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Dihydrochalcones (DHCs) are specialised metabolites with a limited natural distribution, found in significant amounts in Malus x domestica Borkh. (cultivated apple) and wild Malus species. Among them, M. x domestica accumulates significant amounts of phloridzin, whilst trilobatin and sieboldin are abundant in some wild relatives. DHCs have demonstrated a wide range of bioactive properties in biomedical models. Some DHCs have also been reported to act as flavour enhancers. Phloridzin may act as an anti-diabetic compound by blocking sodium-linked glucose transport and renal reabsorption of glucose in kidneys. Despite the protective effects reported in mammal models, little is known about how these metabolites are biosynthesised and what is their function in planta, where it has been hypothesised a role for phloridzin in plant growth. The biosynthetic pathway leading to DHC formation has been proposed in apple, and some steps have been characterised recently. DHC pathway diverts from the main phenylpropanoid pathway most probably from 4-coumaroyl-CoA by the action of a yet unknown reductase that would produce 4-dihydrocoumaroyl-CoA. Then, chalcone synthase (CHS) catalyses its condensation to form phloretin. Phloretin can be directly glycosylated at position 2′- or 4′ by the previously characterised 2′- and 4′-O-UDP-glycosyltransferases PGT1 and PGT2, to produce phloridzin or trilobatin, respectively. However, sieboldin has been postulated to derive from hydroxylation in position 3 of phloretin before been glycosylated, and the key responsible enzyme producing 3-hydroxyphloretin has not been yet discovered. The main aim of this PhD proposal was to provide a better understanding of the physiological functions of DHCs in apple, as well as to contribute to the elucidation of the biosynthetic pathway as the molecular basis for future genetic engineering in apple. Towards this aim, functional characterisation was conducted in MdPGT1 knockdown apple lines by RNAi silencing and CRISPR/Cas9 genome editing to assess the physiological effect of targeting a key biosynthetic gene involved in phloridzin biosynthesis. In addition, molecular, transcriptomic and metabolomic analyses were integrated to evaluate candidate genes accounting for 3-hydroxylase activity involved in DHC biosynthesis in wild Malus species accumulating sieboldin. Moreover, a de novo transcriptome assembly was carried out in an intergeneric hybrid between M. x domestica and Pyrus communis L. known to accumulate intermediate levels of DHCs compared to apple, in order to identify additional genes potentially involved in DHC pathway. We compared the physiological effect of reducing phloridzin through PGT1 knockdown by RNAi silencing and CRISPR/Cas9 genome editing. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology as reported in literature, whereas genome edited lines exhibited normal growth despite reduced foliar phloridzin. Bioactive brassinosteroids and gibberellins were found to be key players involved in the contrasting effects on growth observed following phloridzin reduction. Moreover, a cytochrome P450 from wild M. toringo (K. Koch) Carriere syn. sieboldii Rehder, and M. micromalus Makino was identified as dihydrochalcone 3-hydroxylase (DHCH), proving to produce 3-hydroxyphloretin and sieboldin in yeast. Different DHCH allele isoforms found in domesticated apple and M. toringo and M. micromalus correlated with sieboldin accumulation in a Malus germplasm collection. Finally, the assembled de novo transcriptome of the intergeneric apple/pear hybrid integrated to functional annotation and metabolomic analysis resulted in the identification of genes potentially involved in DHC biosynthesis, providing the basis for future biochemical characterisation. Altogether these results contribute to get insight into the roles of DHCs in apple and to illustrate how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple. Furthermore, the present PhD thesis contributes to the state-of-the-art by elucidating key missing steps in the biosynthesis of DHCs, which could be relevant for future establishment of genetic engineered lines that contribute to assess physiological effects of altering DHCs content, as well as to establish heterologous expression systems to produce de novo DHCs.

George MacDonald's last major work of fiction, Lilith, was published in 1895, but the first version of the romance was written in March of 1890. Lilith is an account of the unintentional journey of the protagonist into another world populated by both mythological figures drawn from the Judeo-Christian tradition and by horrific personifications of the psychological horrors of the protagonist's own mind. The story of Lilith describes the protagonist's experiences in this other world which bring him to the point of repentance. The manuscript of the first version, known now as Lilith A, is housed in the British Library along with seven other typed revisions and printer's proofs. Taken together, the A-H manuscripts of Lilith represent the complete production history textual evolution of what is arguably MacDonald's greatest literary work. The body of this paper contains the 161 page transcription of Lilith A produced from the original manuscript and a microfilm photographic reproduction provided by the British Library. The introduction of this paper outlines the history of Lilith A, describes it's similarities and differences with the published version, provides a bibliographic description of the manuscript, and outlines the editorial principles used in producing the transcript of the text. The introduction is followed by a transcription of the title page created for the manuscripts of Lilith by Winifred Louisa, Lady Troup, who was MacDonald's daughter and amanuensis. This title page is followed by the transcription of Lilith A. / Master of Arts

Thesis advisor: Jia Niu / In the last ten years, programmable CRISPR-Cas systems have been widely-used as genome editing tools for gene manipulation, epigenetic functionalization, and transcriptional regulation. Among them, fusing effector proteins directly to the Cas protein allows the resulting CRISPR machinery to direct these effector proteins to multiple sites of the same gene or multiple genes at once. Although they can be used to target multiple genetic loci simultaneously, these methods are often limited to applying one regulatory function (e.g., activation or repression) at a time. On the other hand, recruiting effector proteins via RNA aptamer-RNA-binding protein (RBP) recognition enabled multiplexed and multi-modular gene manipulations simultaneously. However, there are only a limited set of aptamer-RBP pairs that can function orthogonally and intracellularly, e.g., MS2 RNA aptamer with MS2 coat protein (MCP), and PP7 RNA aptamer with PP7 coat protein (PCP). The scarcity of orthogonal intracellular aptamer-RBP pairs imposes severe constraints on the CRISPR-mediated multifunctional manipulations of the genome and the epigenome. We established an intracellular selection platform for RNA aptamers, named CRISPR-Hybrid, and expanded the scope of aptamer-RBP toolkit for CRISPR transcription regulators. Using CRISPR-Hybrid, we successfully identified a highly active and specific aptamer for bacteriophage Qβ coat protein (QCP) in vivo, and characterized its binding affinity and specificity in vitro. We further validated the orthogonality of selected aptamer with QCP to other available intracellularly functional aptamer-RBP pairs including MS2-MCP and PP7-PCP in mammalian cells. Finally, we demonstrated the utility of this orthogonal pair in multiplexed and multi-modular regulations of endogenous genes. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Technical interviews have become a popular method for recruiters in the tech industry to assess job candidates' proficiency in both soft skills and technical skills as programmers. However, these interviews can be stressful and frustrating for interviewees. One significant cause of the negative experience of technical interviews was the lack of feedback, making it difficult for job seekers to improve their performance progressively by participating in technical interviews. Although there are open platforms like Leetcode that allow job seekers to practice their technical proficiency, resources for conducting mock interviews to practice soft skills like communication are limited and costly to interviewees. To address this, we investigated how professional interviewers provide feedback if they were conducting a mock interview and the difficulties they face when interviewing job seekers by running mock interviews between software engineers and job seekers. With the insights from the formative studies, we developed a new system for technical interviews aiming to help interviewers conduct technical interviews with less cognitive load and provide context-rich feedback. An evaluation study on the usability of using our system to conduct technical interviews further revealed the unresolved cognitive loads of interviewers, underscoring the requirements for further improvement to facilitate easier interview processes and enable peer-to-peer interview practices. / Master of Science / Technical interview is a common method used by tech companies to evaluate job candidates. During these interviews, candidates are asked to solve algorithm problems and explain their thought processes while coding. Running these interviews, recruiters can assess the job candidate's ability to write codes and solve problems in a limited time. At the same time, the requirements for interviewees to talk aloud help interviewers evaluate their communication and collaboration skills. Although technical interviews enable employers to assess job applicants from multiple perspectives, they also introduce interviewees to stress and anxiety. Among the many complaints about technical interviews, one significant difficulty of the interview process is the lack of feedback from interviewers. As a result, it is difficult for interviewees to improve progressively by participating in technical interviews repeatedly. Although there are platforms for interviewees to practice code writing, resources like mock interviews with actual interviewers for job seekers to practice communication skills are costly and rare. Our study investigated how professional programmers run mock technical interviews and provide feedback when required. The mock interview observations helped us understand the standard procedure and common practices of how practitioners run these interviews. At the same time, we concluded the potential cause of cognitive loads and difficulties for interviewers to run such interviews. To answer the difficulties of conducting technical interviews, we developed a new system that enabled interviewers to conduct technical interviews with less cognitive load and provide enriched feedback. After rerunning mock interviews with our system, we noted that while some features in our system helped make the interview process easier, additional cognitive loads are unresolved. Looking into these difficulties, we suggested several directions for future studies to improve our design to enable an easier interview process for interviewers and support interview rehearsals between job seekers.

Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR gene. A significant number of mutations (~13%) alter the correct splicing of the CFTR gene, causing the transcription of aberrant transcripts resulting in the production of a non-functional CFTR channel. We focus our research on two intronic CF causing mutations, 3272-26A>G and 3849+10kbC>T that create a new acceptor and donor splice site, respectively, generating in the inclusion of intronic portions into the mRNA. We developed a new genome editing approach to permanently correct the abovementioned mutations by means of CRISPR nucleases. We exploited the use of either Streptococcus pyogenes Cas9, SpCas9, or Acidaminococcus sp. BV3L6, AsCas12a, to edit the aberrant splicing sites and restore the production of the correct transcript, avoiding modifications of the CFTR coding sequence. A comparative analysis between SpCas9 and AsCas12a revealed that the use of AsCas12a with a single crRNA efficiently edits the target loci, producing correctly spliced mRNAs in both 3272-26A>G and 3849+10kbC>T mutations. Furthermore, this genetic repair strategy proved to be highly specific, exhibiting a strong discrimination between the mutated and the wild-type allele and no detectable off-target activity with genome-wide analysis. The selected crRNAs were tested in patients derived primary airway cells and intestinal organoids compound heterozygous for the 3272-26A>G or 3849+10kbC>T mutations, that are considered relevant CF models for translational research. The efficient splicing repair and the complete recovery of CFTR channel activity observed confirmed the goodness of the proposed gene editing strategy. These results demonstrated that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

Hollywood film editing remains on the theoretical margins of contemporary film scholarship, and the cause of this is three-fold. First, despite advances in collaborative authorship studies, the Hollywood film director is still largely regarded as the sole creative lynchpin upon which the film’s success or failure ultimately lies. Second, Classical Hollywood film editing—commonly referred to as the continuity aesthetic—is considered successful if it remains unnoticed, if it remains invisible. Therefore, within this continuity aesthetic, the editor’s ultimate goal is to hide his or her own labor. Third, determining exactly how and where a film editor contributed to a film text during post-production is an incredibly difficult task. So, what is the solution? This dissertation explores how film archives can contribute to knowledge about the cinematic post-production process. My central research questions are: what kinds of information do film archives contain regarding the creative collaboration between the director and the editor? And, what does available archive material tell us about the changes and creative revisions in post-production? To answer these questions, I conducted original archival research on the following Hollywood Renaissance films: Bonnie and Clyde (1967), The Conversation (1974), Annie Hall (1977), and Raging Bull (1980). These films reflect a highly creative era in the Hollywood industry and are well-known for the collaborative relationship between the directors and the editors. To determine how and where collaborative authorship occurred in these films, I compared archival documents such as the storyboards and shooting scripts to the final film texts. These documents contain explicit instructions about how the scenes should be lit, decorated, and shot and how the film itself should be edited together. Therefore, I argue that any editing discrepancies between these documents and the final films were the result of a creative collaboration between the director and the editor. Ideally, this model of “script-to-screen” archival research will inspire other academics to investigate how and where a film’s creative revision occurs during post-production—and to what effect. / text

Este trabalho é um estudo sobre a montagem audiovisual. A pesquisa se estrutura em torno das relações entre os elementos fundamentais do ofício de se montar imagens e sons: o tempo, o espaço, a montagem, as ferramentas de trabalho, media pipelines e o mapa. Para tanto, investigamos a natureza algorítmica e tecnológica da montagem audiovisual, as formas de sua aplicação na produção industrial e também as possibilidades de criação formas singulares de montagem que se originam a partir da exploração de fissuras (cracks) decorrentes das características algorítmicas da montagem audiovisual. Esta pesquisa também abrange a análise de um conjunto de produtos televisivos seriados, dos filmes Zidane: A 21st Century Portrait, Azul é a Cor Mais Quente, Blue, entre outros. / This research is a study on audiovisual montage. Time, space, montage, tools, media pipelines and maps are the key elements of the craft of editing sound and images and this research is structured around these fundamentals. The algorithmic nature of montage and its use in industrial media production and the possibility of creating unique montage styles that explores the cracks in this algorithm are investigated in this thesis. This research also analyses a set of television series, the feature films Zidane: A 21st Century Portrait, Blues is the Warmest Color, Blue, among others.