THE ATTIC DOOR is the feature-length film co-written and directed by Danny Daneau while pursuing a Master of Fine Arts at the University of Central Florida. Many challenges had to be met to produce a feature-length motion picture utilizing digital technology on an ultra-low budget as part of a graduate education. Beyond gaining a profound understanding of the physical, financial, and emotional strength it takes to complete a feature-length motion picture, Daneau experienced the creative challenges that all filmmakers must meet when applying the principles of filmmaking theory to an actual work of self-expression. The production process for an original narrative film under the guidelines established by the university has pushed him to make a motion picture that is both a highly personal work of film art and evidence of the educational journey he has taken for the past three years.

The recent call to reconstruct a detailed picture of the tree of life for all organisms has forever changed the field of molecular phylogenetics. Sequencing technology has improved to the point that scientics can now routinely sequence complete plastid/mitochondrial genomes and thus, vast amounts of data can be used to reconstruct phylogenies. These data are accumulating in DNA sequence repositories, such as GenBank, where everyone can benefit from the vast growth of information. The trend of generating genomic-region rich datasets has far outpaced the expasion of datasets by sampling a broader array of taxa. We show here that expanding a dataset both by increasing genomic regions and species sampled using GenBank data, despite the inherent missing DNA that comes with GenBank data, can provide a robust phylogeny for the plant order Caryophyllales (angiosperms). We also investigate the utility of trnK intron in phylogeny reconstruction at relativley deep evolutionary history (the caryophyllid order) by comparing it with rapidly evolving matK. We show that trnK intron is comparable to matK in terms of the proportion of variable sites, parsimony informative sites, the distribution of those sites among rate classes, and phylogenetic informativness across the history of the order. This is especailly useful since trnK intron is often sequenced concurrently with matK which saves on time and resources by increasing the phylogenetic utility of a single genomic region (rapidly evolving matK/trnK). Finally, we show that the inclusion of RNA edited sites in datasets for phylogeny reconstruction did not appear to impact resolution or support in the Gnetales indicating that edited sites in such low proportions do not need to be a consideration when building datasets. We also propose an alternate start codon for matK in Ephedra based on the presense of a 38 base pair indel in several species that otherwise result in pre-mature stop codons, and present 20 RNA edited sites in two Zamiaceae and three Pinaceae species. / Ph. D.

More than 300 human conditions, ranging from cancer predisposition to developmental and neurological mendelian disorders, are caused by haploinsufficiency (HI), a genetic condition by which mutational inactivation of a single allele leads to reduced protein levels and is enough to produce the disease phenotype. Therefore, translational enhancement of the spare allele could exert a therapeutic effect. Here we propose a novel approach for the potential rescue of haploinsufficiency disease loci based on the insertion of specific single nucleotide changes in the Kozak sequence. Since this sequence controls translation by regulating start codon recognition, we aimed at identifying and introducing specific nucleotide variations to enhance translation and rescue haploinsufficiency. To do so, we used CRISPR-Cas base editors, able to generate single nucleotide changes in genomic DNA without the need of a donor DNA and without creating double-strand breaks. We performed a high-throughput screening to evaluate the strength of the Kozak sequences of 231 haploinsufficient genes. We compared the translational efficiency of each wild-type sequence to that of several variants using FACS-seq, which combines fluorescence-activated cell sorting and high-throughput DNA sequencing. We thus selected 5 candidate genes (PPARGC1B, FKBP6, GALR1, NRXN1, and NCF1) and several nucleotide variations able to up-regulate translation. Finally, we used CRISPR-Cas base editors to reproduce the most efficient variants of NCF1 in a cell model relevant for the associated haploinsufficient disease and verified the increase of protein levels. This study proposes a novel therapeutic strategy to rescue haploinsufficiency and sheds new insights into the regulatory mechanisms underlying the translational process. On a broader level, the possibility of modulating gene expression by acting exclusively on translation expands the CRISPR-Cas genome editing applications.

The development of a preimplantation embryo is a stepwise process consisting of morphological, biochemical, and genomic changes. Much remains unknown about the attainment of embryo competency to develop and establish pregnancy. To investigate this, we compared methods of selection at the oocyte or embryo level for improved blastocyst production. Brilliant cresyl blue staining was used to sort oocytes by their growth status (not fully grown vs. fully grown) and the timing of the first embryonic cell division to sort embryos. We found that an embryo's cleavage kinetics are more indicative of their competency than the growth status of the oocyte that gave rise to that embryo. We further investigated the cryopreservation survival of embryos with fast or slow cleavage kinetics and found no significant differences in their ability to hatch post-thawing. Next, we used the complete sequence of the cattle Y chromosome to identify oligonucleotides for efficient sexing of samples. These materials may be used to understand sexual dimorphism as a biological factor in future experiments. Finally, we designed a new method to induce targeted DNA sequence deletions and mRNA cleavage in zygotes using CRISPR-Cas. We targeted the gene OCT4, since the literature shows variable knockout outcomes. Our method improved deletion efficiency while accounting for preexisting or maternally inherited mRNA of the target gene. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes. / Master of Science / The development of an early embryo involves many biological and structural changes. Much remains unknown about the influences on embryo quality and ability to successfully develop. To investigate this, we compared methods for selecting the highest quality cattle eggs or embryos. We found that the observation of an embryo's development speed is better for selecting high quality embryos than egg quality. We further investigated the freezing survival of embryos with fast or slow growth. We found that the freezing survival of fast and slow growing embryos is not different. Next, we used the complete sequence of the cattle Y chromosome to identify PCR primers for determining sample sex. These resources can help us understand how an individual's sex can influence biological differences. Finally, we designed a new method for removing the total function of a gene in embryos. For this, we deleted segments of DNA and cut RNAs. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24912号 / 農博第2575号 / 新制||農||1102(附属図書館) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 大門 高明, 教授 松浦 健二, 准教授 小野 肇 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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