The dissertation consists of a critical preface and excerpts from the novel Patrol. The preface explores how the novel Patrol utilizes characters that engage with tropes of the Romantic Genius in order to establish their subjectivity while navigating the standardizing mechanisms of twenty-first century information technologies. The preface analyzes how the rise of the organic food movement, the usage of biotech genetic engineering, and the tactics of Big Data-era marketing all inform the critical underpinnings of Patrol, situating the novel in conversation with works of fiction and nonfiction that also explore the interplay of these topics with contemporary American culture. Set primarily in Cincinnati, Ohio, the bifurcated narrative of the novel Patrol enlists the perspectives of both a science-tech father from the Boomer generation, Tim Smith, and his millennial public relations-major daughter, Sarah Smith. Both work in industries that seek to utilize the concept of the individual genius in service of quantification. Tim and Sarah’s interactions with Alexandra Smith, a family member who transitions from female to male over the course of the novel, cause both protagonists to recognize that their own identities are malleable, and this discovery goads each into reexamining their career choices and personal relationships. The plot depicts the outcome of these explorations, culminating in a series of choices for Tim and Sarah that showcase the fundamental change in each character. Unable to simply quantify themselves and those around them, Tim and Sarah instead adopt a more nuanced view of the world that seeks to find a balance between the individualistic conceit of the Romantic genius and the quantifying mandates of technology.

Synthetic genetic regulatory networks (or genetic circuits) can operate in complex biochemical environments to process and manipulate biological information to produce a desired behavior. The ability to engineer such genetic circuits has wide-ranging applications in various fields such as therapeutics, energy, agriculture, and environmental remediation. However, engineering multilevel genetic circuits quickly and reliably is a big challenge in the field of synthetic biology. This difficulty can partly be attributed to the growing complexity of biology. But some of the predominant challenges include the absence of formal specifications -- that describe precise desired behavior of these biological systems, as well as a lack of computational and mathematical frameworks -- that enable rapid in-silico design and synthesis of genetic circuits. This thesis introduces two major frameworks to reliably design genetic circuits. The first implementation focuses on a framework that enables synthetic biologists to encode Boolean logic functions into living cells. Using high-level hardware description language to specify the desired behavior of a genetic logic circuit, this framework describes how, given a library of genetic gates, logic synthesis can be applied to synthesize a multilevel genetic circuit, while accounting for biological constraints such as 'signal matching', 'crosstalk', and 'genetic context effects'. This framework has been implemented in a tool called Cello, which was applied to design 60 circuits for Escherichia coli, where the circuit function was specified using Verilog code and transformed to a DNA sequence. Across all these circuits, 92% of the output states functioned as predicted. The second implementation focuses on a framework to design complex genetic systems where the focus is on how the system behaves over time instead of its behavior at steady-state. Using Signal Temporal Logic (STL) -- a formalism used to specify properties of dense-time real-valued signals, biologists can specify very precise temporal behaviors of a genetic system. The framework describes how genetic circuits that are built from a well characterized library of DNA parts, can be scored by quantifying the 'degree of robustness' of in-silico simulations against an STL formula. Using formal verification, experimental data can be used to validate these in-silico designs. In this framework, the design space is also explored to predict external controls (such as approximate small molecule concentrations) that might be required to achieve a desired temporal behavior. This framework has been implemented in a tool called Phoenix. / 2021-02-28T00:00:00Z

The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.

The methylotrophic yeast, Pichia past oris, is one of the most respected and widely used systems today. The ability of this yeast to produce large masses of protein and metabolize methanol as a sole source of carbon and energy is attributed to the highly induceable Alcohol Oxidase I promoter (AOXI). Despite of the disperse popularity and use of this promoter over the last 15 years, little is known about the transcription controls at a molecular level. A 5'>3' deletion analysis of the AOXI promoter was perrormed to gain understanding of the promoter's regulation and provided insight to the approximate locations of the important regulatory regions. A total of 10 truncations were made unveiling two areas ofhigh activity located between positions, -257 to-235, and, -235 to -188. In addition, a 14-base pair internal deletion was made between positions, -215 to -201. This region was shown to be necessary for transcriptional activation by deletion analysis. Sufficiency studies suggested that this 14-base pair element could serve as an activator sequence in both glucose and methanol.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12948号 / 論農博第2820号 / 新制||農||1035(附属図書館) / 学位論文||H27||N4935(農学部図書室) / 32207 / (主査)教授 三芳 秀人, 教授 加納 健司, 教授 三上 文三 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

In mehrjährigen Feldversuchen wurde der Anbau von Bt-Mais mit unbehandelt angebautem Mais und einer Anbauvariante, die mit Insektiziden gegen den Maiszünsler behandelt wurde, verglichen. Untersucht wurden Maiszünslerbefall, entstandene Ertragsverluste, Futterwert, Futterqualität, Polleneintrag in Bienenvölker und Auskreuzung. Ökonomische Vorteile von Bt-Mais zeigen sich bei Körnermais ab Ertragsverlusten von 3 - 7 dt/ha und bei Silomais ab 5 Prozent Ertragsverlust gegenüber der konventionellen Sorte. Eine Veränderung der Futterqualität bei Bt-Mais oder der behandelten Variante konnte nicht festgestellt werden. Bt-Maispolleneintrag in Honig wurde im Spurenbereich nachgewiesen. Die Kennzeichnungsschwelle von 0,9 Prozent wird dabei deutlich unterschritten. Bt-Maispollenanteile im Honig sind in einer Entfernung von 3 km nachweisbar. In Bodenproben konnte keine Anreicherung mit Cry1Ab-Protein, das im Bt-Mais (MON 810) auf den Maiszünsler toxisch wirkt, nachgewiesen werden.

Over 90% of human genes produce precursor mRNA (pre-mRNA) that undergoes splicing, an RNA processing mechanism. Alternative splicing (AS) of pre-mRNA allows a gene to generate multiple coding and non-coding isoforms by removing introns and ligating distinct exonic combinations. It is a mechanism that plays a major role in driving molecular diversity in mammals. This process is tightly regulated to determine the types and levels of protein products expressed in specific cellular contexts. Cis-acting splicing regulatory elements (SREs) found within the pre-mRNA are recognized and bound by RNA-binding proteins that either assist or interfere with the recruitment of the spliceosome. In the field of splicing, a long-standing goal has been to develop a “splicing code”, or a set of rules to understand the splicing patterns of a gene in a predictable manner. It is essential to highlight the significance of sequence context for SREs and the potential impact that distal intronic elements can have on splice site selection to better understand splicing. Given the importance of sequence context and the involvement of distal intronic regions in splicing, future approaches aimed at identifying SREs should consider these factors.This thesis will describe the process of the identification and validation of two novel distal intronic SREs located in critical disease exons. Importantly, these findings were made by combining experimental and computational approaches and through the development of a high-throughput SRE screening methodology. Chapter 1 will provide a general context for splicing, in particular AS, as an important mechanism among a plethora of RNA regulatory functions. The significance of AS regulation will be explored, as it plays a key role in the occurrence of physiological events and incorrect regulation can trigger disease. I will also introduce several methods used to study SREs, with experimental efforts primarily focusing on exonic and proximal intronic sequences. Additionally, as mis-splicing is associated with disease, there is a high interest in modulating splicing with novel therapeutic interventions, the development of which benefits from an increased understanding of SREs. Specifically, I will provide a landscape view of the splicing research field for the genes spinal muscular atrophy 2 (SMN2) and microtubule-binding protein tau (MAPT), given their relevance to our discoveries. As there is currently a paucity of high-throughput methods for studying SREs, especially those that allow for analysis of SREs in a near-native sequence context, I will introduce the CRISPR-Cas system (dCas13d) as a potential splicing modulator. This system will form the foundation for developing a tool to help us understand splicing regulation. Chapter 2 will discuss the discovery of a distal SRE regulating MAPT exon 10 splicing divergence in the primate lineage. Our lineage-specific AS analysis found that MAPT exon 10 shows a two-step evolutionary shift in the Catarrhine and hominoid lineages. The previously identified splicing regulatory elements cannot explain this evolutionary shift. Instead, a key splicing factor, muscleblind-like (MBNL), was found to be a major contributor to the observed splicing pattern divergence. Further mechanistic dissection revealed divergent, distal regulatory sequences in intron 10 that are recognized by MBNL. Based on this finding, we also demonstrated the potential of developing a therapeutically compatible strategy to target the MBNL binding sites by a steric hindrance to modulate exon 10 splicing effectively. Chapter 3 will discuss the development of a method allowing for a more unbiased, high-throughput screening of SREs, including those in the distal intronic regions. This method relies on the nuclease-inactive dRfxCas13d to modulate splicing. We use a dual-color fluorescent splicing reporter to identify the impact of splicing in a high-throughput manner. For our proof of concept, we use SMN2 exon 7 to identify SREs that influence the splicing of this exon and corroborate our findings with known SMN2 SREs. We performed a screen on the SMN2 dual-color splicing reporter using a gRNA library and obtained highly reproducible results. The screen also correctly identified gRNAs targeting known SREs, including the well-studied exonic regions and the downstream ISS-N1 element, the target of the ASO therapeutic known as nusinersen. Importantly, this screen also discovered novel splicing inhibiting gRNAs in a more distal region of the downstream intron, suggesting that a robust splicing enhancer was targeted. Previous studies likely overlooked this region due to its distance from the exon. This novel approach allows for the simultaneous screening of sizeable genetic regions using a large-scale gRNA library.

Indiana University-Purdue University Indianapolis (IUPUI) / Transcription and alternative splicing play important roles in HIV-1 gene expression and replication and mandate complicated but coordinated interactions between the host and the virus. Studies from our group have shown that a HIV-1 Tat-interacting protein of 110 kDa, Tip110 synergies with Tat in Tat-mediated HIV-1 gene transcription and replication. However, the underlying molecular mechanisms were not fully understood and are the focus of the dissertation research. In the study, we first demonstrated that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) in a direct and specific manner. We then showed that Tip110 was detected at the HIV-1 long terminal repeat (LTR) promoter and associated with increased phosphorylation of serine 2 within the RNAPII C-terminal domain (CTD) and increased recruitment of positive transcription elongation factor b (P-TEFb) to the LTR promoter. Consistent with these findings, we demonstrated that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. During these studies, we also found that Tip110 altered HIV-1 mRNA alternative splicing and increased tat mRNA production. Subsequent analysis indicated that Tip110 selectively increased tat exons 1-2 splicing by activating HIV-1 A3 splice site but had no function in tat exons 2-3 splicing. We then showed that the preferential splicing activity of Tip110 resulted from Tip110 complex formation with hnRNP A1 protein, a negative splicing regulator that binds to the ESS2 element within tat exon 2, and as a result, blocked the complex formation of hnRNP A1 with ESS2 and subsequently activated HIV-1 A3 splice site. Taken together, these results show that Tip110 functions to regulate HIV-1 transcription elongation and HIV-1 RNA alternative splicing. These findings not only add to our understanding of Tip110 biology and function but also uncover a new potential target for development of anti-HIV intervention and therapeutic strategies.

Bacillus anthracis is a Gram positive, spore forming, non-motile, rod shaped, soil bacterium, and is endemic worldwide. Currently, the biology of B. anthracis is poorly understood. B. anthracis is one of many biological weapons used today. A -/- mutant strain of B. anthracis that lacks the pathogenic plasmids was created by serial culture at 42°C. Key DNA replication genes were identified by homology as targets. The dnaB gene, essential for B. subtilis initiation of DNA replication, was my focus. A vector system was created by polymerase chain reaction (PCR) with the pMUTIN4 integration vector and the promoter region of dnaB to study the genetics of B. anthracis. An electro-transformation system was formulated to knock-out the -/- B. anthracis dnaB gene. We have successfully incorporated the pMUTIN4 vector into the chromosomal DNA of B. anthracis. We also have formulated an electro-transformation system and vector system for use in B. anthracis.

The Corral de Piedra community is located in Chiquimula, Guatemala. The main agricultural activities are corn and bean production, small-scale gardens, and small animal production, such as poultry and pigs. The crop areas are predominantly located on lands with slopes between 12 and 55%, presenting low fertility conditions and poor moisture retention capabilities. In addition, local crop varieties have low productivity potentials and the winter season is characterized by poorly distributed and erratic periods of rain. The Benson Institute introduced a technology transfer model to increase and diversify agricultural production, while preserving current natural resources. An integral part of this model included educating the community on nutrition and health topics. The school was considered to be the most appropriate place to introduce training and education in agriculture among students and community leaders, thus contributing to community development.