miRNAs are small regulatory RNAs, which function as post-transcriptional mRNA regulators. They direct ribonucleoprotein complexes to cognate mRNA to repress them by translational inhibition and degradation. miRNAs regulate thousands of mRNAs in mammals and have been recognized as regulatory factors in most cellular and developmental processes. Dysregulation of the miRNA pathway can lead to severe defects and diseases. Interestingly, a unique situation exists in mouse oocytes, where all the miRNA pathway components are present, yet the pathway is dispensable and nonfunctional, the molecular foundation of this phenomenon and its significance still remain unclear. In spite of the pronounced effects of the miRNA pathway in gene regulation in somatic cells, study strategies of the pathway bare limitations. Current methods for studying the activity of the miRNA pathway employ corelative studies (such as NGS) or reporter assays, which have relatively low throughput and are prone to artifacts. Here, I present design and development of a new strategy for directly monitor global miRNA pathway activity and integrity in near physiological conditions in living cells, which could also be employed in vivo for studies of mouse oocytes. The strategy is based on fluorescently tagged endogenous proteins of the...

abstract: A novel clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool for simultaneous gene editing and regulation was designed and tested. This study used the CRISPR-associated protein 9 (Cas9) endonuclease in complex with a 14-nucleotide (nt) guide RNA (gRNA) to repress a gene of interest using the Krüppel associated box (KRAB) domain, while also performing a separate gene modification using a 20-nt gRNA targeted to a reporter vector. DNA Ligase IV (LIGIV) was chosen as the target for gene repression, given its role in nonhomologous end joining, a common DNA repair process that competes with the more precise homology-directed repair (HDR). To test for gene editing, a 20-nt gRNA was designed to target a disrupted enhanced green fluorescent protein (EGFP) gene present in a reporter vector. After the gRNA introduced a double-stranded break, cells attempted to repair the cut site via HDR using a DNA template within the reporter vector. In the event of successful gene editing, the EGFP sequence was restored to a functional state and green fluorescence was detectable by flow cytometry. To achieve gene repression, a 14-nt gRNA was designed to target LIGIV. The gRNA included a com protein recruitment domain, which recruited a Com-KRAB fusion protein to facilitate gene repression via chromatin modification of LIGIV. Quantitative polymerase chain reaction was used to quantify repression. This study expanded upon earlier advancements, offering a novel and versatile approach to genetic modification and transcriptional regulation using CRISPR/Cas9. The overall results show that both gene editing and repression were occurring, thereby providing support for a novel CRISPR/Cas system capable of simultaneous gene modification and regulation. Such a system may enhance the genome engineering capabilities of researchers, benefit disease research, and improve the precision with which gene editing is performed. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2018

Made available in DSpace on 2015-05-07T14:46:39Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1183621 bytes, checksum: b37fc0d4ae2e17bcfe0d2e1da4b88821 (MD5) Previous issue date: 2012-03-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study aims to understand the production of Brazilian documentaries of today from one of its technical processes: the montage. We want this work to study the relationship between this stage of production and the construction of the narrative, since it is the montage process that the raw material takes shape, it is composed of images recorded for the film or image file, giving rise to the audiovisual work finalized. Undergirded by the main theories of montage and documentary filmmaking, we analyze how is the construction of their own voice in each of the films that comprise the corpus of this thesis. In the first case, we observed the relationship between the montage and construction of their own voice of the film based on the spoken word in the form of interviews and voice-over narration from the example of O fim e o princípio (2005), by Eduardo Coutinho. We take the device used in this film to apply the concepts of mounting evidence and modes of representation of Nichols (2009). In the second case we turn to the expressive use of montage features in the construction of a narrative content more poetic, this time analyzing the film Nós que aqui estamos por vós esperamos (1998), by Marcelo Masagão, where we observe the role of montage in process of "reframing" of archive images that make up the whole movie (BERNARDET, 1999). Thus, starting from two distinct forms of montage, we sought to understand the most comprehensive way possible to mount as the place of construction of the narrative in an attempt to unite the technique to the theory of media in order to give our contribution to the studies applied to contemporary documentary . / Neste estudo buscamos compreender a produção dos documentários brasileiros dos dias atuais a partir de um dos seus processos técnicos: a montagem. Queremos neste trabalho estudar a relação existente entre esta etapa da produção e a construção da narrativa, uma vez que é na montagem que o material bruto ganha forma, seja ele composto por imagens gravadas para o filme ou por imagens de arquivo, dando origem à obra audiovisual finalizada. Embasados pelas principais teorias da montagem e do cinema documentário, analisamos como se dá a construção da voz própria em cada um dos filmes que compõem o corpus desta dissertação. No primeiro caso, observamos a relação entre a montagem e a construção da voz própria do filme com base na palavra dita sob a forma de entrevistas e narração em voz over, a partir do exemplo de O fim e o princípio (2005), de Eduardo Coutinho. Aproveitamos o dispositivo adotado neste filme para aplicarmos os conceitos de montagem de evidência e modos de representação de Nichols (2009). No segundo caso nos atemos ao uso expressivo dos recursos de montagem na construção de uma narrativa de teor mais poético, desta vez analisando o filme Nós que aqui estamos por vós esperamos (1998), de Marcelo Masagão, onde observamos o papel da montagem no processo de ressignificação das imagens de arquivo que compõem todo o filme (BERNARDET, 1999). Assim, partindo de duas formas distintas de montagem, buscamos compreender da maneira mais abrangente possível a montagem enquanto lugar de construção da narrativa, em uma tentativa de unir a técnica à teoria do audiovisual no intuito de dar nossa contribuição para os estudos aplicados ao documentário contemporâneo.

Edição semidiplomática de corpus selecionado a partir do códice ?Ofícios do Vice-Rei para o Governador da Capitania 1793/98?, visando a fornecer fonte fidedigna para futuros estudos filológicos, lingüísticos, históricos e culturais do período. Trata-se também de um estudo mais detalhado desse códice catarinense que contextualiza historicamente os documentos que o compõem, assim como aborda aspectos paleográficos, codicológicos e diplomáticos com o propósito de responder a questionamentos e hipóteses surgidos durante a edição acerca de sua composição, criação e origem. Este trabalho descreve codicológica e paleograficamente o códice em geral, apresenta uma demonstração de diferentes punhos e procura levantar traços de natureza fonológica de um manuscrito dentro do códice. Consta também deste trabalho uma tentativa de identificação tipológica mais adequada através da composição de um verbete descritivo do corpus. / Semidiplomatic edition of the selected corpus from the codex ?Oficios do Vice Rei para o Governador da Capitania 1793/98? (Memorandums from the Vice-king to the Governor of the Province 1793/98?) seeking to provide a trustworthy source to further philological, linguistical, historical and cultural studies of such period. It is also a thorough study of this Catarinense (from Santa Catarina) codex that put its documents into a historical perspective. It touches its paleographic, codicological and diplomatic aspects aiming to address questionings and hypotheses raised during the editing process concerning its composition, how it was assembled, and its origin. This work describes the codex general paleography and codicology. It presents a demonstration of different handwritings and attempts to raise traces of phonological aspects from the codex. This project also proposes a more adequate typological identification of the documents, through descriptive summaries of each memo.

A produção de proteínas terapêuticas recombinantes compreende moléculas complexas e de alto valor agregado, incluindo a enzima glucocerebrosidase (GCase). Sua deficiência resulta na Doença de Gaucher, passível de tratamento por meio da terapia de reposição enzimática. A forma ativa da GCase recombinante usada na terapia apresenta resíduos terminais de manose expostos no seu perfil de glicosilação. Perfil este que espera-se ser reproduzido por meio da construção de uma linhagem de Pichia pastoris com um padrão de glicosilação humanizado, por meio da deleção de dois genes envolvidos no sistema de glicosilação da levedura: alg3 e och1, responsáveis pela posterior hiper-manosilação característica desse organismo. Assim, a expressão da GCase será usada como modelo no desenvolvimento desta linhagem de Pichia pastoris que permita a expressão de glicoproteínas com um perfil humanizado específico de glicosilação. Além da produção da linhagem mutante pela técnica de CRISPR/Cas9, propomos a construção de duas linhagens controle: uma expressando a proteína GCase para análise do seu padrão selvagem de glicosilação em P. pastoris e outra expressando a proteína Cas9 de Streptoccocus pyogenes (SpCas9). A linhagem P. pastoris/GCase foi construída testando-se duas sequências sinal de secreção diferentes: fosfatase alcalina (PHO1) e albumina humana (Alb). Resultados de western blot mostraram a GCase no lisado celular e baixos níveis de proteína secretada no sobrenadante de cultura, sendo mais expresso na linhagem contendo a sequência PHO1. A linhagem P. pastoris/SpCas9 foi construída e a enzima SpCas9 foi detectada via western blot no lisado celular após indução com metanol. Para a produção da linhagem com padrão de glicosilação humanizado propôs-se a deleção dos genes alg3 e och1 e a inserção, pela via de reparo por recombinação homóloga (HDR), de marcas de resistência aos antibióticos higromicina ou canamicina. Para tal, propusemos a construção de dois vetores finais de expressão do sistema CRISPR/Cas9 em P. pastoris, cada um contendo a enzima SpCas9 e os RNAs guia (gRNAs) para deleção do gene alg3 ou och1, e também a construção de dois fragmentos para HDR contendo o gene de resistência ao antibiótico flanqueado por regiões de 1Kb de homologia com a região de deleção do gene alg3 ou och1. A construção dos vetores e fragmentos para HDR foram inicialmente feitas por meio de técnicas de clonagem clássica. No entanto, apesar de inúmeras tentativas, resultados de PCR e sequenciamento mostraram o insucesso das construções. Partiu-se então para a técnica de Gibson Assembly®, através da qual os dois fragmentos para HDR foram construídos. Porém, os vetores de expressão contendo SpCas9 e os gRNAs ainda apresentam dificuldades na sua construção. Esforços ainda estão sendo feitos para a construção dos vetores e consequente tentativa de estabelecimento das linhagens mutantes. O sucesso no estabelecimento de um sistema de expressão de proteínas heterólogas com este padrão de glicosilação humano específico permitirá a obtenção e possível comercialização da GCase em sua forma terapêutica. Além disso, permitirá possíveis edições genômicas futuras para um padrão de maior complexidade de glicosilação humanizado, criando uma plataforma nacional para produção de outras glicoproteínas terapêuticas de interesse biotecnológico. / The production of therapeutic recombinant protein comprises complex and high valued molecules, including the glucocerebrosidase enzyme (GCase). Its deficiency results in Gaucher Disease, susceptible of treatment by enzymatic replacement therapy. The active form of recombinant GCase employed in therapy presents exposed terminal mannose residues in its glycosylation pattern. We hope to reproduce such pattern by constructing a Pichia pastoris strain with a specific human glycosylation pattern through the deletion of two genes involved in yeast glycosylation system, alg3 and och1, responsible for the final hyper-mannosylation characteristic of this organism. Therefore, the expression of GCase will be a case model for the development of the recombinant Pichia pastoris strain that could allow the expression of glycoproteins with a specific humanized glycosylation profile. Despite the establishment of the mutant strain using the CRISPR/Cas9 technique, we propose the construction of two control strains: one expressing the GCase protein for analysis of its wild type glycosylation pattern and another one expressing the Cas9 protein from Streptoccocus pyogenes (SpCas9). The P. pastoris/GCase strain was constructed testing two different secretion signal sequences: alkaline fosfatase (PHO1) and human albumin (Alb). Western blot results have shown GCase in cell lysate and in low expression levels in culture supernatant, being more expressed in the strain containing the PHO1 signal sequence. P. pastoris/SpCas9 strain was constructed and SpCas9 enzyme was detected via western blot in cell lysate after the induction with methanol. To produce the strain with the humanized glycosylation pattern, the deletion of alg3 and och1 genes was proposed along with the insertion, by homology directed repair pathway (HDR), of hygromycin and kanamycin antibiotics resistance marks. In order to do so, we have proposed the construction of two final expression vectors of the CRISPR/Cas9 system in P. pastoris, each one containing SpCas9 enzyme and the guide RNAs (gRNAs) for deletion of alg3 or och1, and also the construction of two fragments for HDR containing the antibiotics resistance gene flanked by 1Kb regions of homology with the deleted regions of alg3 or och1. Vectors and HDR fragments constructions were initially performed using classic cloning techniques. However, despite numerous tries, PCR and sequencing results have shown the failure of the constructions. Then, we moved on to the Gibson Assembly® technique, through which the two HDR fragments were built. Still, the expression vectors containing SpCas9 and the gRNAs presented difficulties in its assembly. Efforts continue to be made to successfully construct the remaining vectors and to establish the mutant lineage. Success in the establishment of a heterologous protein expression system with specific human glycosylation pattern will allow the obtainment and possible commercialization of the therapeutic form of GCase. Furthermore, it will also allow possible future genomic editing to a high complexity human glycosylation pattern, creating a national platform for the production of other therapeutic glycoproteins of biotechnological interest.

São muitas as parcerias no cinema, mas a que relaciona o diretor de cena ao diretor de fotografia é certamente a principal. A proposta desta dissertação de mestrado é analisar o estilo de fotografia criado por Eduard Tissé em três dos filmes que trabalhou com Eisenstein. A metodologia teórica da pesquisa baseia-se em Roland Barthes a partir da análise do artefato primeiro do cinema, que é o fotograma. As dimensões sociais ligadas ao materialismo dialético e suas aplicações ao filme, na forma como o espectador promove a leitura de sentidos, preocupavam o diretor soviético. Com isso evidencia-se o princípio conceitual de Eisenstein, desenvolvido pela célula de montagem, na visão da sua missão sócio-cultural em busca da fotografia do conflito. / There are many partnerships in the movie business, but that between director and director of photography is certainly the main one. The purpose of this dissertation is to analyze the style of photography created by Eduard Tissé in three of the movies he worked with Eisenstein. The theoretical research methodology is based on Roland Barthes from the analysis of the first film artifact: the frame. The social dimensions related to the dialectical materialism and its applications to the cinema - the way the audience reads the film sense - worried the soviet director. With that, the conceptual principle developed by Eisenstein is highlighted in search of the conflict cinematography.

A montagem cinematográfica foi um advento que mudou os rumos da história do audiovisual. O processo de edição e montagem possui uma importância ímpar na constituição da linguagem audiovisual. Modificada desde Méliès e aperfeiçoada ao longo do tempo, a montagem cinematográfica se tornou um dos pilares sobre os quais se sustentam os estudos de diversos autores dentro da história do Audiovisual, especificamente na história e teoria do cinema. Através da leitura de inúmeras dessas obras, em conjunto com a análise fílmica, podemos constatar que a manipulação do tempo e do espaço durante o processo de montagem de uma obra audiovisual permite a construção de múltiplos sentidos discursivos. Considerando essa relevância da montagem na construção da narrativa fílmica, esse projeto visa refletir sua importância discursiva por meio da análise de um recurso empregado constantemente na montagem: o faux raccord. A evidência do aparato e a adição de sentidos na etapa da pós-produção através da utilização do faux raccord faz com que esse elemento seja fulcral na análise da importância da montagem discursiva. Assim, essa pesquisa busca postular uma precisa definição para o termo, bem como situá-lo na produção teórica e audiovisual ao longo da história. Compreendendo seus usos e aplicações e averiguando de que modo esse elemento gesta nos filmes sentidos discursivos e propicia ao espectador a percepção dessa construção de sentido através da evidência do corte. / Film editing was a invention that changed the course of audiovisual\'s history. The process of editing has a unique importance in the creation of audiovisual language. Modified since Méliès and improved over time, film editing became one of the pillars in film studies, specifically in history and film theory. By reading many theoretical works, combined with film analysis, we note that the manipulation of time and space during the film editing process of an audiovisual work allows the construction of multiple discursive meanings. Considering the importance of editing in the construction of film narrative, this project is a reflection of discursive importance through the analysis of a resource constantly used in film editing: faux raccord. Therefore, this research seeks to postulate a precise definition for the term, and place it in the theoretical and audiovisual production throughout history. Understanding its uses and applications, and reflecting how this element generates discursive meanings in films and provides the spectator the perception of meaning construction through the evidence of the cut.

En 1967, sous l’influence du jeune cinéma européen, Hollywood accède à son tour à la modernité : le « Vieil Hollywood » laisse place à un « Nouvel Hollywood ». Ce mouvement de renouvellement du cinéma américain, symboliquement inauguré par Bonnie and Clyde d’Arthur Penn et Le lauréat de Mike Nichols, est, explique-t-on, le fait d’une génération nouvelle de cinéastes, parmi lesquels, outre Penn et Nichols, Francis Ford Coppola, Brian De Palma, George Lucas, William Friedkin,Michael Cimino, Martin Scorsese, Robert Altman, Bob Rafelson, Hal Ashby... Il est aussi le fait d’une génération nouvelle de monteurs, qui, en complicité avec leurs réalisateurs, éprouvèrent de nouvelles formes d’expressivité esthétique et narrative, sans craindre de contrevenir aux règles conventionnelles instituées par leurs aînés ; parfois même en réaction contre ces règles. On peut notamment citer, parmi ces fidèles et précieux collaborateurs, Dede Allen, Walter Murch, Sam O’Steen, Verna Fields, Alan Heim, Paul Hirsch, Robert C. Jones, Richard Marks, ou encore Ralph Rosenblum. Nous veillons à déterminer, dans cette thèse, la part de responsabilité, tant collective qu’individuelle, de cette nouvelle génération de monteurs dans le mouvement de renouvellement esthétique du cinéma américain. Et, au-delà des personnes, d’apprécier ce style de montage néohollywoodien, d’évaluer son importance dans la constitution du style neuf des films américains des années 1970, de même que l’étendue de ses innovations. / In 1967, under the influence of European art films, Hollywood underwent important changes in the course of the 1960s : « Old Hollywood » was taken over by a « New Hollywood ». This process of renewal of American cinema, which has been symbolically launched by Arthur Penn’s Bonnie and Clyde and Mike Nichols’ The Graduate, is due, it has been said, to the arrival of a new – mostly film-school educated – generation of filmmakers, such as Francis Ford Coppola, Brian De Palma,George Lucas, William Friedkin, Michael Cimino, Martin Scorsese, Robert Altman, Bob Rafelson or Hal Ashby. It is also due to the arrival of a new generation of film editors who did not fear to challenge the doctrine of classical Hollywood editing and break the established rules in order to take a chance and try new ways of telling a story. These films editors are, amongst others, Dede Allen, Walter Murch, Sam O’Steen, Verna Fields, Alan Heim, Paul Hirsch, Robert C. Jones, Richard Marks, or Ralph Rosenblum. Our thesis examines the share of responsability, on both a collective and individual level, of this new generation of film editors in this process of renewal of American cinema in the 1960s and 1970’s. Its goal is, more generally, to determine and appreciate this new editing style, realize its importance and its influence on the Hollywood Renaissance style, as well as the wide range of its innovations.

Xanthomonas oryzae pv. oryzae (Xoo), l'agent causal de bactériose vasculaire du riz (BLB), injecte des protéines de liaison à l'ADN, appelées Transcription Activator-Like Effectors (TALEs) dans les cellules hôtes afin de moduler l'expression de gènes cibles. Certains TALEs agissent comme des facteurs de virulence majeurs, indispensables à la mise en place du BLB et ciblent des gènes de sensibilité du riz. Les TALEs majeurs de Xoo ciblent universellement les gènes de sensibilité de la famille SWEET. Il existe dans la nature un polymorphisme des séquences ADN des gènes SWEET reconnues par les TALEs qui confère une résistance à la maladie. L’utilisation de la technologie TALEN a permis d'introduire artificiellement ce type de mutation dans le promoteur du gène de sensibilité SWEET14 le rendant insensible aux TALEs et conférant une résistance à certaines souches de Xoo asiatiques. La caractérisation des populations de Xoo africaines montrent qu'elles sont distinctes de celles d’Asie. L’objectif du projet de thèse était de créer à l'aide des technologies d'édition des génomes des sources de résistances efficaces contre un large panel de Xoo maliennes.Dans une première partie, l'édition des boites ADN de SWEET14 ciblées par des TALEs majeurs de souches africaines a effectivement permis d'obtenir des résistances contre les souches utilisant le TALE TalF (initialement appelé Tal5) mais pas contre celles utilisant TalC. La caractérisation du répertoire de TALEs des souches maliennes par des approches fonctionnelles (sensibilité des lignées éditées, expression de SWEET14 et autres gènes cibles de TALEs) et in silico (séquençage du génome de 8 souches) à révélé une diversité fonctionnelle de ces répertoires et la présence simultanée quasi systématique de versions actives et redondantes de TalF et TalC. La caractérisation de la sensibilité de variétés de riz locales aux souches de Xoo maliennes a montré que ces dernières possèdent un large spectre de virulence et qu'à une exception près, toutes les variétés testées sont sensibles au BLB. L'édition en multiplex par la technique CRISPR/Cas9 des boites TalF et TalC a aboli l'induction de SWEET14 en réponse à une souche malienne. Cependant, les lignées correspondantes sont restées sensibles à cette souche. Dans la dernière partie, pour expliquer ce résultat, nous avons postulé l'existence d'au moins un gène de sensibilité, cible de TalC et redondant avec SWEET14. Une approche bioinformatique a permis d'identifier un locus dont plusieurs caractéristiques en faisaient un candidat intéressant. Ce locus, nommé ATAC (pour Alternative TalC Target) est composé de deux gènes, ATAC1 et ATAC2 induits de façon bidirectionnelle par TalC. Nous avons montré qu'ATAC2, qui code pour un facteur de transcription bHLH atypique potentiellement impliqué dans l’élongation cellulaire et l'immunité chez le riz, se comporte comme un locus de sensibilité lorsqu'il est induit par des TALE artificiels dans un système gain de fonction. Nous avons édité simultanément le promoteur SWEET14 et le locus ATAC. Ces éditions devraient empêcher la reconnaissance du promoteur SWEET14 et du locus ATAC par TalC et TalF afin de conférer une résistance large au BLB au Mali. / Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight of rice (BLB), injects DNA binding proteins called Transcription Activator-Like Effectors (TALEs) into host cells to manipulate plant genes expression. Some TALEs behave as major virulence factors essential for BLB to occur by binding directly to target DNA boxes of rice susceptibility genes and inducing their expression. Xoo major TALEs universally target susceptibility genes of the SWEET family. In nature, polymorphism in the DNA sequence of SWEET genes recognized by TALEs confers resistance to BLB. Using the TALEN technology, this type of mutations has been artificially introduced in the promoter of the SWEET14 susceptibility gene to make it TALE-unresponsive and confer resistance to some Asian Xoo. The characterizations of Malian Xoo populations show that they are distinct from the Asian ones. The PhD project aimed to create broad tailored BLB resistance against Malian Xoo using genome editing technologies.First, editing SWEET14 DNA boxes targeted by major TALEs of African strains indeed yielded resistance against strains relying on TalF (initially named Tal5) but not against those relying on TalC. The characterization of Malian strains TALE repertoires using functional (edited lines susceptibility assays, SWEET14 and other TALE target expression studies) and in silico (genome sequencing of 8 strains) approaches uncovered functional diversity in these repertoires and, the almost systematic, simultaneous presence of active and redundant versions of TalF and TalC. In susceptibility assays of local rice varieties, Malian Xoo strains exhibited a broad virulence spectrum and, with one exception, all tested varieties were susceptible to BLB. Multiplex editing of TalF and TalC target boxes with the CRISPR/Cas9 technology abolished SWEET14 induction in response to a Malian strain. However the corresponding rice lines remained susceptible to this strain. Finally, to explain these results, we postulated the existence of, at least, a TalC target susceptibility gene redundant with SWEET14. Bioinformatics analysis identified a rice locus with several features electing it as a high priority candidate. This locus named ATAC (Alternative TalC Target) is composed of two genes, ATAC1 and ATAC2, bidirectionally upregulated by TalC. We further showed that ATAC2 which is predicted to code for an atypical bHLH transcription factor potentially involved in rice cell elongation and immunity, behaves as a susceptibility gene upon artificial TALEs-mediated induction in a gain of function assay. We used the CRISPR/Cas9 system to simultaneously edit the SWEET14 promoter and the ATAC locus. These mutations should prevent the recognition of the SWEET14 promoter and the ATAC locus by TalC and TalF, compromise their transcriptional induction and ultimately provide broad BLB resistance in Mali.

This submission falls into two sections: a thesis and a screenplay. My thesis presents an original approach to screenwriting using storytelling dynamics found in film editing; I call this “writing-for-the-cut”. This section also contains my software experiments that hold the promise of innovative digital tools for screenwriters. In the second section I apply both my editing strategy and my software experiments in the production of an original screenplay called Rush the Sky. In the history of the screenplay, the advent of the master scene format, which gained fairly wide circulation from the early 1950s (Price 11), marked a moment of separation of the screenwriter from the film production process. Up to this point most screenwriters worked closely with studios and were steeped in the contiguous crafts of filming and editing. But the master scene format freed the script from all references to the ‘factory’ and in so doing fundamentally transformed film writing culture; now a new generation of largely non-specialists were writing for the big screen. To fill the ‘film school’ void occasioned by the loss of studio apprenticeships and mentoring, a lively market in guru screenwriting manuals emerged, particularly from around the 1970s. Taking their cue from the ‘no camera angles’ injunction on screenplays, the manual-writers tended to delineate the territory of screenwriting as a craft detached from production; in this way manual-readers have been discouraged from any serious consideration of the follow-on crafts (filming and editing) as potential modifiers of the screenplay. The perhaps unintended consequence has been that ‘manual culture’ has come to foster a view of film as a finished, projected product: we are ‘writing for cinema’. I propose an alternative strategy: the edit suite, not the cinema, is the real destination for our screenplay. This is a view of film as a constructed product: we are ‘writing for the cut’. This idea finds its roots in the lively theories and debates advanced by the early Soviet filmmakers such as Lev Kuleshov and Sergei Eisenstein in the 1910s and 1920s. For them, as Pudovkin declared “The foundation of film art is editing” (Pudovkin xiii). They viewed editing as a juxtapositional dynamic, one that engaged the inductive capacities of the audience to ‘discover’ the story. From this Hegelian notion of juxtaposition to its more nuanced application today, I identify three kinds of editorial juxtaposition that are essential to cinematic storytelling: poetry, puzzle, and kinesis. I suggest that these juxtapositions are interrelated and on axes of intensity: Poetry to Prose, Puzzle to Exposition, and Kinesis to Stasis. Finally, I identify how each of these editing terms can be adapted for use by screenwriters. In the second part of this submission, Rush the Sky is a demonstration of how the techniques of writing-for-the-cut can be applied in practice. This is a fast moving thriller in the style of British Indie films such as Trainspotting (wr: John Hodge dir: Danny Boyle1996), Sexy Beast (wrs: Louis Mellis, David Scinto dir: Johanthan Glazer 2000), and Dead Man’s Shoes (wrs: Paddy Considine, Shane Meadows dir: Shane Meadows 2004). Rush the Sky tells the story of two adrenaline-addicted lovers: Ella and Luke. Luke is a young base-jumper who has witnessed a gangland murder. Desperate to escape the mob and the police, he climbs a high mast and base-jumps into a storm cloud. Struck by lightning, he falls to earth in a coma. Ella joins forces with Luke’s feral brother Jared and together they ‘rescue’ Luke from hospital and attempt to wake him up. Rush the Sky is a non-linear story that interweaves a present-day road movie with a darkly euphoric backstory. Some of the specific editing figures I employ include parallel action, ‘split-edit’, non-linear shuffle, scene-scripted montage, and ellipsis. In the development of the treatment, I devised a hybrid writing-editing interface that allowed me to ‘mount’ and sequence the beats of my story. This is a kinetic environment where the beats are displayed as text, proxy images and film clips. In this way the familiar write/read/revise process of screenwriting moved closer to the play/watch/edit process of the cutting room. I strongly believe this approach could herald a fresh way of both composing a screenplay and ‘proving’ the cinema-worthiness of the story before filming commences.