No description available.

Single-particle tracking is a technique that allows for quantitative analysis of the localization and movement of particles. In this technique, trajectories are constructed by determining and connecting the positions of individual particles from consecutive images. Recent advances have made it possible to track hundreds of particles in an individual cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time. Single-particle tracking can be used to study the diffusion of particles. Here, we use intracellular single-particle tracking and trajectory simulations to study the diffusion of the fluorescent protein mEos2 in living Escherichia coli cells. Our data are consistent with a simple model in which mEos2 diffuses normally at 13 µm2 s−1 in the E. coli cytoplasm. Our approach can be used to study the diffusion of intracellular particles that can be labeled with mEos2 and are present at high copy numbers. Single-particle tracking can also be used to determine whether an individual particle is bound or free if the free particle diffuses significantly faster than its binding targets and remains bound or free for a long time. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that, unlike bound subunits, free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with co-transcriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

abstract: Measles is a contagious, vaccine-preventable disease that continues to be the leading cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins. Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects. Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23). These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker. / Dissertation/Thesis / Masters Thesis Biology 2016

The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.

No description available.

The mRNA cap added to the 5’ end of nascent transcripts is required for the efficient gene expression in eukaryotes. In vertebrates, the guanosine cap is methylated at N7 position by RNMT, which is in complex with its activating subunit RAM. Additionally, the first and second transcribed nucleotides can be methylated at ribose O2 position by CMTR1 and CMTR2 respectively. The mRNA cap protects transcripts from degradation and recruits cap-binding factors to promote pre-mRNA processing, nuclear export and translation initiation. In mouse embryonic stem cells (mESCs), high levels of RAM maintain expression of pluripotency factors. Differentiation of mESCs to neural progenitors is accompanied by a suppression of RAM, resulting in downregulation of pluripotency factors and efficient formation of neural cells. Here, I demonstrated that the suppression of RAM during neural differentiation is promoted via ubiquitination and proteasomal degradation. Concurrently, neural differentiation is associated with an increase in CMTR1 expression, creating a developmental cap methyltransferase switch. Moreover, differentiation into endodermal and mesodermal lineages exhibited distinct changes in the mRNA capping enzymes expression. In mESCs, RAM promotes expression of translation-associated proteins and promotes global loading of mRNA on ribosomes. RAM contributes to the ESC-specific gene expression program, by maintaining optimal expression of pluripotency-associated transcripts and inhibiting expression of neural genes. Chromatin immunoprecipitation revealed that RAM, RNMT and CMTR1 promote binding of RNA polymerase II at gene loci. In RAM-repressed cells, RNA polymerase II binding was reduced at pluripotency-associated genes, but relatively increased at neural genes. Moreover, knock-down of RNMT or CMTR1 induced increased or decreased accumulation of RNA polymerase II at promoter proximal regions respectively. In naïve T cells, Rnmt or Cmtr1 conditional knock-outs caused downregulation of translation-related transcripts and upregulation of cell cycle transcripts. Furthermore, many transcripts were specifically dependent on RNMT or CMTR1 for expression, demonstrating distinct roles of these cap methyltransferases. Thus, the mRNA cap methylation emerges as an important regulator of pluripotency and differentiation, modulating gene expression at transcriptional and post-transcriptional levels.

Les systèmes toxine-antitoxine (TA) sont de petits modules génétiques largement présents dans les génomes bactériens. Ils codent pour une petite protéine toxique et une antitoxine. Ils sont classés en six types en fonction de la nature et du mode d'action de l'antitoxine. Ce travail a porté sur l'étude du type I, pour lequel l'antitoxine est un ARN antisens qui cible l'ARNm de la toxine afin de réprimer son expression. Au cours de cette thèse, nous avons étudié le système aapA3/IsoA3, codé sur le chromosome du pathogène gastrique humain Helicobacter pylori. À ce jour, la plupart des systèmes TA ont été étudiés à l'aide de systèmes d'expression artificiels, qui ne permettent pas de caractériser la régulation transcriptionnelle ou post-transcriptionnelle. En utilisant la létalité induite par l’expression chromosomique de la toxine obtenue en absence d’antitoxine, nous avons développé une sélection génétique de mutants suppresseurs révélés par séquençage haut-débit. Cette approche, appelée FASTBAC-Seq, nous a permis de cartographier une myriade de déterminants de toxicité localisés dans les régions codantes et non codantes du gène de la toxine AapA3. En particulier, certaines de ces mutations ont révélé l'existence de tige-boucles ARN transitoires qui agissent de manière co-transcriptionnelle pour empêcher l'initiation de la traduction pendant la synthèse de l'ARNm codant pour la toxine. Ces structures ARN métastables fonctionnelles sont nécessaires pour découpler les processus de transcription et de traduction et permettent la présence de ces gènes toxiques sur le chromosome bactérien. Bien que les ARNm non traduits deviennent rapidement instables, nos travaux ont également révélé l'existence de deux tige-boucles protectrices situées aux deux extrémités de l'ARNm. Ces structures secondaires empêchent des activités exonucléolytiques agissant en 5' et 3'. Dans l’ensemble, notre travail met en évidence les conséquences de la forte pression de sélection pour limiter l'expression des toxines sous laquelle évoluent les systèmes TA. Cela nous a permis de mieux comprendre l’influence du repliement secondaire des ARNm, non seulement lors de la régulation posttranscriptionnelle, mais aussi co-transcriptionnelle de l’expression de cette famille particulière de gènes. Ces caractéristiques de régulation basées sur l'ARN peuvent être exploitées à l'avenir pour des applications biotechnologiques (p. ex., production accrue de protéines par stabilisation d'ARNm) ou biomédicales (p.ex., développement de stratégies antimicrobiennes alternatives pour l'activation de la synthèse de toxines). / Toxin-antitoxin (TA) systems are small genetic modules widely present in bacterial genomes. They usually code for a small toxic protein and its cognate antitoxin and can be classified into six types depending on the nature and mode of action of the antitoxin. This work focuses on the study of type I, for which the antitoxin is an antisense RNA that targets the toxin mRNA to inhibit its expression. We characterized the aapA3/IsoA3 system, encoded on the chromosome of the human gastric pathogen Helicobacter pylori. To date, most TAs have been studied using artificial expression systems, which do not allow the characterization of transcriptional or post-transcriptional regulation. Taking advantage of the lethality induced by the toxin chromosomal expression in the absence of antitoxin, we developed a high-throughput genetic selection of suppressor mutations revealed by Next-Generation Sequencing. This approach, named FASTBAC-Seq, allowed us to map a myriad of toxicity determinants located in both, coding and noncoding regions, of the aapA3 toxic gene. More precisely, some suppressor mutations revealed the existence of transient RNA hairpins that act co-transcriptionally to prevent translation initiation while the toxinencoding mRNA is being made. Such functional RNA metastable structures are essential to uncouple the transcription and translation processes and allow the presence of these toxic genes on bacterial chromosomes. Although untranslated mRNAs become rapidly unstable, our work also revealed the presence of two protective stem-loops located at both mRNA ends that prevent from both, 5’ and 3’ exonucleolytic activity. Altogether, our work evidenced the consequences of the strong selection pressure to silence toxin expression under which the TAs evolve, and highlighted the key role of mRNA folding in the co- and post-transcriptional regulation of this family of genes. These RNA-based regulatory mechanisms may be exploited in the future for biotechnological (e.g., increased protein production through mRNA stabilization) or biomedical (e.g., development of alternative antimicrobial strategies aiming at the activation of toxin synthesis) applications.

Continuous Curatorial Conversations is a practice-led exploration of conversation, both as a medium and as a tool for capturing supplementary histories of the curatorial. The primary question of this research project is how the medium of conversation can be explored to write supplementary histories of the curatorial which thus far have been omitted from extant publications on the subject. Three important sub questions guide this exploration. First, what is and has been the role of conversation within the curatorial? What are the possibilities and limitations within the medium of conversation? What roles do conviviality and hospitality play within the process of conversation? This thesis reflects upon a series of curated projects that explore the sp/pl/ace for curatorial conversation and also reviews a collection of one-to-one recorded conversations conducted by the author, including conversations with Alfredo Cramerotti, Hedwig Fijen, Mel Gooding, William Furlong and Sarah Lowndes. Sites of fieldwork include: the 54th Venice Biennale; Manifesta 8, The European Biennial of Contemporary Art; and Glasgow International Festival of Visual Art 2012. Through these projects and related recordings it unpicks the norms and possibilities of what and when one can record on the subject of the curatorial. The hypothesis of this study is that a great deal of curatorial activity is locked up in conversation, yet a disproportion makes it to the pages of the history of the field. Furthermore, in its clean transcribed form it misrepresents the fragility and nuance of the original exchange. The theoretical context of this research looks at Nicolas Bourriaud’s notion of Relational Aesthetics, the writing of Maria Lind and Paul O’Neill, with a focus on Audio Arts. A new methodology relating to curatorial conversation and its recording has therefore been identified as ‘critical conviviality’. The writing relating to Continuous Curatorial Conversations research takes the form of four books. The book ‘An Introduction’ comprises the PhD thesis and sits next to a bespoke online platform www.continuous-curatorial-conversations.org which hosts a selection of audio recordings collated during the research process. The books ‘Continuous’, ‘Curatorial’, and ‘Conversations’ unpack the lineage and context of Alexandra C.M. Ross’s practice and projects conducted during her research and are to be read in no strict order. The new knowledge resulting from this thesis and relating practice is the attention to the subtleties of conversation and its capture as it relates to the instigation, recording and presentation of semi-private matters in semi-public contexts.

Les dinoflagellés sont des eucaryotes unicellulaires retrouvés dans la plupart des écosystèmes aquatiques du globe. Ces organismes amènent une contribution substantielle à la production primaire des océans, soit en tant que membre du phytoplancton, soit en tant que symbiontes des anthozoaires formant les récifs coralliens. Malheureusement, ce rôle écologique majeur est souvent négligé face à la capacité de certaines espèces de dinoflagellés à former des fleurs d'eau, parfois d'étendue et de durée spectaculaires. Ces floraisons d'algues, communément appelées "marées rouges", peuvent avoir de graves conséquences sur les écosystèmes côtiers, sur les industries de la pêche et du tourisme, ainsi que sur la santé humaine. Un des facteurs souvent corrélé avec la formation des fleurs d'eau est une augmentation dans la concentration de nutriments, notamment l’azote et le phosphore. Le nitrate est un des composants principaux retrouvés dans les eaux de ruissellement agricoles, mais également la forme d'azote bioaccessible la plus abondante dans les écosystèmes marins. Ainsi, l'agriculture humaine a contribué à magnifier significativement les problèmes associés aux marées rouges au niveau mondial. Cependant, la pollution ne peut pas expliquer à elle seule la formation et la persistance des fleurs d'eau, qui impliquent plusieurs facteurs biotiques et abiotiques. Il est particulièrement difficile d'évaluer l'importance relative qu'ont les ajouts de nitrate par rapport à ces autres facteurs, parce que le métabolisme du nitrate chez les dinoflagellés est largement méconnu. Le but principal de cette thèse vise à remédier à cette lacune. J'ai choisi Lingulodinium polyedrum comme modèle pour l'étude du métabolisme du nitrate, parce que ce dinoflagellé est facilement cultivable en laboratoire et qu'une étude transcriptomique a récemment fourni une liste de gènes pratiquement complète pour cette espèce. Il est également intéressant que certaines composantes moléculaires de la voie du nitrate chez cet organisme soient sous contrôle circadien. Ainsi, dans ce projet, j'ai utilisé des analyses physiologiques, biochimiques, transcriptomiques et bioinformatiques pour enrichir nos connaissances sur le métabolisme du nitrate des dinoflagellés et nous permettre de mieux apprécier le rôle de l'horloge circadienne dans la régulation de cette importante voie métabolique primaire. Je me suis tout d'abord penché sur les cas particuliers où des floraisons de dinoflagellés sont observées dans des conditions de carence en azote. Cette idée peut sembler contreintuitive, parce que l'ajout de nitrate plutôt que son épuisement dans le milieu est généralement associé aux floraisons d'algues. Cependant, j’ai découvert que lorsque du nitrate était ajouté à des cultures initialement carencées ou enrichies en azote, celles qui s'étaient acclimatées au stress d'azote arrivaient à survivre près de deux mois à haute densité cellulaire, alors que les cellules qui n'étaient pas acclimatées mourraient après deux semaines. En condition de carence d'azote sévère, les cellules arrivaient à survivre un peu plus de deux semaines et ce, en arrêtant leur cycle cellulaire et en diminuant leur activité photosynthétique. L’incapacité pour ces cellules carencées à synthétiser de nouveaux acides aminés dans un contexte où la photosynthèse était toujours active a mené à l’accumulation de carbone réduit sous forme de granules d’amidon et corps lipidiques. Curieusement, ces deux réserves de carbone se trouvaient à des pôles opposés de la cellule, suggérant un rôle fonctionnel à cette polarisation. La deuxième contribution de ma thèse fut d’identifier et de caractériser les premiers transporteurs de nitrate chez les dinoflagellés. J'ai découvert que Lingulodinium ne possédait que très peu de transporteurs comparativement à ce qui est observé chez les plantes et j'ai suggéré que seuls les membres de la famille des transporteurs de nitrate de haute affinité 2 (NRT2) étaient réellement impliqués dans le transport du nitrate. Le principal transporteur chez Lingulodinium était exprimé constitutivement, suggérant que l’acquisition du nitrate chez ce dinoflagellé se fondait majoritairement sur un système constitutif plutôt qu’inductible. Enfin, j'ai démontré que l'acquisition du nitrate chez Lingulodinium était régulée par la lumière et non par l'horloge circadienne, tel qu'il avait été proposé dans une étude antérieure. Finalement, j’ai utilisé une approche RNA-seq pour vérifier si certains transcrits de composantes impliquées dans le métabolisme du nitrate de Lingulodinium étaient sous contrôle circadien. Non seulement ai-je découvert qu’il n’y avait aucune variation journalière dans les niveaux des transcrits impliqués dans le métabolisme du nitrate, j’ai aussi constaté qu’il n’y avait aucune variation journalière pour n’importe quel ARN du transcriptome de Lingulodinium. Cette découverte a démontré que l’horloge de ce dinoflagellé n'avait pas besoin de transcription rythmique pour générer des rythmes physiologiques comme observé chez les autres eukaryotes. / Dinoflagellates are unicellular eukaryotes found in most aquatic ecosystems of the world. They are major contributors to carbon fixation in the oceans, either as free-living phytoplankton or as symbionts to corals. Dinoflagellates are also infamous because some species can form spectacular blooms called red tides, which can cause serious damage to ecosystems, human health, fisheries and tourism. One of the factors often correlated with algal blooms are increases in nutrients, particularly nitrogen and phosphorus. Nitrate is one of the main components of agricultural runoffs, but also the most abundant bioavailable form of nitrogen in marine environments. Thus, agricultural activities have globally contributed to the magnification of the problems associated with red tides. However, bloom formation and persistence cannot be ascribed to human pollution alone, because other biotic and abiotic factors are at play. Particularly, it is difficult to assess the relative importance of nitrate addition over these other factors, because nitrate metabolism in dinoflagellate is mostly unknown. Filling part of this gap was the main goal of this thesis. I selected Lingulodinium polyedrum as a model for studying nitrate metabolism, because this dinoflagellate can easily be cultured in the lab and a recent transcriptomic survey has provided an almost complete gene catalogue for this species. It is also interesting that some molecular components of the nitrate pathway in this organism have been reported to be under circadian control. Thus, in this project, I used physiological, biochemical, transcriptomic and bioinformatic approaches to enrich our understanding of dinoflagellate nitrate metabolism and to increase our appreciation of the role of the circadian clock in regulating this important primary metabolic pathway. I first studied the particular case of dinoflagellate blooms that occur and persist in conditions of nitrogen depletion. This idea may seems counterintuitive, because nitrogen addition rather than depletion, is generally associated with algal blooms. However, I discovered that when nitrate was added to nitrogen-deficient or nitrogen-sufficient cultures, those that had been acclimated to nitrogen stress were able to survive for about two months at high cell densities, while non-acclimated cells died after two weeks. In conditions of severe nitrogen limitation, cells could survive a little bit more than two weeks by arresting cell division and reducing photosynthetic rates. The incapacity to synthesize new amino acids for these deprived cells in a context of on-going photosynthesis led to the accumulation of reduced carbon in the form of starch granules and lipid bodies. Interestingly, both of these carbon storage compounds were polarized in Lingulodinium cells, suggesting a functional role. The second contribution of my thesis was to identify and characterize the first nitrate transporters in dinoflagellates. I found that in contrast to plants, Lingulodinium had a reduced suite of nitrate transporters and only members of the high-affinity nitrate transporter 2 (NRT2) family were predicted to be functionally relevant in the transport of nitrate. The main transporter was constitutively expressed, which suggested that nitrate uptake in Lingulodinium was mostly a constitutive process rather than an inducible one. I also discovered that nitrate uptake in this organism was light-dependent and not a circadian-regulated process, as previously suggested. Finally, I used RNA-seq to verify if any transcripts involved in the nitrate metabolism of Lingulodinium were under circadian control. Not only did I discovered that there were no daily variations in the level of transcripts involved in nitrate metabolism, but also that there were no changes for any transcripts present in the whole transcriptome of Lingulodinium. This discovery showed that the circadian timer in this species did not require rhythmic transcription to generate biological rhythms, as observed in other eukaryotes.

As depicted in Chapter I, 2-oxoglutarate- (2OG) dependent oxygenases are ubiquitous in living systems and display a wide range of cellular functions, spanning metabolism, transcription, and translation. Although functionally diverse, the 2OG oxygenases share a high degree of structural similarities between their catalytic sites. From a medicinal chemistry point of view, the combination of biological diversity and structural similarity presents a rather challenging task for the development of selective small molecules for functional studies in vivo. The non-selective metal chelator 8-hydroxyquinoline (8HQ) was used as a template for the generation of tool compound <b>I</b> for the KDM4 subfamily of histone demethylases via application of the Betti reaction. Structural analogue <b>II</b> was used as the corresponding negative control (Figure A). These compounds were characterised in vitro against a range of 2OG oxygenases and subsequently used for studies in cells. <b>I</b> displays selectivity for KDM4 and increases the level of the H3K9me3 histone mark in cells. It has an effect on the post-translational modification pattern of histone H3, but not other histones, and reduces the viability of lung cancer cells, but not normal lung cells, derived from the same patient. <b>I</b> also stabilises hypoxia-inducable factor HIF in cells via a mechanism which seems to be independent from prolyl hydroxylase inhibition. This work is described in Chapters II and III. The chemical biology research in epigenetics is complemented by qualitative analysis conducted in the social sciences at Said Business School. With a global view on how innovation occurs and may actively be fostered, Chapter IV focuses on the potential of epigenetics in drug discovery and how this process may actively be promoted within the framework of open innovation. Areas of focus include considerations of incremental and disruptive technology; how to claim, demarcate, and control the market; how knowledge brokering occurs; and insights about process, management, organisation, and culture of open innovation. In contrast to the open-skies approach adopted for the development of a tool compound in Chapters II and III, a focused-library approach was taken for the generation of a tool compound for the OGFOD1 ribosomal prolyl hydroxylase. The development of a suitable in vitro activity assay for OGFOD1 in Chapter V enabled the development of lead compound <b>III</b> in Chapter VI. <b>III</b> is selective for OGFOD1 against the structurally closely related prolyl hydroxylase PHD2.