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Several tools were developed to help researchers facilitate clinical translation of the use of engineered nucleases towards their disease gene of interest. Two major issues addressed were the inability to accurately predict nuclease off-target sites by user-friendly \textit{in silico} methods and the lack of a high-throughput, sensitive measurement of gene editing activity at endogenous loci. These objectives were accomplished by the completion of the following specific aims. An online search interface to allow exhaustive searching of a genome for potential nuclease off-target sites was implemented. Previously discovered off-target sites were collated and ranking algorithms developed that preferentially score validated off-target sites higher than other predictions. HEK-293T cells transfected with newly developed TALENs and ZFNs targeting the beta-globin gene were analyzed at the off-target sites predicted by the tool. Many samples of genomic DNA from cells treated with different ZFNs and TALENs were analyzed for off-target effects to generate a greatly expanded training set of bona fide off-target sites. Modifications to the off-target prediction algorithm parameters were evaluated for improvement through Precision-Recall analysis and several other metrics. An analysis pipeline was developed to process SMRT reads to simultaneously measure the rates of different DNA repair mechanisms by directly examining the DNA sequences. K562 cells were transfected with different types of nucleases and donor repair templates in order to optimize conditions for repairing the beta-globin gene. This work will have significant impact on future studies as the methods developed herein allow other laboratories to optimize nuclease-based therapies for single gene disorders.

<p>Duchenne muscular dystrophy (DMD) is a severe hereditary disorder caused by a loss of dystrophin, an essential musculoskeletal protein. Decades of promising research have yielded only modest gains in survival and quality of life for these patients and there have been no approved gene therapies for DMD to date. There are two significant hurdles to creating effective gene therapies for DMD; it is difficult to deliver a replacement dystrophin gene due to its large size and current strategies to restore the native dystrophin gene likely require life-long administration of a gene-modifying drug. This thesis presents a novel method to address these challenges through restoring dystrophin expression by genetically correcting the native dystrophin gene using engineered nucleases that target one or more exons in a mutational hotspot in exons 45-55 of the dystrophin gene. Importantly, this hotspot mutational region collectively represents approximately 62% of all DMD mutations. In this work, we utilize various engineered nuclease platforms to create genetic modifications that can correct a variety of DMD patient mutations.</p><p>Initially, we demonstrate that genome editing can efficiently correct the dystrophin reading frame and restore protein expression by introducing micro-frameshifts in exon 51, which is adjacent to a hotspot mutational region in the dystrophin gene. Transcription activator-like effector nucleases (TALENs) were engineered to mediate highly efficient gene editing after introducing a single TALEN pair targeted to exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from DMD patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. We show that our engineered TALENs have minimal cytotoxicity and exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein coding regions of the genome, as predicted by in silico target site analysis. </p><p>In an alternative approach, we capitalized on the recent advances in genome editing to generate permanent exclusion of exons by using zinc-finger nucleases (ZFNs) to selectively remove sequences important in specific exon recognition. This strategy has the advantage of creating predictable frame restoration and protein expression, although it relies on simultaneous nuclease activity to generate genomic deletions. ZFNs were designed to remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript, a method that can potentially restore the dystrophin reading frame in up to 13% of DMD patients. Nucleases were assembled by extended modular assembly and context-dependent assembly methods and screened for activity in human cells. Selected ZFNs had moderate observable cytotoxicity and one ZFN showed off-target activity at two chromosomal loci. Two active ZFN pairs flanking the exon 51 splice acceptor site were transfected into DMD patient cells and a clonal population was isolated with this region deleted from the genome. Deletion of the genomic sequence containing the splice acceptor resulted in the loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin expression in vitro. Furthermore, transplantation of corrected cells into the hind limb of immunodeficient mice resulted in efficient human dystrophin expression localized to the sarcolemma. </p><p>Finally, we exploited the increased versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system to enable a variety of otherwise challenging gene correction strategies for DMD. Single or multiplexed sgRNAs were designed to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing either intraexonic small insertions and deletions, or large deletions of one or more exons. Significantly, we generated a large deletion of 336 kb across the entire exon 45-55 region that is applicable to correction of approximately 62% of DMD patient mutations. We show that, for selected sgRNAs, CRISPR/Cas9 gene editing displays minimal cytotoxicity and limited aberrant mutagenesis at off-target chromosomal loci. Following treatment with Cas9 nuclease and one or more sgRNAs, dystrophin expression was restored in Duchenne patient muscle cells in vitro. Human dystrophin was detected in vivo following transplantation of genetically corrected patient cells into immunodeficient mice. </p><p>In summary, the objective of this work was to develop methods to genetically correct the native dystrophin as a potential therapy for DMD. These studies integrate the rapid advances in gene editing technologies to create targeted frameshifts that restore the dystrophin gene around patient mutations in non-essential coding regions. Collectively, this thesis presents several gene editing methods that can correct patient mutations by modification of specific exons or by deletion of one or more exons that results in restoration of the dystrophin reading frame. Importantly, the gene correction methods described here are compatible with leading cell-based therapies and in vivo gene delivery strategies for DMD, providing an avenue towards a cure for this devastating disease.</p> / Dissertation

Thesis (MPhil)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Theses and dissertations constitute a substantial platform for the documentation and dissemination of research findings, and the professional presentation of such findings is crucial for maintaining scientific integrity. Highly effective fact finders may lack writing skills and experience, or they may simply encounter barriers when expressing ideas, and thus perhaps inadequately present what they have so adequately found. In short, adequate editing of theses and dissertations is essential. Whereas a multitude of guidelines is available for thesis and dissertation writing, there is little guidance available on the editing of such works. Thus, with the latter objective in mind, this thesis is dedicated to developing a practical model to editing postgraduate research papers. Despite a notable lack of theory in the field of thesis editing, which became apparent while reviewing the respective literature, the most suitable sources of theory were selected to provide a basis for developing a model for thesis editing. These sources, combined with insights from a practical dissertation editing assignment, allowed for the design of a model for the practical editing process of postgraduate research texts. The editing model is based on a process-oriented approach, i.e. one which focuses on the learning process of the student. Moreover, the model promotes a level of editorial intervention that conforms to the current perception of ethical intervention in thesis editing. Ethical intervention is currently being negotiated against the backdrop of such standards as the purpose of thesis writing as well as the requirement of originality of theses and dissertations. In a testing phase the model was applied in a thesis editing assignment and emerged as a valuable guide in the process of editing. It also proved practicable in all its major aspects. Nevertheless, since a single testing assignment is not sufficient to prove the general practicality of any model, the model is still to be considered a prototype and may have to undergo further refinement after additional comprehensive testing. / AFRIKAANSE OPSOMMING: Tesisse en verhandelinge is 'n belangrike basis vir die optekening en verspreiding van navorsingsbevindinge, en die professionele aanbieding van sodanige bevindinge is noodsaaklik vir die behoud van wetenskaplike integriteit. Tog is kom hoogs doeltreffende navorsers soms minder bedrewe of ervare skrywers, of hulle bloot voor hindernisse te staan wanneer hulle hul gedagtes moet verwoord, wat tot die ontoereikende aanbieding van bevredigende bevindinge lei. Kortom die toereikende redigering van tesisse en verhandelinge is van die allergrootste belang. Hoewel daar etlike riglyne vir die skryf van tesisse en verhandelinge bestaan, is daar weinig leiding beskikbaar vir die redigering daarvan. Gedagtig hieraan is hierdie tesis daarop toegespits om 'n praktiese model vir die redigering van nagraadse navorsingstekste te ontwikkel. Ondanks 'n merkbare gebrek aan teorie op die gebied van tesisredigering, wat baie duidelik uit 'n oorsig van die betrokke literatuur blyk, is die mees toepaslike teoretiese bronne as grondslag vir die ontwikkeling van 'n model vir tesisredigering gekies. Met behulp van hierdie bronne, tesame met die insigte verkry uit 'n praktiese redigeeropdrag, kon 'n praktiese model vir die redigering van nagraadse navorsingstekste ontwerp word. Die redigeermodel berus op 'n prosesgerigte benadering, dit wil sê 'n benadering wat op die student se leerproses konsentreer. Daarbenewens argumenteer die model ten gunste van redaksionele ingrepe wat met huidige opvattings oor etiese tesisredigering strook. Dit geskied teen die agtergrond van die huidige gesprek oor etiese intervensie, wat onder meer teen die agtergrond van standaarde soos die doel van die tesis sowel as die oorspronklikheidsvereiste vir tesisse en verhandelinge gevoer word. Die model is tydens 'n toetsfase in 'n tesisredigeringsopdrag toegepas en blyk nuttige riglyne vir die redigeerproses te bied. Ook het al die kernkomponente daarvan geblyk prakties bruikbaar te wees. Aangesien 'n enkele toetsopdrag nie voldoende is om die algemene bruikbaarheid van 'n model te bewys nie, word die model steeds as 'n prototipe beskou en dit sal waarskynlik ná bykomende omvattende toetsing verder verbeter word.

This work will give a wide survey for various techniques that are present in the field of character computer animation, which concentrates particularly on those techniques and problems involved in the production of realistic character synthesis and motion. A preliminary user study (including Questionnaire, online publishing such as flicker.com, interview, multiple choice questions, publishing on Android mobile phone, and questionnaire analysis, validation, statistical evaluation, design steps and Character Animation Observation) was conducted to explore design questions, identify users' needs, and obtain a "true story" of quality character animation and the effect of using animation as useful tools in Education. The first set of questionnaires were designed to accommodate the evaluation of animation from candidates from different walks of life, ranging from animators, gamers, teacher assistances (TA), students, teaches, professionals and researchers using and evaluating pre-prepared animated character videos scenarios, and the study outcomes has reviewed the recent advances techniques of character animation, motion editing that enable the control of complex animations by interactively blending, improving and tuning artificial or captured motions. The goal of this work was to augment the students learning intuition by providing ways to make education and learning more interesting, useful and fun objectively, in order to improve students’ respond and understanding to any subject area through the use of animation also by producing the required high quality motion, reaction, interaction and story board to viewers of the motion. We present a variety of different evaluation to the motion quality by measuring user sensitivity, observations to any noticeable artefact, usability, usefulness etc. to derive clear useful guidelines from the results, and discuss several interesting systematic trends we have uncovered in the experimental data. We also present an efficient technique for evaluating the capability of animation influence on education to fulfil the requirements of a given scenario, along with the advantages and the effect on those deficiencies of some methods commonly used to improve animation quality to serve the learning process. Finally, we propose a wide range of extensions and statistical calculation enabled by these evaluation tools, such as Wilcoxon, F-test, T-test, Wondershare Quiz creator (WQC), Chi square and many others explained with full details.

電視新聞利用剪接將影像轉化成為敘述工具，由於剪接者手法上的差異，使得電視新聞可以利用不同的方式來敘述故事。這種多元的敘事風格，讓觀眾可以透過不同的影像形式抒發情感，其中「感動」是電視新聞中最常運用之情感效果。因此，本研究主要針對電視新聞中的影像與其引發感動的原因作為研究重點，以電視新聞專題做為研究對象，而發展出以下三個問題：一、電視新聞專題中的感動：要素為何？二、電視新聞專題畫面如何表現感動要素？三、電視新聞專題的剪接如何表現感動？ 由於感動是一個心裡描述的過程，任何的情緒皆無法透過數字來檢視它的強弱程度，因為它的形成過程中有許多複雜的因素，這些都必須藉由參與者的描述才可以得知。而感動的面貌隱藏於影像之中，電視新聞中的感動影像也不可能經由問卷中的量表來制定。因此本研究採用質性研究之深度訪談與文獻分析作為研究工具。 電視新聞中的感動藉由影像的敘述方式來呈現，透過訪談與分析，本研究得到以下結論：一、電視新聞以影像作為表達情緒的工具，利用既有的新聞故事，透過當中的人、事、物將情感展現出來，其中的感動來自於真實的情感與人物之間彼此的互動。二、影像利用剪接串聯情感時，必須強調其中的因果關係，沒有因果關係的影像，不會讓人感動。三、除了因果關係與共同經驗外，美也是造成感動的因素之一。在許多的藝術形式中，美經常被用來製造感動，影像當然也不例外，在電視新聞當中，美感的塑造是在既有敘事結構之外的影像運用。四、感動來自於一個有系統的故事，任何片段的感動，必定由其結構性的故事情節所引起，而一個故事能夠透過影像完整呈現，依賴的便是剪接。 關鍵字：電視新聞、影像、剪接、感動 / The image of television news is presented by editing. Because of the different techniques of editors, television news could be presented in various ways. The diversified modes of image allow the audience to off-load their feelings. To move them is the frequent technique used. This research focuses on the special television news subject.The researcher attempts to figure out the reasons that image evoke feeling. There are three main topics. One topic is to find out what is the element of touching in special television news subject. Then the following is to analyze the elements. Finally, the researcher tries to find out how the editing expresses affection. Touching is the process of inside description. The feeling is impossible to examine the degree by figures. The constructing process includes of many complicated factors, which have to be expressed by the actors. Furthermore, because the touching is concealed from the image, the touching image is not possible to quantify by questionnaire. The research proceeds by interview and document analyze. Touching of television news is presented by the expressing of image. By interview and analyze, this research concludes in four aspects. First, image is the instrument to express emotion in television news. A news story is constructed by the events, the actors, and the objects. The touching comes from the interaction of all the subjects. Second, editing has to emphasize on the cause and effect to connect the feeling. Without the emphasis, the image is not touchable. Third, in addition to cause and fact and the common experience, beauty is the important factor to touch people. In many forms of arts, beauty is applied to evoke emotion. Image is no exception. In television news, the application of beauty is beyond description. Finally, touching is evoked from a systematic story. Any part of touching must have arose from the constructive plots. A story which can be presented in complete relies on the editing. Keywords: television news、image、editing、touching

This thesis involves both editorial practice and literary analysis. In order to establish an editorial framework for the electronic scholarly edition of Dr. Ralph Barnes Grindrod's pamphlet <em>Slaves of the Needle</em>, I examine current issues in electronic textual editing. In the electronic scholarly edition, approximately twelve of the pamphlet's thirty-five pages are transcribed and encoded using TEI-based code. The second aspect of my master's thesis concerns the depiction of seamstresses in nineteenth-century British literature. <em>Slaves of the Needle</em> provides a non-fiction counterpart to the fictional seamstresses of mid-nineteenth-century literature. Using <em>Slaves of the Needle</em> as a basis for evaluating the accuracy of mid-nineteenth-century characterizations of seamstresses, I show that authors such as Charles Dickens, Elizabeth Gaskell, Ernest Jones, and Charlotte Elizabeth Tonna were familiar with the working conditions of seamstresses. By conducting a close reading of certain representations of the seamstress in both fiction and non-fiction, I develop a theory of why the depiction of some aspects of the seamstress story are more accurate than others.

The transcriptome of the mammalian brain is extensively modified by adenosine to inosine (A-to-I) nucleotide conversion by two adenosine deaminases (ADAR1 and ADAR2). As adenosine and inosine have different base pairing properties, A-to-I RNA editing shapes the functional output of both coding and non-coding RNAs (ncRNAs) in the brain. The aim of this thesis was to identify editing events in small regulatory ncRNAs (miRNAs) and to determine their temporal and spatial editing status in the developing and adult mouse brain. To do this, we initially analyzed the editing status of miRNAs from different developmental time points of the mouse brain. We detected novel miRNA substrates subjected to A-to-I editing and found a general increase in miRNA editing during brain development, implicating a more stringent control of miRNAs as the brain matures. Most of the edited miRNAs were found to be transcribed as a single long consecutive transcript from a large gene cluster. However, maturation from this primary miRNA (pri-miRNA) transcript into functional forms of miRNAs is regulated individually, and might be influenced by the ADAR proteins in an editing independent matter. We also found that edited miRNAs were highly expressed at the synapse, implicating a role as local regulators of synaptic translation. We further show that the increase in editing during development is explained by a gradual accumulation of the ADAR enzymes in the nucleus. Specifically for ADAR2, we found a developmentally increasing interaction with two factors, importin-α4 and Pin1, that facilitate nuclear localization of the editing enzyme. We have also found that selectively edited stem loops often are flanked by other long stem loop structures that induce editing in cis. This may explain why multiple pri-miRNAs are edited within the same cluster. In conclusion, this thesis has significantly increased the understanding of the dynamics of both editing substrates and enzymes in the developing and mature brain. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

In this article, the reader will find methodological guidelines on how to write a college textbook. It offers procedures for setting up a work team responsible for the preparation of textbooks; explains how to define the topics to be addressed, as well as procedures for information management, and detection of appropriate literature. Additionally, it emphasizes theoretical research types, interpretation of theories, theoretical conclusions and the pedagogical function of the college textbook. Finally, it provides some editorial guidelines, such as textbook structure, notes, bibliographical references, images and charts, authors’ biographies, correction stage, and final review up to distribution. / En el presente trabajo, el lector encontrará pautas metodológicas para la realización de un libro de texto universitario; se sistematizan procedimientos para la conformación del equipo de trabajo encargado de la elaboración de los libros de texto; se explica cómo delimitar las temáticas que pueden ser abordadas; se exponen los procedimientos de gestión de la información, así como la detección de la literatura apropiada. Además, se enfatiza en las formas de indagación teórica, interpretación de teorías, conclusiones teóricas y las funciones didácticas del libro de texto. Por último, se exponen algunas pautas editoriales que van, desde la estructura del libro de texto, notas, referencias bibliográficas, tratamiento de imágenes o tablas, semblanza de los autores, etapa de corrección; hasta la revisión final y puesta en valor de la obra.

<p>Glycogen storage disease type Ia is an autosomal recessive disorder caused by a mutation in the glucose-6-phosphatase (G6Pase) catalytic subunit, encoded in humans by G6PC. G6Pase dephosphorylates glucose-6-phosphate (G6P) in the liver to generate glucose that can be shuttled to the bloodstream to maintain normoglycemia. Patients with GSD Ia typically present at 6 months of age with sever hypoglycemia, which is lethal if untreated. The current treatment is a strict dietary regimen in which children must be fed every 2 hours overnight or given nasogastric tube feeding, and adults must consume uncooked cornstarch around the clock to maintain normal blood sugar levels. This treatment maintains survival but fails to prevent other symptoms related to metabolism of the excess G6P, and patients develop hepatic adenomas that may become hepatocellular carcinoma later in life, in addition to progressive renal complications.</p><p>To overcome the problems persisting during dietary therapy, the Koeberl lab has sought to develop gene therapy approaches that use adeno-associated virus (AAV) vectors to replace the G6pase activity, restoring normoglycemia and normal metabolic processes. However, the vast majority of AAV-delivered genetic material exists as episomes that do not replicate as cells divide, so the effects of AAV gene therapy on GSD Ia mouse and dog models have proven temporary. We hypothesized that driving integration of therapeutic vector genomes into an affected individual's genome would improve beneficial effects' longevity.</p><p>We tested several approaches to accomplish this, and have found positive effects using a zinc finger nuclease (ZFN) that targets the mouse safe harbor ROSA26 locus to induce homologous recombination of the G6PC donor vector into the mouse genome. We were able to see an improvement in mouse survival to 8 months of age, an increase in G6Pase activity at 3 months of age, and a decrease in glycogen accumulation at 3 months of age, when the ZFN vector is administered alongside the G6PC vector, compared with mice that received the G6PC vector alone.</p><p>We have also taken an alternative approach to overcoming the long-term complications of the current dietary treatment, which would augment rather than replace the current treatment. We have examined several drugs known to induce autophagy in other disease models or cell culture systems, to determine if we could manipulate autophagic activity in G6PC knockdown hepatocytes or GSD Ia mice. We have found positive results using rapamycin, a well-studied MTOR inhibitor, in mice and cells, and have screened several other drugs as well, finding positive effects for bezafibrate, mifepristone, carbamazepin, and lithium chloride, in terms of lipid reduction (which accumulates as a symptom of GSD Ia) and/or LC3-II enhancement, which is reduced in GSD Ia due to downregulation of autophagy during G6P accumulation.</p> / Dissertation