Bacterial transcriptomes are highly complex, comprising not only protein-coding RNAs and translation-related non-coding RNAs, but also non-coding RNAs that function as regulators of gene expression. The post-transcriptional modification of RNA sequences by RNA editing enzymes, which has recently been shown to affect diverse RNA substrates in several bacteria, can magnify this complexity further still. However, little is known about RNA editing and non-coding RNAs in Streptomyces venezuelae, a model organism for studying complex bacterial development and specialized metabolism. This thesis investigates RNA editing and non-coding regulatory RNAs in S. venezuelae using RNA sequencing data from wild type and mutant strains at various stages of development and under several laboratory-controlled conditions. We identified hundreds of adenosine-to-inosine editing events throughout the transcriptome and predicted the potential impact of the edits occurring in protein-coding RNAs. The potential role of the adenosine deaminase enzyme TadA in facilitating these RNA editing events is also considered. Additionally, we detected thousands of transcripts that are expressed from unannotated regions of the S. venezuelae genome, many of which we predict are non-coding RNAs. Furthermore, we highlight our efforts to characterize a highly expressed putative non-coding RNA that exhibits considerable sequence conservation in other streptomycetes. This work provides new insights into the transcriptomic complexity of S. venezuelae and expands our understanding of RNA-based regulation in bacteria. / Thesis / Master of Science (MSc) / All living things have DNA, which contains the instructions for maintaining life in the form of genes. These genes are copied into RNAs, and some of these RNA molecules are used to make proteins, which are the building blocks and machinery of cells. However, not all RNAs make proteins; some act as regulators, controlling which genes and proteins are active. Additionally, some proteins edit the instructions contained by RNA molecules after they are made, adding another layer of complexity to how cells regulate their activities. This thesis investigates these processes in Streptomyces venezuelae, a soil-dwelling bacterium known for its complex development and metabolism. We found hundreds of cases where RNA molecules are edited, potentially affecting their functions in the cell, and discovered thousands of non-protein-coding RNAs that may regulate genes or proteins. Our findings expand our understanding of how Streptomyces bacteria manage their complex genetic activities at the RNA level.

Oomycetes are fungi-like eukaryotic microorganisms, which are actually phylogenetic relatives of diatoms and brown algae, within the kingdom Stramenopila. Many oomycete species, mainly in the genera Phytophthora, Pythium and downy mildews, are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Some oomycetes are also animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. Phytophthora species, represented by the Irish Potato Famine pathogen P. infestans and the soybean pathogen P. sojae, are arguably the most destructive pathogens of dicotyledonous plants among the oomycete species and thus have been extensively studied. This dissertation focuses on the model oomycete pathogen P. sojae to investigate specific aspects of its molecular biology and establish an efficient genetic manipulation tool. Specifically, in Chapter 1, I briefly introduce the basic concepts of oomycete biology and pathology, and summarize the experimental techniques used for studies of oomycete genetics over the past two decades. Because the approach to studying fungi and oomycetes are similar (indeed they were incorrectly placed in the same taxonomic group until recently), a special section reviews the emerging genome editing technology CRISPR/Cas system in these organisms together. Chapter 2 and Chapter 3 focus on one of the most important intracellular activities, nuclear localization of proteins, and describe the characterization of nuclear localization signals (NLSs) in P. sojae. This focus stemmed from my early work on genome editing in P. sojae, when I discovered that conventional NLS signals from SV40 used to target the TAL effector nuclease (TALEN) to the nucleus worked poorly in P. sojae. In the first part of this work (Chapter 2), I used confocal microscopy to identify features of nuclear localization in oomycetes that differ from animals, plants and fungi, based on characterization of two classes of nuclear localization signals, cNLS and PY-NLS, and on characterization of several conserved nuclear proteins. In the second part (Chapter 3), I determined that the nuclear localization of the P. sojae bZIP1 transcription factor is mediated by multiple weak nuclear targeting motifs acting together. In Chapter 4 and Chapter 5, I describe my implementation of nuclease-based technology for genetic modification and control of P. sojae. In Chapter 4, I describe the first use of the CRISPR system in an oomycete, including its use to validate the function of a host specificity gene. This is of particular importance because molecular techniques such as gene knockouts and gene replacements, widely used in other organisms, were not previously possible in oomycetes. The successful implementation of CRISPR provides a major new research capability to the oomycete community. Following up on the studies described in Chapter 4, in Chapter 5, I describe the generalization and simplification of the CRISPR/Cas9 expression strategy in P. sojae as well as methods for mutant screening. I also describe several optimized methodologies for P. sojae manipulation based on my 5 years of experience with P. sojae. / Ph. D. / Oomycetes (water molds) are eukaryotic microorganisms that resemble filamentous fungi (molds), but are actually relatives of diatoms and brown algae, within a different kingdom of life named Stramenopila. The functional relationship between oomycetes and fungi is similar to that between fish and dolphins, which also acquired similar functions via different evolutionary paths. Many families of oomycetes are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Other families of oomycetes are animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. <i>Phytophthora</i> species, represented by the Irish Potato Famine pathogen <i>P. infestans</i> and the soybean pathogen <i>P. sojae</i>, are among the most destructive oomycete pathogens of plants and thus have been extensively studied. This dissertation is focused on the model oomycete pathogen <i>P. sojae</i>. It investigates specific aspects of its molecular biology and establishes an efficient genetic manipulation tool. All complex organisms (eukaryotes) package their genetic material in nuclei, which contain proteins as well as DNA. In the first part of my research (Chapter 2 and Chapter 3), I focused on the mechanisms used by <i>P. sojae</i> to target nuclear proteins into the nucleus, particularly the tags (called nuclear localization signals, or NLSs) that are identify the proteins that must travel to the nucleus. I showed that nuclear targeting mechanisms in oomycetes differ in distinct ways from well-studied eukaryotes such as humans. In particular, the nuclear targeting signals in <i>P. sojae</i> proteins are diffused over multiple sites on the proteins, whereas in human proteins there’s usually just a single signal. For one particular oomycete protein, a transcription factor, nuclear targeting involves four weak signals that cooperate synergistically. Two of these four weak signals define a new class of nuclear localization signal. In the second part of my research (Chapter 4 and Chapter 5), I implemented and further optimized a genome editing technology for genetic modification and control of <i>P. sojae</i>. This technology is based on the CRISPR system that has revolutionized genome editing in plants and animals over the last three years. This is of particular importance because genome editing techniques were not previously possible in oomycetes. The successful implementation of CRISPR technology in <i>P. sojae</i> has provided a major new research capability to the oomycete community. In Chapter 5, I also describe several optimized methodologies for <i>P. sojae</i> genetic manipulation based on my 5 years of experience with <i>P. sojae</i>.

Im experimentellen Ansatz sollte mithilfe der CRISPR-Cas9-Methode eine gerichtete ALK1-Rezeptor-Eliminierung in myoblastischen C2C12-Zellen durchgeführt werden. Nach erfolgreicher Klonierung der jeweiligen, für den Typ-I-Rezeptor ALK1-kodierenden, gRNA-Sequenzen in die Puro- und GFP-CRISPR-Plasmide gelang der mittels Lipofektion durchgeführte Transfer der vier klonierten Plasmide in die C2C12-Zellen. Parallel aufgetaut wurden C2C12*ALK2- sowie ALK3-Knockout-Zelllinien, welche zuvor durch die Masterandin L. Wiesmann, ebenfalls mithilfe der CRISPR-Cas9-Methode, induzierte Knockouts der jeweiligen Rezeptoren ALK2 sowie ALK3 enthielten. Anschließend erfolgte die Puromycin-Selektion der mit den Puro-Klonen transfizierten C2C12*ALK1-3, ALK1-4-, ALK2- sowie ALK3-KO-Zellpopulationen. Die Zellen der C2C12*ALK1-3-KO-Population überlebten die Selektion trotz erneuter Durchführung der Transfektion sowie Selektion nicht. Somit erfolgte die Kultivierung der verbliebenen Zellen der C2C12*ALK1 4-, C2C12*ALK2- sowie C2C12*ALK3-KO-Population. Anschließend galt es zu untersuchen, wie responsiv die einzelne KO-Zelle für verschiedene Liganden ist. Im Rahmen der Durchführung differenter, zellbasierter Versuche wie der qPCR, des Western Blots und des ALP-Assays wirkten verschiedene BMPs auf die KO-Populationen ein. Somit konnten die BMP-induzierten, nachfolgenden Ereignisse wie die mRNA-Expression, die SMAD-Phosphorylierung sowie die Induktion der ALP-Expression innerhalb der KO-Populationen genauer betrachtet werden. Es ist allgemein bekannt, dass ALK1 sowohl bei der Angiogenese als auch bei der kardio-vaskulären Homöostase eine wichtige Rolle übernimmt. ALK1 ist vermutlich für die Gefäßneubildung in manchen Tumoren verantwortlich und auch die vaskuläre Erkrankung „Hereditäre hämorrhagische Teleangiektasie (HHT)“ steht im Zusammenhang mit einer Mutation des ALK1-Rezeptorgens. BMP9 beeinflusst als ALK1-bindender Ligand neben der Tumorentwicklung und der Angiogenese auch die osteogene Differenzierung mesenchymaler Stammzellen. Im Hinblick auf zukünftige Versuche sind daher weitere, noch aussagekräftigere Ergebnisse erstrebenswert, allerdings unter der Verwendung von ausschließlich homozygoten KO-Zelllinien. Weitere Erkenntnisse über die Rolle des ALK1-Rezeptors in BMP-vermittelter Signaltransduktion könnten für therapeutische Ansätze bei der Behandlung von vaskulären Erkrankungen und Tumorprogression sowie bei der Förderung der Knochenregeneration und -heilung hilfreich sein. / In the experimental approach, targeted ALK1 receptor elimination was to be performed in myoblastic C2C12 cells using the CRISPR-Cas9 method. After successful cloning of the respective gRNA sequences coding for the type I receptor ALK1 into the Puro and GFP-CRISPR plasmids, the four cloned plasmids were transferred into the C2C12 cells by lipofection. In parallel, C2C12*ALK2 and ALK3 knockout cell lines were thawed, which previously contained knockouts of the respective ALK2 and ALK3 receptors induced by the master student L. Wiesmann, also using the CRISPR-Cas9 method. Puromycin was then used to select the C2C12*ALK1-3, ALK1-4, ALK2 and ALK3-KO cell populations transfected with the Puro clones. The cells of the C2C12*ALK1-3-KO population did not survive the selection despite renewed transfection and selection. The remaining cells of the C2C12*ALK1-4, C2C12*ALK2 and C2C12*ALK3-KO populations were therefore cultivated. It was then necessary to investigate how responsive the individual KO cells are to different ligands. By performing different cell-based experiments such as qPCR, Western blot and ALP assay, different BMPs affected the KO populations. Thus, the BMP-induced downstream events such as mRNA expression, SMAD phosphorylation and induction of ALP expression within the KO populations could be analysed precisely.

Thesis (MDram)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This thesis sets out to provide comparative analyses of selected play texts and their film adaptations in order to demonstrate the significant role that editing techniques play in translating the play text’s dramatic elements into the visual language of film. The purpose of a film adaptation is to present a new interpretation of the play text that audiences will find engaging. In order to establish how the film medium is potentially able to enhance or alter the audience’s understanding of the original source text, the study turns to the field of semiotics to determine how the play text’s themes, plot and characters – embodied in a verbal sign system – are adapted into the audio-visual sign system of film. While cinematography, production design and music are critical elements in film making, editing can be regarded as the distinctive and fundamental signifying practice in the construction of meaning in a film. This will be the point of departure in analysing how meaning is “translated” from one sign system into another in the process of adaptation. By manipulating the key relations between shots, editing is able to guide the audience’s understanding of the film narrative, amplify character development, and generate intellectual and emotional responses. Different editing conventions have therefore been developed to amplify the dramatic effect of the narrative and the filmmaker’s vision. The different effects that editing conventions create in the interpretation of a play text are demonstrated by comparing two cinematic versions of William Shakespeare’s Romeo and Juliet. The use of continuity editing techniques in Franco Zeffirelli’s 1968 film adaptation of Romeo and Juliet is compared to Baz Luhrmann’s use of modern MTV conventions in his William Shakespeare’s Romeo + Juliet (1996). Zeffirelli and Luhrmann both employ different editing conventions to amplify their “readings” of Shakespeare’s play text, thereby presenting an adaptation that their target audience will find engaging. The film adaptations of Shakespeare’s Romeo and Juliet therefore demonstrate the significance of different editing techniques in conveying meaning within a specific reception context. The series of reinterpretations of Christopher Isherwood’s Goodbye to Berlin (1939) illustrates how editing techniques are able to transfer Isherwood’s themes and political commentary on the rise of Nazism in Weimar Berlin across various texts and mediums, which include the film adaptation I am a Camera (1955) directed by Henry Cornelius, the Broadway musical Cabaret (1966) directed by Joe Masteroff, and finally Bob Fosse’s musical film Cabaret (1972). The comparative analyses of the above-mentioned source texts and their subsequent film adaptations demonstrate how different editing techniques are able to highlight new perspectives on the source material. Editing conventions are therefore highly significant in the creation of cinematic representations of the play text as they lead audiences to “read” the dramatic narrative within new contexts, using the visual language of film to create new insights that will complement the audience’s understanding and appreciation of the play. / AFRIKAANSE OPSOMMING: Vergelykende analises tussen gekose speeltekste en hul verwerkings vir film word in hierdie tesis uiteengesit om die betekenisvolle rol wat redigeertegnieke in die vertolking van die speelteks se dramatiese elemente in die visuele styl van die film speel, te demonstreer. Die doel met ’n filmverwerking is om ’n nuwe interpretasie van die speelteks aan te bied wat gehore vasgevang sal hou. Om te bepaal hoe die filmmedium die gehoor se begrip van die oorspronklike teks potensieel kan versterk of verander, gebruik hierdie studie die veld van semiotiek om vas te stel hoe die speeltekste se temas, intrige en karakters – beliggaam in ’n verbale simboolstelsel – aangepas word in die oudiovisuele simboolstelsel van die film. Terwyl filmfotografie, produksie-ontwerp en musiek kritiese elemente in die vervaardiging van films is, word redigering as die onderskeidende en fundamentele belangrike praktyk in die konstruksie van betekenis in ’n film geag. Hierdie is die vertrekpunt in die analisering van hoe betekenis “vertaal” word van een simboolstelsel na ’n ander tydens die verwerkingsproses. Redigering kan deur middel van manipulering van die sleutelverwantskappe tussen skote die gehoor lei om die narratief van die film te verstaan, karakterontwikkeling uit te brei en intellektuele en emosionele reaksies te skep. Onderskeie redigeerkonvensies is dus ontwikkel om die dramatiese effek van die narratief en die filmvervaardiger se visie te versterk. Die verskillende resultate wat deur middel van hierdie tegnieke in die interpretasie van ’n speelteks verkry word, word toegelig deur die twee filmweergawes van William Shakespeare se Romeo and Juliet te vergelyk. Die gebruik van kontinuïteit-redigeertegnieke in Franco Zeffirelli se 1968 filmverwerking van Romeo and Juliet word vergelyk met Baz Luhrmann se gebruik van moderne MTV-konvensies in sy William Shakespeare’s Romeo + Juliet (1996). Beide Zeffirelli en Luhrmann gebruik verskillende redigeerkonvensies om hulle “lees” van Shakespeare se speelteks toe te lig en daarmee ’n verwerking wat hulle teikengehoor vasgevang sal hou, te bied. Die filmverwerkings van Shakespeare se Romeo and Juliet demonstreer dus die belang van verskillende redigeertegnieke in die oordra van betekenis binne ’n spesifieke konteks waarin dit ontvang word. Die reeks herinterpretasies van Christopher Isherwood se Goodbye to Berlin (1939) illustreer hoe redigeertegnieke in staat is om Isherwood se temas en politieke kommentaar aangaande die opkoms van Nazisme in Weimar Berlyn oor verskeie tekste en mediums oor te dra. Insluitend hierby is die filmverwerking I am a Camera (1955) onder regie van Henry Cornelius, die Broadway musiekblyspel Cabaret (1966) onder regie van Joe Masteroff, en laastens Bob Fosse se musiekfilm Cabaret (1972). Die vergelykende analise van bogenoemde tekste en hul daaropvolgende filmverwerkings demonstreer hoe verskillende redigeertegnieke nuwe perspektiewe op die oorspronklike materiaal na vore kan bring. Redigeerkonvensies is uiters betekenisvol in die skep van filmiese voorstellings van die speelteks aangesien die gehoor daarmee gelei word om die dramatiese narratief binne nuwe konteks te “lees” deur gebruik te maak van die visuele styl van die film om nuwe insig te skep wat die gehoor se verstaan en waardering van die stuk aanvul.

<p>Although several systems with structure editors have been built, no model exist to formally describe the edit operations used in such editors. This thesis introduces such a model --- a formalism to describe general structure edit operations for text oriented documents. The model allows free bottom-up editing for any tree-based structural document with a textual content. It can also handle attribute and erroneous structures. Some classes of common structures have been identified and structure editor specifications constructed for them, which can be used and combined in the creation of other structure editors.</p>

This paper reports work in progress on an experimental text editor called CREF, the Cross Referenced Editing Facility. CREF deals with chunks of text, called segments, which may have associated features such as keywords or various kinds of links to other segments. Text in CREF is organized into linear collections for normal browsing. The use of summary and cross-reference links in CREF allows the imposition of an auxiliary network structure upon the text which can be useful for "zooming in and out" or "non-local transitions." Although it was designed as a tool for use in complex protocol analysis by a "knowledge Engineer's Assistant," CREF has many interesting features which should make it suitable for a wide variety of applications, including browsing, program editing, document preparation, and mail reading.

Although several systems with structure editors have been built, no model exist to formally describe the edit operations used in such editors. This thesis introduces such a model --- a formalism to describe general structure edit operations for text oriented documents. The model allows free bottom-up editing for any tree-based structural document with a textual content. It can also handle attribute and erroneous structures. Some classes of common structures have been identified and structure editor specifications constructed for them, which can be used and combined in the creation of other structure editors.

The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable—ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.

No description available.

"November, 1994" / Thesis (PhD)--Macquarie University, School of English & Linguistics, 1995. / Bibliography: leaves 199-212. / Introduction -- Bilingual editing of magazines -- Bilingual editing of magazines in Hong Kong -- Survey and interviews -- Grammatical bases for textual analysis -- Textual analysis -- Discussion and conclusion. / In cross-cultural or intercultural encounters of the modern age, mass communication has become a daily feature of our technological civilisation, and mass media have facilitated effective international information flow. Bilingual editing becomes an important medium of mass communication. The effectiveness of such communication rests upon the grammatical, lexical, sociolinguistic, socio-cultural, discourse and strategic competence of participants (editors, writers, translators and readers). It rests upon their ability to creatively use and to sensitively respond to language. In this dynamic process of communication, a bilingual editor not only plays the role of translator but also acts as a mediator; as Hatim and Mason (1990:223) suggest, s/he "has not only a bilingual ability but also a bi-cultural vision". -- In view of the diversity of usage of bilingual editing in the media, this research delves into the role of translation from English to Chinese in the bilingual editing of magazines in Hong Kong. This area is of interest for four reasons: first, since the press medium engages most translation practitioners, a study in this area may help future practitioners to have a better understanding of this science and art and its practice; second, text types are highly diverse, allowing room for discussion of translation devices; third, Hong Kong is a typical meeting place of the East and West and bilingual editing serves as a tool of information flow; fourth, the rising status of Chinese in Hong Kong approaching 1997 will enhance the role of bilingual editing. -- The study focuses on translation only from English and Chinese, or vice versa. In as much as there is very little academic attention to bilingual editing and its nature, processes and techniques, or to the role of translation in bilingual editing, it is believed that this research will help facilitate cross-cultural communication between Westerners and Chinese. -- The objective of this study is to derive new insights into the translation process with the support of contemporary approaches, and to descant on different lexical, grammatical and cultural features between English and Chinese; and most important, to elicit from the above features a set of parameters which may promote consistency and precision in the discussion of translating articles of the abovementioned press medium. -- Editors and theorists agree that an understanding of the source language text is essential. To review the basis for understanding the source language correctly, a text analysis of an English text and its Chinese translation will be performed. This analysis will take a functional approach which is based on Halliday's model of analysing the functional grammar of English. The first concern is with the analysis of clause complexes. The thinking behind this concern is influenced by Bell's approach to the clause. Such a functional approach is applied to the Chinese text. This does not' mean a complete application of Halliday's functional model to the Chinese language, but the functional approach will be used as a tool to reveal the relationship between the two languages as well as to analyse the source language. -- Case studies developed from the textual analysis of different types of magazines and from discussions with the translators or bilingual editors about their views of the translating process will be presented. Samples of articles illustrating the difficulties and challenges are also cited. / Mode of access: World Wide Web. / viii, 212 [28] leaves ill