No description available.

abstract: The research question explored in this thesis is how CRISPR mediated editing is influenced by artificially opened chromatin in cells. Closed chromatin poses a barrier to Cas9 binding and editing at target genes. Synthetic pioneer factors (PFs) are a promising new approach to artificially open condensed heterochromatin allowing greater access of target DNA to Cas9. The Haynes lab has constructed fusions of enzymatic chromatin-modifying domains designed to remodel chromatin and increase Cas9 editing efficiency. With a library of PFs available, this research focuses on analyzing the behavior of Cas9 in chromatin that has been artificially opened by PFs. The types and frequency of INDELs (insertions & deletions) were determined after non-homologous end joining (NHEJ) in PF and Cas9-treated cells using quantitative Sanger sequencing and Synthego’s ICE software. Furthermore, NOME-seq analysis was carried out to map nucleosome position in PF and Cas9 treated cells. Although this experiment was unsuccessful, the heat map generated with data obtained from Synthego ICE predicts a possible presence of nucleosome in the vicinity suggesting that perhaps a fully open chromatin state was not achieved. Linear Regression analysis with certain assumptions confirms that with the increase in distance downstream of cut-site, the editing frequency decreases exponentially. Nevertheless, further experimental work should be carried out to investigate this hypothesis. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2019

L’arrivée de la traduction automatique (TA) bouleverse les pratiques dans l’industrie de la traduction et soulève par le fait même des questions sur l’utilité et l’utilisation de cette technologie. Puisque de nombreuses études ont déjà porté sur son utilisation dans un contexte où elle est imposée aux traducteurs, nous avons choisi d’adopter la perspective toute particulière des traducteurs pour examiner son utilité et son utilisation volontaire dans l’environnement de traduction (ET). Notre recherche visait à répondre à trois grandes questions : les traducteurs utilisent-ils la TA dans leurs pratiques? Les traducteurs croient-ils que les données de sortie sont utiles et utilisables? Les traducteurs utilisent-ils concrètement ces données de sortie dans le processus de traduction? Pour répondre à ces questions, nous avons d’abord diffusé un sondage à grande échelle afin de mesurer l’utilisation de la TA en tant qu’outil, de recueillir des données sur le profil des répondants et d’évaluer leur perception d’utilité par rapport aux données de sortie et aux divers types de phénomènes que nous avions identifiés au préalable avec l’aide de deux traducteurs professionnels. Ensuite, nous avons réalisé une expérience avec d’autres traducteurs professionnels où nous leur avons demandé de procéder à la traduction de courts segments et avons examiné s’ils utilisaient ou non ces données de sortie pour produire leur traduction. Notre analyse était fondée sur le principe que, dans un contexte d’utilisation volontaire, l’utilisation d’une donnée de sortie permet d’induire une perception d’utilité et d’examiner, par le fait même, l’utilité de la TA. Dans l’ensemble, nous avons trouvé que la TA n’est habituellement pas utilisée de façon volontaire, que les perceptions des traducteurs sont peu favorables à une telle utilisation, que la perception des traducteurs quant à l’utilité des données de sortie est aussi plutôt négative, mais que les données de sortie semblent être beaucoup plus utiles et utilisables que ce que ne pourraient le croire les traducteurs, car ils les ont généralement utilisées dans le processus de traduction. Nous avons aussi examiné les facteurs déterminants de l’utilité et de l’utilisation de la TA et des données de sortie.

Intrigued by the English language and its far-reaching applications worldwide as a standard means of communication, I begin my disquisition with a focus on the meaning and derivation of grammar and its place in the trivium of ancient and modern study. I stress the need to reemphasize college-level instruction in grammar and mechanics as a complement to rhetoric and logic by studying and teaching editing, which involves semantics, syntax, phonology, morphology, conventions, mechanics (spelling, punctuation and format), in writing centers and classrooms. Noting growing nationwide illiteracy, I research the pedagogies and writing of experts in the field of rhetoric and composition to develop and share a balanced philosophy of learning and teaching the art, science, and mathematics of writing with a focusing on conscientiously creating sentences with an Isocratean sense of perfection. Continually learning methods to reintroduce grammar in a novel way, I present antidotal information, statistics, and expert opinions and interpretations of pedagogists and rhetoricians of both sides of the Grammar Debate, a polemic over the effectiveness or ineffectiveness of teaching grammar. From my experience as a composition teacher at the University of South Florida and Hillsborough Community College in Florida, I present suggestions from students, who through their questions and overwhelming documented requests for grammar help and attention to sentence-level concerns, helped me rediscover myself through the reflective and recursive aspects of writing. Teaching students Standard English for academic discourse and for writing with computers across the curriculum, I share the power of words and explain the negative effects of errors and how to eliminate the serious ones. Graphs and tables of data collected from conference information forms and questionnaires filled out by students in writing centers or classrooms reveal the objectives and viewpoints of students, those whom institutions and teachers serve. Having developed a "polypedagogy," I share the knowledge I have gathered from others with innovative and creative ideas for teaching the historically boring or abstract subject of grammar.

The fern genus Pteridium comprises a number of closely related species distributed throughout the world. Collectively they are called bracken ferns and have historically been treated as a single species, Pteridium aquilinum. Bracken is notorious as a toxic weed that colonizes open fields and poisons livestock. Bracken is also easily cultured and has become one of the most intensively studied ferns. Bracken has been used as a model system for the study of the fern life cycle, fern gametophyte development, the pheromonal mechanism of sex determination, toxicology, invasion ecology, and climate change. This dissertation places bracken within a global evolutionary perspective and establishes bracken as an emerging system for evolutionary genomics in ferns. Bracken samples from around the world were examined for chloroplast DNA variation to infer historical phylogenetic and biogeographic evolutionary events. New high-throughput DNA sequencing technologies and bioinformatic approaches were used to determine the complete chloroplast genome sequence in bracken, to identify novel RNA editing sites in chloroplast transcripts, and to identify gene sequences that are expressed in the gametophyte stage of the fern life cycle. These data represent an important genomic resource in ferns and were examined within a functional and evolutionary perspective. Several novel approaches and analyses were developed in the course of this research. Results presented in this dissertation provide novel insights into fern biology and land plant evolution.

With advancements in CRISPR-cas9 broadening the potential paths for clinical usage of genetic editing, conversations about genetic editing have grown to outside simply scientific communities and into mainstream conversations. This study focuses specifically on Christian discourse of genetic editing and locates four major tensions for many Christians when they think about genetic editing: beginning of life, Creator-human relationship, imago Dei, and stewardship. With these major concerns in mind, I identify epigenetics, somatic cell genetic editing, and in vivo genetic editing research as important research paths to pursue as they can potentially produce techniques that more Christian individuals would feel comfortable using. I pursue one of these paths and conclude with an experimental proposal for an analysis of in vivo and in vitro CIRSPR-Cas9 efficiency in regards to on- and off-target rates.

Les β-hémoglobinopathies (β-thalassémies et drépanocytose) sont des anémies génétiques qui touchent des milliers de nouveaux nés chaque année dans le monde. Ces maladies sont causées par des mutations affectant l'expression de l'hémoglobine chez l'adulte. Le seul traitement disponible est la transfusion sanguine à vie, associée à une chélation du fer. Pour les patients les plus touchés, la greffe de cellule souche hématopoïétique (CSH) demeure le seul traitement curatif. Néanmoins, la transplantation autologue de cellules souches génétiquement corrigées représente une alternative thérapeutique pour les patients dépourvus de donneur compatible. Certaines délétions naturelles comprenant les gènes de la β- et δ- globine dans le locus de l'hémoglobine sont corrélées à une persistance de l'expression de l'hémoglobine fœtale (HPFH) à l'âge adulte. Ainsi il a été démontré que un taux élevé d'hémoglobine fœtale (HbF) améliore l'évolution clinique de ces deux pathologies. Afin d'identifier les régions régulatrices potentielles de la γ-globine, nous avons combiné les données issues d'analyses de mutations rencontrées chez des patients HPFH avec les sites d'hybridation de facteur de transcription. Sur la base de cette analyse, en ayant recours à la technologie CRISPR/CAS9, nous avons développé un protocole permettant de générer: (i) la délétion d'un potentiel suppresseur de l'HbF situé entre les gènes des globines δ et γ, ciblé par le répresseur de l’HbF BCL11A chez les érythroblastes adultes; (ii) la plus courte délétion associée à des taux élevés d’HbF (délétion Corfu) chez les patients β-thalassemiques; (iii) une délétion de 13.6-kb rencontrée fréquemment chez les patients HPFH et incluant les gènes des globines β et δ ainsi que le potentiel suppresseur de l'HbF. Notre travail a montré que la délétion de la région génomique de 13.6-kb entraîne une forte production de HbF et une réduction concomitante de l'expression de la β-globine soit dans des lignées cellulaires érythroïdes humaines soit dans des érythroblastes primaires dérivées des cellules souches et progéniteurs hématopoïétiques (CSPH). Par ailleurs, nous avons montré que la génération de cette délétion sur des CSPHs issus de patients drépanocytaires entraîne une augmentation de la transcription de la γ-globine dans une proportion significative d'érythroblastes, conduisant à une amélioration du phénotype drépanocytaire. Enfin, nous avons exploré le mécanisme menant à la réactivation de l'expression de la γ-globine. Nous avons évalué des changements dans la conformation de la chromatine et des modifications épigénétiques dans le locus de la β-globine lors de la délétion ou de l'inversion de la région de 13.6 kb. Dans l'ensemble, cette étude contribue à la connaissance des mécanismes favorisant l'échange de l'hémoglobine fœtale à l'adulte et fournit des indices pour une approche d'édition du génome dans le traitement de la β-thalassémies et de la drépanocytose. / Β-hemoglobinopathies (β-thalassemias and sickle cell disease) are genetic anemias affecting thousands of newborns annually worldwide. β-thalassemias and sickle cell disease (SCD) are caused by mutations affecting the adult hemoglobin expression and are currently treated by red blood cell transfusion and iron chelation regiments. For patients affected by severe β-hemoglobinopathies, allogenic hematopoietic stem cell (HSCs) transplantation is the only definitive therapy. However, transplantation of autologous, genetically corrected HSCs represents an alternative therapy for patients lacking a suitable HSC donor. Naturally occurring large deletions encompassing β- and δ-globin genes in the β-globin gene cluster, defined as Hereditary Persistence of Fetal Hemoglobin (HPFH) traits, lead to increased fetal hemoglobin (HbF) expression ameliorating both thalassemic and SCD clinical phenotypes. In this study, we integrated transcription factor binding site analysis and HPFH genetic data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy disrupting: (i) a putative δγ-intergenic HbF silencer targeted by the HbF repressor BCL11A in adult erythroblasts; (ii) the shortest deletion associated with elevated HbF levels (“Corfu” deletion) in β-thalassemic patients, encompassing the putative δγ-intergenic HbF silencer; (iii) a 13.6-kb genomic region including the δ- and β-globin genes and the putative intergenic HbF silencer. Targeting the 13.6-kb region, but not the Corfu and the putative δγ-intergenic regions, caused a robust HbF re-activation and a concomitant reduction in β-globin expression in an adult erythroid cell line and in healthy donor hematopoietic stem/progenitor cells (HSPC)-derived erythroblasts. We provided a proof of principle of this potential therapeutic strategy: disruption of the 13.6-kb region in HSPCs from SCD donors favored the β-to-γ globin switching in a significant proportion of HSPC-derived erythroblasts, leading to the amelioration of the SCD cell phenotype. Finally, we dissected the mechanisms leading to HbF de-repression demonstrating changes in the chromatin conformation and epigenetic modifications within the β-globin locus upon deletion or inversion of the 13.6-kb region. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult hemoglobin switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia.

Nuclear magnetic resonance (NMR) is a common tool utilized in core analysis. NMR can reveal important information about pore structure, fluid configuration and wettability. However, standard NMR core analysis techniques look at the sample as a whole or only at thin slices. Two NMR pulse sequences are introduced that allow for the determination of relaxation time or diffusion-relaxation time distributions as a function of sample height. One-dimensional T 2 and D-T 2 profiles can be determined with a low-field Maran Ultra spectrometer by implementing nuclear magnetic resonance imaging techniques. Frequency encoding gradients impart spatial resolution to the measurements and allow for the creation of T, and D-T 2 profiles without having to perform multiple slice selective measurements. The first technique, denoted as RARE, relies solely on resolving transverse relaxation, T 2 , as a function of height. The second method, D-T 2 profiling, allows for the determination of both the diffusion coefficient, D, and T 2 as a function of height. The ability to resolve D in addition to T 2 allows fluids with overlapping relaxation times to be distinguished, and therefore it is not necessary to use D 2 O in order to differentiate the water signal from the oil signal. Implementation of these two methods allows for the determination of porosity and saturation profiles. Experiments were performed with a sandpack in order to demonstrate the applicability of these two techniques, and saturation profiles of a sandpack were determined at various stages of the flooding process.

ScoreSVG implements a three-tiered software architecture that generates musical scores in scalable vector graphics (SVG), something no other known music editor has done. SVG is non-proprietary XML-based format that renders graphical content into a web browser equipped with a SVG plugin. Scores in SVG can be scripted to produce interactive music theory examples, or make other graphical score changes impossible in any other format. Large music score editors such as Finale and Sibelius output to proprietary file formats, or Postscript (.ps) files that are not interchangeable or modifiable once created. Open source efforts such as LilyPond or Guido require the user to learn a new text-based music format to get a PostScript music score. ScoreSVG converts the data from a user-friendly visual front end to GuidoXML, capturing the semantic meaning of the score, and then outputs the result in SVG using an XSL stylesheet and the Saxon 8.4 XSLT processor.

In the transition to a fully integrated database at Marion College in Marion, Indiana, data elements needed more rigorous definitions both in terms of the relationships existing between data elements and attributes assigned to each between files. This data dictionary was thenused to access the attributes of each data element and the links existing and verify data elements within the database to locate illegal and inconsistent data items. From this data verification, weaknesses in procedures and software for loading information into the database were discovered and corrected.Ball State UniversityMuncie, IN 47306