm livestock and companion animals. Resistance to currently deployed human and veterinary anthelmintic drugs is a growing problem. Therefore, new chemical anthelmintic lead compounds are urgently needed. One of the fastest routes to a novel therapeutic lead is to screen libraries of drugs which are either already approved for human use or have already been part of clinical trials. We have pursued this approach to anthelmintic lead discovery using an invertebrate automated phenotyping platform (INVAPP) for screening chemicals and the well-established nematode genetic model organism Caenorhabditis elegans. The 400 compound Medicines for Malaria Venture (MMV) Pandemic Response Box library was screened with each compound tested initially at 1.0x10-4 M. We identified 6 compounds (MMV1593515 (vorapaxar), MMV102270 (diphyllin), MMV1581032 (ABX464), MMV1580796 (rubitecan), MMV1580505 and MMV1593531) active in both an L1-L4 growth/motility assay and in an L4 motility assay. For vorapaxar, an EC50 of 5.7x10-7 M was observed, a value comparable to those of some commercial anthelmintics. Although not a parasite, the ease with which high-throughput screens can be pursued on the free-living nematode C. elegans makes this a useful approach to identify chemical leads and complements the often lower-throughput experiments on parasitic nematode models

Anthelmintic drug discovery: target identification, screening methods and the role of open science

Helminths, including cestodes, nematodes and trematodes, are a huge global health burden, infecting hundreds of millions of people. In many cases, existing drugs such as benzimidazoles, diethylcarbamazine, ivermectin and praziquantel are insufficiently efficacious, contraindicated in some populations, or at risk of the development of resistance, thereby impeding progress towards World Health Organization goals to control or eliminate these neglected tropical diseases. However, there has been limited recent progress in developing new drugs for these diseases due to lack of commercial attractiveness, leading to the introduction of novel, more efficient models for drug innovation that attempt to reduce the cost of research and development. Open science aims to achieve this by encouraging collaboration and the sharing of data and resources between organisations. In this review we discuss how open science has been applied to anthelmintic drug discovery. Open resources, including genomic information from many parasites, are enabling the identification of targets for new antiparasitic agents. Phenotypic screening remains important, and there has been much progress in open-source systems for compound screening with parasites, including motility assays but also high content assays with more detailed investigation of helminth physiology. Distributed open science compound screening programs, such as the Medicines for Malaria Venture Pathogen Box, have been successful at facilitating screening in diverse assays against many different parasite pathogens and models. Of the compounds identified so far in these screens, tolfenpyrad, a repurposed insecticide, shows significant promise and there has been much progress in creating more potent and selective derivatives. This work exemplifies how open science approaches can catalyse drug discovery against neglected diseases

New Technologies’ Promise to the Self and the Becoming of the Sacred: Insights from Georges Bataille’s Concept of Transgression

This article draws on Georges Bataille’s concept of transgression, a key element in Bataille’s theory of the sacred, to highlight structural implications of the way the self-empowerment ethos of new technologies suffuses the digital tracking culture. Pointing to the original conceptual stance of transgression, worked out against prohibition, I first argue that, beyond a critique of new technologies’ promise of self-empowerment as coming at the expense of an acknowledgement of the ultimate taboo—death—is the problem of the sanitizing of the tension between the crossing of the line of the symbolic taboo and prohibition; this undermines a “libidinal investment” towards the sacred, which is central in Bataille’s theory. Second, focussing on “eroticism”, since this embodies the emancipative potential of the Bataillean sacred, I argue that while a fear of eroticism marks out the digital technological realm, this is covered up by the blurring of boundaries between pleasure, fun and sex(iness) that currently governs our experience with technological devices

Population genetics of abnormal haemoglobins in Burkina Faso, West Africa

Les fréquences géniques des hémoglobines A (HbA), HbS et HbC, ont été étudiées au Burkina-Faso (BF) ainsi que dans une région avoisinante du Niger : Ayorou. La fréquence d'HbS est plus élevée dans le Sahel (Nord du BF et Ayorou) que dans la zone de savane. L'inverse est trouvé pour HbC. Les résultats essentiels de ce travail sont : (a) la confirmation d'un pic de la fréquence génique de HbC dans le centre du BF (plateau Mossi); (b) une corrélation négative entre les fréquences de HbS et HbC. Cavalli-Sforza et Bodmer ont observé que cette corrélation atteint une intensité significativement différente de celle qui est attendue, par suite de la relation allélique entre HbS et HbC; (c) la comparaison de ces résultats avec les données rassemblées par Livingstone montre une modification de la valeur sélective des différents génotypes au cours des dernières années. Les individus AS ont une valeur sélective plus basse et les AA et SS en ont une plus haute. Nos données sont favorables à l'hypothèse du relâchement partiel de la sélection dans cette région. (Résumé d'auteur

Alternaria species associated with early blight epidemics on tomato and other Solanaceae crops in northwestern Algeria

Early blight is a common disease of Solanaceae crops worldwide. The occurrence of Alternaria spp. was studied during three epidemics on tomato in northwestern Algeria. Alternaria was detected in more than 80 % of the diseased plant samples and accounted for more than 50 % of the total fungal isolates recovered from these samples. Morphological and molecular investigations revealed that small-spored isolates producing beaked conidia, i.e. belonging to the section alternaria, were prominent in most of the surveyed locations representing more than 80 % of the total Alternaria isolates in three locations (Mascara, Ain Témouchent and Sidi Belabbèsse). Based on their sporulation patterns they were recognized as A. alternata and A. tenuissima. Small-spored isolates producing conidia without beak and assigned to A. consortialis were also found at a low frequency (< 1 %). Large-spored isolates producing conidia ended by typical long beaks and identified as A. linariae (syn. A. tomatophila), A. solani and A. grandis were also recovered from all the sampled areas and represented 33.8 %, 6.3 % and 1.3 % of the total Alternaria isolates, respectively. Pathogenicity tests on tomato with a selection of 85 strains representative of the isolates collection revealed that all the tested isolates were able to produce extending lesions on inoculated leaves albeit with variable intensity. Large-spored species included the most aggressive isolates. Small-spored Alternaria, although less aggressive than large-spored Alternaria, had the ability to provoke brown necrotic spots and circumstantially developed synergistic interactions in mixed infections with moderately aggressive isolates of A. linariae

Status of QCD

I have been asked to discuss the status of QCD. It seems to me that there are three main points to be made about the present status of QCD: $\bullet$ QCD is right, and we can do many beautiful things with it. $\bullet$ There are several important concrete problems that lie just beyond the edge of our current understanding. $\bullet$ There are some foundational issues in QCD, and some recent developments, that may point toward entirely new directions. These points will, I believe, emerge quite clearly from the following more detailed discussion. The discussion will be in three parts. I'll first discuss elementary processes, then more complicated processes, and then finally foundational issues.Comment: 28 pages, use Phyzzx, figures available by FAX or mail on request, IASSNS-HEP-93/6