La rétine est un tissu spécialisé dans le traitement de l'information visuelle par l'intermédiaire des photorécepteurs, cônes et bâtonnets, et des neurones de deuxième ordre, les cellules bipolaires et les cellules ganglionnaires dont les axones forment le nerf optique. Notre groupe s'intéresse à élucider les mécanismes génétiques impliqués dans les maladies rares stationnaires, comme dans la cécité nocturne congénitale stationnaire (CNCS), ou progressives comme dans la dystrophie de type bâtonnet-cône (DBC). Cette thèse apporte de nombreuses connaissances sur la physiologie rétinienne. D'une part, nous avons identifié GPR179, un nouveau gène impliqué dans la CNCS complète, étudié la localisation de la protéine et la physiopathologie des protéines mutantes. Nous avons également créé et caractérisé fonctionnellement un nouveau modèle souris invalidé pour GPR179 qui nous a permis de mieux approcher la première synapse rétinienne entre les photorécepteurs et les cellules bipolaires adjacentes. D'autre part, nous avons caractérisé le génotype et le phénotype de l'un des modèles les plus utilisés de la DBC, le rat P23H. Nous avons ensuite développé une approche d'édition génomique pour invalider les mutants RHO ayant un effet dominant négatif en testant in vitro, ex vivo et in vivo les meganucleases, TALEN (Transcription Activator-Like Effector Nuclease) puis le système CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9). / The first steps in vision occur in the retina when rod and cone photoreceptors transform light into a biochemical signal, which gets processed by bipolar cells, ganglion cells and finally by the brain. Our group investigates genetic causes and mechanisms involved in inherited stationary and progressive retinal diseases as congenital stationary night blindness (CSNB), and rod-cone dystrophy (RCD), also called retinitis pigmentosa. This thesis gives several insights on the retinal physiology. On one hand, we identified GPR179, a new gene mutated in complete CSNB, studied the localization and the physiopathology of missense and splice-site mutations. We also delivered a new knock-out mouse model which we functionally characterized, and studied GPR179 partners to provide a better understanding of the first visual synapse between photoreceptors and ON-bipolar cells. On the other hand, we genotypically and phenotypically characterized one of the most popular RCD model, the P23H rat model. There is currently no treatment for RCD and different therapeutic strategies are under investigation. We wanted to deliver the basis for a genome editing approach for RHO mutations, acting as a dominant negative effect, which cannot be addressed by current gene replacement strategies. We opened the field by performing in vitro, ex vivo and in vivo genome editing experiments using meganucleases, TALEN (Transcription Activator-Like Effector Nuclease) and finally CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) and revealed how challenging the setting of genome editing strategies was.

Abstract Heart rate variability (HRV) is used in the assessment of cardiovascular health. However, often contradictory results have impeded the efficient use of HRV in clinical practice. HRV signals can contain artifacts leading to errors in the interpretation of HRV results. Various methods have been used for artifact editing, but there is relatively little information on how the actual editing can influence the HRV measures. The main aim of this thesis was to improve the reliability of HRV analysis by concentrating on the HRV signal preprocessing methods. The effects of three editing methods on the HRV of short (512 R-R) and long-term (24-hour) R-R interval data were studied with non-edited and edited data from healthy subjects (n=10) and patients with acute myocardial infarction (AMI) (n=10). The effects of ectopic beats on short (α1) and long-term (α2) fractal scaling exponents were studied by inserting artificial ectopic beats into the HRV signals of 20 healthy subjects and 20 AMI patients. The prognostic significance of edited and non-edited α1 and α2 was studied in random elderly (n=84) and post-AMI (n=84) populations. A new method to quantify respiratory sinus arrhythmia (RSA) was developed based on the HRV signals of 13 healthy subjects. A new measure, the RSA index, was defined to evaluate the risk to sudden cardiac death (SCD) in 1631 AMI patients. Lastly, a new algorithm was developed in order to edit heart rate (HR) turbulence occurring immediately after a ventricular premature beat (VPB). The effects of HR turbulence editing on the HRV analysis were studied in 267 AMI patients. Editing had distinct effects on the HRV analysis depending on the editing method and data type. Deletion editing was found to be unsuitable for the HRV spectrum analysis. There was no universal editing method for the time and frequency domain HRV analyses. Unedited ectopic beats increased the randomness of short-term R-R interval dynamics, especially in AMI patients. However, unedited α1 differed significantly between survivors and those who died during the follow-up. Ectopic beats do not necessarily need to be edited if fractal analysis is used in the risk evaluation. A depressed RSA index was found to be a strong predictor of SCD but a weak predictor of non-SCD in AMI patients. Editing of HR turbulence affected differently the various HRV measures. ULF and VLF components were most clearly influenced by HR turbulence removal. The amount of VBPs had an important impact on the results. When the VBPs/hour were >50, ULF and VLF were >30% lower after turbulence removal. The results of this thesis highlight the importance of editing the erroneous or irrelevant R-R interval oscillation in an HRV analysis. The careful choice of preprocessing method is essential if one wishes to obtain reliable HRV analyses for clinical purposes.

In this paper, I will catalog and describe my process involved in the creation of my thesis film If Not Now. In the main body of the paper I will cover the topics of Writing, Casting, Directing, Production Design, Cinematography, Editing, and Sound, as well as Technology and Workflow. Special emphasis will be placed on Writing, Directing, Editing, and Sound. The Analysis section will discuss the overall effectiveness of my goals to communicate a story about self-identity and community, as well as the film's artistic merit and quality.

M.Tech. / RNA interference (RNAi) is a gene silencing mechanism that uses short RNA duplexes to block gene expression. This mechanism has been widely explored to determine functions of genes. Furthermore, this phenomenon has been used to silence unwanted genes such as viral genes. RNAi has been successfully employed in non-mammalian organisms such as plants, where long dsRNAs (more than 30 bp) have been used without inducing non-specific effects. However, in mammalian cells, cytoplasmic dsRNAs of more than 30 bp trigger non-specific induction of many genes, which may result from the activation of dsRNA-dependent protein kinase (PKR) and 2’,5’-oligoadenylate synthetase (2’,5’-OAS), via the interferon response pathway. In this study, we describe a novel and simple strategy to overcome nonspecific effects induced by longer RNA duplexes. This strategy uses sodium bisulphite which is a mutagen that deaminates cytosine residue to uracil residues in order to introduce mutations in the sense strand of the duplex. Introduction of these mutations results in the formation of G:U pairings between the sense and antisense strands of the long hairpin RNA. RNA duplexes with mismatches have been shown to be able to prevent interferon induction in mammalian cells. According to the obtained results, long hairpins RNA with and without mismatches were unable to inhibit the expression of the target region, which was the U5 region of the HIV-1 subtype C LTR. The U5 region of the LTR is actively involved in the reverse transcription of HIV-1. Therefore silencing of this region would have led to the inhibition or reverse transcription blockage. Furthermore, data showed that the interferon response was induced when using these long hairpin RNA duplexes. Due to the sensitivity of mammalian cells, the action of sodium bisulphite could have stimulated certain genes of the interferon pathway. Even though hairpins constructed in this study were unable to prevent the induction of the interferon response pathway and also could not silence the target, this strategy of using sodium bisulphite has a great potential as shown by its ability to induce changes in cytosine residues and leaving other nucleotides unchanged.

Dans cette thèse, nous abordons deux problèmes soulevés par les systèmes distribués décentralisés - le placement de réseaux logiques de façon compatible avec le réseau physique sous-jacent et la construction de cohortes d'éditeurs pour dans les systèmes d'édition collaborative. Bien que les réseaux logiques (overlay networks) été largement étudiés, la plupart des systèmes existant ne prennent pas ou prennent mal en compte la topologie du réseau physique sous-jacent, alors que la performance de ces systèmes dépend dans une grande mesure de la manière dont leur topologie logique exploite la localité présente dans le réseau physique sur lequel ils s'exécutent. Pour résoudre ce problème, nous proposons dans cette thèse Fluidify, un mécanisme décentralisé pour le déploiement d'un réseau logique sur une infrastructure physique qui cherche à maximiser la localité du déploiement. Fluidify utilise une stratégie double qui exploite à la fois les liaisons logiques d'un réseau applicatif et la topologie physique de son réseau sous-jacent pour aligner progressivement l'une avec l'autre. Le protocole résultant est générique, efficace, évolutif et peut améliorer considérablement les performances de l'ensemble. La deuxième question que nous abordons traite des plates-formes d'édition collaborative. Ces plates-formes permettent à plusieurs utilisateurs distants de contribuer simultanément au même document. Seuls un nombre limité d'utilisateurs simultanés peuvent être pris en charge par les éditeurs actuellement déployés. Un certain nombre de solutions pair-à-pair ont donc été proposées pour supprimer cette limitation et permettre à un grand nombre d'utilisateurs de collaborer sur un même document sans aucune coordination centrale. Ces plates-formes supposent cependant que tous les utilisateurs d'un système éditent le même jeu de document, ce qui est peu vraisemblable. Pour ouvrir la voie à des systèmes plus flexibles, nous présentons, Filament, un protocole décentralisé de construction de cohorte adapté aux besoins des grands éditeurs collaboratifs. Filament élimine la nécessité de toute table de hachage distribuée (DHT) intermédiaire et permet aux utilisateurs travaillant sur le même document de se retrouver d'une manière rapide, efficace et robuste en générant un champ de routage adaptatif autour d'eux-mêmes. L'architecture de Filament repose sur un ensemble de réseaux logiques auto-organisées qui exploitent les similarités entre jeux de documents édités par les utilisateurs. Le protocole résultant est efficace, évolutif et fournit des propriétés bénéfiques d'équilibrage de charge sur les pairs impliqués. / In this thesis, we address two issues in the area of decentralized distributed systems: network-aware overlays and collaborative editing. Even though network overlays have been extensively studied, most solutions either ignores the underlying physical network topology, or uses mechanisms that are specific to a given platform or applications. This is problematic, as the performance of an overlay network strongly depends on the way its logical topology exploits the underlying physical network. To address this problem, we propose Fluidify, a decentralized mechanism for deploying an overlay network on top of a physical infrastructure while maximizing network locality. Fluidify uses a dual strategy that exploits both the logical links of an overlay and the physical topology of its underlying network to progressively align one with the other. The resulting protocol is generic, efficient, scalable and can substantially improve network overheads and latency in overlay based systems. The second issue that we address focuses on collaborative editing platforms. Distributed collaborative editors allow several remote users to contribute concurrently to the same document. Only a limited number of concurrent users can be supported by the currently deployed editors. A number of peer-to-peer solutions have therefore been proposed to remove this limitation and allow a large number of users to work collaboratively. These decentralized solution assume however that all users are editing the same set of documents, which is unlikely to be the case. To open the path towards more flexible decentralized collaborative editors, we present Filament, a decentralized cohort-construction protocol adapted to the needs of large-scale collaborative editors. Filament eliminates the need for any intermediate DHT, and allows nodes editing the same document to find each other in a rapid, efficient and robust manner by generating an adaptive routing field around themselves. Filament's architecture hinges around a set of collaborating self-organizing overlays that utilizes the semantic relations between peers. The resulting protocol is efficient, scalable and provides beneficial load-balancing properties over the involved peers.

This thesis draws the line of a rise and a fall, an ironic pattern whereby the English stage of the long eighteenth century, in its relation to Shakespeare in particular, first acquired powerful influence, and then, through the very effects of that power, lost it. It also shows what contemporary literary criticism might learn from the activities that constitute this arc of evolution. My first chapter interrogates the relationship between text and performance in vernacular writings about acting and editing from the death of Betterton in 1710 to the rise of Garrick in the middle decades of the century. From the status of a distinct tradition, performance comes to rely on text as a basis for the intimate, personal engagement with Shakespeare believed necessary to the work of the sentimental actor. Such a reliance grants the performer new potential as a literary critic, but also prepares a fall. The performer becomes another kind of reader, and so is open to accusations of reading badly. My second chapter analyses the evolving definition of Shakespeare as a dramatic author from Samuel Johnson onwards. An untheatrical definition of the dramatic (Johnson's) is answered by one which recognises the power and vitality of the stage, especially in its representation of sympathetic character (Montagu and Kenrick). Yet that very recognition leads to a set of altered critical priorities in which the theatre is, once more, relegated (Morgann and Richardson). My third and fourth chapters consider the practices and critical implications of theatrical performance of Shakespeare during Garrick's career. I focus on the acting of emotion, the portrayal of what Aaron Hill called 'the very Instant of the changing Passion', and show that performance of this time, attentive to the striking moment and the transitions that power it, required from the actor both attention to the text and preternatural control over his own emotions. In return, it allowed Garrick and others to claim a special affinity with Shakespeare and to capture the public's attention, both in the theatre and outside it. Yet this situation, that of 'twin stars', does not last. French and German responses to English acting, the concern of my last chapter, show its decline particularly well. They also, however, show the power that existed in such a union between page and stage, and equal weight is given in both my third and my fourth chapter to how the theatrical-literary insights of eighteenth-century critical culture might also illuminate modern approaches.

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abstract: Alzheimer’s disease (AD) affects over 5 million individuals each year in the United States. Furthermore, most cases of AD are sporadic, making it extremely difficult to model and study in vitro. CRISPR/Cas9 and base editing technologies have been of recent interest because of their ability to create single nucleotide edits at nearly any genomic sequence using a Cas9 protein and a guide RNA (sgRNA). Currently, there is no available phenotype to differentiate edited cells from unedited cells. Past research has employed fluorescent proteins bound to Cas9 proteins to attempt to enrich for edited cells, however, these methods are only reporters of transfection (RoT) and are no indicative of actual base-editing occurring. Thus, this study proposes a transient reporter for editing enrichment (TREE) and Cas9-mediated adenosine TREE (CasMasTREE) which use plasmids to co-transfect with CRISPR/Cas9 technologies to serve as an indicator of base-editing. Specifically, TREE features a blue fluorescent protein (BFP) mutant that, upon a C-T conversion, changes the emission spectrum to a green fluorescent protein (GFP). CasMasTREE features a mCherry and GFP protein separated by a stop codon which can be negated using an A-G conversion. By employing a sgRNA that targets one of the TREE plasmids and at least one genomic site, cells can be sorted for GFP(+) cells. Using these methods, base-edited isogenic hiPSC line generation using TREE (BIG-TREE) was created to generate isogenic hiPSC lines with AD-relevant edits. For example, BIG-TREE demonstrates the capability of converting Apolipoprotein E (APOE), a gene associated with AD-risk development, wildtype (3/3) into another isoform, APOE2/2, to create isogenic hiPSC lines. The capabilities of TREE are vast and can be applied to generate various models of diseases with specific genomic edits. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2020

The CRISPR-Cas9 gene editing system gives researchers the ability to manipulate and edit DNA with unprecedented ease and precision. It was discovered in bacteria as part of their adaptive immune system, but has been reengineered to target any double stranded DNA. This burgeoning molecular tool has created great excitement as scientists are rapidly adopting it to study fields including human gene therapy, disease modeling, agriculture, gene drive in mosquitos, and many others. This paper will explore the potential impact of CRISPR-Cas9 in human therapeutics. Specifically, the potential of CRISPR-Cas9 to treat Duchenne Muscular Dystrophy will be examined. In several ways, this debilitating degenerative disease is an ideal candidate for gene-editing with CRISPR-Cas9. Recent progress in the lab has demonstrated the gene editing system’s ability to rescue dystrophin protein levels in vivo. Although CRISPR-Cas9 holds great promise for previously incurable diseases, there are still many limitations that must be overcome before the gene editing system can be used in patients. This paper will discuss these barriers as well as recent advancements to overcome them.