REGULATION OF LOW DENSITY LIPOPROTEIN RECEPTOR SPLICING EFFICIENCY

Low density lipoprotein receptor (LDLR) is an apolipoprotein E (apoE) receptor and may play a role in Alzheimer’s disease (AD) development. A single nucleotide polymorphism (SNP), rs688, that has been identified to modulate the splicing efficiency of LDLR exon 12 and is associated with higher cholesterol and AD in some case-control populations. The exon 12 deleted mRNA is predicted to produce a soluble form of LDLR that fails to mediate apoE uptake. To gain additional insights, in this study, I seek to understand the regulation of LDLR splicing efficiency. To identify functional cis-elements within LDLR exon 12, I mutated several conserved putative exonic splicing enhancers (ESEs) to neutralize their affinity to serine/arginine-rich (SR) proteins. Transfection of wild type (WT) or mutant LDLR minigenes in HepG2 cells was performed, and splicing efficiency evaluated by quantitative RT-PCR. The results showed that two functional ESEs within exon 12, near rs688, are critical to LDLR splicing. To identify splicing factors that modulate exon 12 splicing, I co-transfected an LDLR minigene and vectors encoding different SR proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). After quantifying the splicing efficiency, I found that SRp20 and SRp38 increased exon 11- skipping. Moreover, ectopic expression of SRp38-2 and hnRNP G increased exon 11&12-skipping. Interestingly, the actions of hnRNP G did not require its RNA recognition motif (RRM). To further investigate the role of theses splicing factors on LDLR splicing, I quantified the expression level of these splicing factors as well as LDLR splicing efficiency in human brain and liver. I found that SRp38 mRNA expression is associated with LDLR splicing efficiency. In conclusion, this study discovered that rs688 is located close to the two functional ESEs within LDLR exon 12, and revealed a role of SRp38 in LDLR splicing efficiency

Cryo-EM Studies of Bacterial RNA Polymerase

In bacteria, a single RNA polymerase (RNAP) performs all transcription. The overall structure of bacterial RNAP resembles a crab claw with pincers comprising the β’ and β subunits and a large cleft where the active site sits. Structural information about this essential enzyme has mainly been provided by X-ray crystal structures of stable transcription complexes. RNAP crystal structures are difficult to obtain and the captured states may not always represent physiological states of the enzyme due to crystal packing artifacts. Recent advances in electron detectors and software enable near-atomic resolution structures of large biological complexes to be determined by single particle cryo-electron microscopy (cryo-EM). Unlike X-ray crystallography, cryo-EM samples can be directly visualized without crystallization. In this thesis, I optimized and utilized cryo-EM methodologies to structurally characterize several bacterial RNAP complexes from Escherichia coli (Eco) and Mycobacterium tuberculosis (Mtb): (1) Eco σ70-holoenzyme (Eσ70) in complex with the Eco non-coding RNA (ncRNA) 6S RNA; (2) Mtb RNAP bound to the RNAP inhibitor Fidaxomicin (Fdx); (3) Eσ70 bound to the F element-encoded TraR protein; and (4) Eσ70-dependent promoter DNA melting intermediates stabilized by the TraR transcription factor. Using cryo-EM, I captured RNAP structures that were intractable to crystallization, visualized multiple RNAP conformational states populated in solution, deconvoluted RNAP molecular motions, and observed transient complexes. The work in this thesis showcases the power of cryo-EM to examine macromolecular machines in action. (1) Bacterial 6S RNAs globally regulate transcription by RNAP, directly competing with promoter DNA binding. During transitions between exponential and stationary growth phases, Eco 6S RNA plays a key role in the transcriptional reprogramming by interacting specifically with the housekeeping Eσ70. During my initial cryo-EM experiments with the Eco 6S RNA-Eσ70 complex, I encountered a severe particle orientation bias in my samples. I discovered that the zwitterionic detergent 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO) was uniquely effective at solving this issue for bacterial RNAPs. Using this detergent, I determined the cryo-EM structure of the Eco 6S RNA/Eσ70 complex in combination with footprinting and crosslinking approaches in order to elucidate the structural mechanism of 6S RNA mediated inhibition of Eσ70. The structure reveals that 6S RNA is composed of duplex RNA segments that have A-form C3\u27-endo sugar puckers but with widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. The results showed how 6S RNA specifically targets Eσ70 and how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP. (2) Fdx is a RNAP-binding drug that is highly effective against Mtb RNAP in vitro. In collaboration with postdoctoral fellow Hande Boyaci, we solved cryo-EM structures of Mtb σA-holoenzyme (σA-holo) bound to the actinobacteria general transcription factor RbpA and an upstream promoter DNA fork (us-fork) in the presence and absence of Fdx to identify the structural determinants of Fdx binding to RNAP. The results show that Fdx acts like a doorstop fo jam the RNAP clamp module in an open conformation. The structures define the Fdx binding pocket, which includes contacts with RbpA, explaining the drug\u27s strong effect on Mtb. (3) Under starvation conditions, Eco changes the expression of almost one-quarter of its genes within 5 minutes. This global response depends on two factors, the alarmone ppGpp, and the transcription factor DksA. Unlike most transcription factors, ppGpp and DksA bind directly to RNAP but not to DNA. Another transcription factor, TraR, regulates RNAP the same way as ppGpp and DksA. TraR, and ppGpp/DksA, can activate or inhibit transcription initiation depending on the promoter sequence. TraR inhibits Eσ70-dependent transcription from ribosomal RNA and ribosomal protein promoters and activates amino acid biosynthesis and transport promoters in vivo and in vitro. To understand how these factors modulate transcription initiation, I solved cryo-EM structures of Eσ70, with and without TraR, and an open promoter complex (RPo) using a ribosomal promoter that is regulated by TraR, rpsT P2. My cryo-EM structural analyses show that TraR regulates Eσ70 transcription initiation by binding and inducing major conformational changes to mobile RNAP domains that affect RNAP-promoter DNA interactions. (4) Transcription initiation is a multi-step process that leads to the formation of RPo. Using TraR to modulate Eσ70 transcription initiation on the ribosomal protein promoter rpsT P2, I observed promoter DNA melting intermediates by cryo-EM. These structures and supporting biochemical data delineate steps of the RPo formation pathway and provide insight into transient promoter-RNAP interactions that occur along this pathway. These structures span the initial recognition of the duplex promoter in a closed complex (RPc) to the final RPo. Findings from this thesis provide structural and biochemical insight into bacterial RNAP regulation and function. The work in this thesis shows how single-particle cryo-EM can be applied to study transcription factors and small molecules that modulate RNAP as well as transient transcriptional states. This thesis research outlines a framework for future studies of bacterial transcription complexes using cryo-EM

Universal Leadership Model

The Great Commission is the call of Jesus to his people to go out into the world and make disciples. The universal leadership model presents an opportunity for disciples/leaders to step up and fulfill the Great Commission. The church should be involved in discipleship training, outreaches and supporting the issues that are aligned with Christian ideology and important to people in the communities they serve. An effective universal leadership model should include diversity, training, desired change, accountability and effectiveness of change. In so doing, the church can then find parishioners within the church who may be transformed into disciples/leaders and communities will become stronger because of the immersion of disciples into our communities. The researcher utilized a 50-question anonymous survey taken by 161 participants. The questionnaire was presented electronically to Christians and non-Christians over the age of 18. The data gathered should encourage leaders to lead from the top, re-evaluate disciple/leadership training programs and to develop leaders with a heart for fulfilling the Great Commission

Machines That Learn: Aesthetics of Adaptive Behaviors in Agent-based Art

Since the post-war era, artists have been exploring the use of embodied, artificial agents. This artistic activity runs parallel to research in computer science, in domains such as Cybernetics, Artificial Intelligence and Artificial Life. This thesis offers an account of a particular facet of this broader work — namely, a study of the artistic practice of agent-based, adaptive computational artistic installations that make use of Machine Learning methods. Machine Learning is a sub-field of the computer science area of Artificial Intelligence that employs mathematical models to classify and make predictions based on data or experience rather than on logical rules. These artworks that integrate Machine Learning into their structures raise a number of important questions: (1) What new forms of aesthetic experience do Machine Learning methods enable or make possible when utilized outside of their intended context, and are instead carried over into artistic works? (2) What characterizes the practice of using adaptive computational methods in agent-based artworks? And finally, (3) what kind of worldview are these works fostering? To address these questions, I examine the history of Machine Learning in both art and science, illustrating how artists and engineers alike have made use of these methods historically. I also analyze the defining scientific characteristics of Machine Learning through a practitioner’s lens, concretely articulating how properties of Machine Learning interplay in media artworks that behave and evolve in real time. I later develop a framework for understanding machine behaviors based on the morphological aspects of the temporal unfolding of agent behaviors as a tool for comprehending both adaptive and non-adaptive behaviors in works of art. Finally, I expose how adaptive technologies suggest a new worldview for art that accounts for the performative engagement of agents adapting to one another, which implies a certain way of losing control in the face of the indeterminacy and the unintelligibility of alien agencies and their behaviors

The Seine River Basin

This open access book reviews the water-agro-food and socio-eco-system of the Seine River basin (76,000 km2), and offers a historical perspective on the river’s long-term contamination. The Seine basin is inhabited by circa 17 million people and is impacted by intensive agricultural practices and industrial activities. These pressures have gradually affected its hydrological, chemical and ecological functioning, leading to a maximum chemical degradation between the 1960s and the 1990s. Over the last three decades, while major water-quality improvements have been observed, new issues (e.g. endocrine disruptors, microplastics) have also emerged. The state of the Seine River network, from the headwaters to estuary, is increasingly controlled by the balance between pressures and social responses. This socio-ecosystem provides a unique example of the functioning of a territory under heavy anthropogenic pressure during the Anthropocene era. The achievements made were possible due to the long-term PIREN Seine research program, established in 1989 and today part of the French socio-ecological research network “Zones Ateliers”, itself part of the international Long-term Socio-economic and Ecological Research Network (LTSER). Written by experts in the field, the book provides an introduction to the water budget and the territorial metabolism of the Seine basin, and studies the trajectories and impact of various pollutants in the Seine River. It offers insights into the ecological functioning, the integration of agricultural practices, the analysis of aquatic organic matter, and the evolution of fish assemblages in the Seine basin, and also presents research perspectives and approaches to improve the water quality of the Seine River. Given its scope, it will appeal to environmental managers, scientists and policymakers interested in the long-term contamination of the Seine River

Midwife of An-arché: Toward a Poetics of Becoming-with-Woman

This project explores the connections between midwifery and the ethical demands attendant to poetic practice. Through verse and prose, I unfold a figuration of the midwife that traverses the boundaries between Levinasian heteronomy and Deleuzian heteromorphism, and is a constitutive factor in sites of resistance to the biomedical territorialisation of the creative body. Chief archival and methodological components that inform the thesis include: a historiography of childbirth - tracing the development of ‘holistic’ and ‘interventionist’ paradigms, and the ideological underpinnings of the phallocratic takeover of the birthing room in certain Western countries; idiographic insights gathered from dialogues with maternal practitioners and mothers, including residents of The Farm in Tennessee - where I participated in a midwifery workshop week; an experiential inquiry into Holotropic Breathwork - to facilitate access to non-ordinary states of consciousness; and a negotiation between Marxist-feminist and poststructuralist articulations of ethico-political agency. Subject matter ranges from a consideration of the ethical import of the placental economy to the bio-intelligent tissue of the psoas, the banishment of Anne Hutchinson from Massachusetts Bay to the legacy of the ‘Twilight Sleep’ movement. Sustained critical attention is devoted to Mina Loy’s “Parturition”, and contemporary poets that have acknowledged Loy as an influence, such as Lara Glenum. I suggest that, despite the absence of a birth attendant on the symbolic level, Loy’s poem resonates with the investments of midwifery, instating a ‘subjectin- process’ that woks through and against abstruse and instrumental discourses, defying both the technocratic erasure of maternal knowing and the fetishistic reduction of labour to an end-product. Art’s capacity for opening up a corporeallycharged zone of between-ness is further elaborated in an essay on Andrei Tarkovsky’s Stalker - through which the treatment of spatiotemporality is aligned with the imperatives of midwifery guardianship.

Images under control: pessimism, humour and stupidity in the digital age

This thesis offers a periodization of the present according to which contemporary art and visual culture are understood to be symptomatic of an increasingly pervasive pessimistic social, political and ecological outlook. This pessimism I will claim is what is authentically new about our contemporary cultural forms, which are directed towards a particular form of humour and stupidity. Core elements in the periodization include the limitation of imaginative horizons expressed in the well-known remark of Fredric Jameson’s that it is easier to imagine the end of the world than the end of capitalism, as well as the pervasive sense that nature is in a state of perpetual and endemic crisis and the idea that modern computing technology is making us stupider than we have ever been before. I argue that these issues are symptomatic of what Gilles Deleuze, in 1990, termed the societies of Control – a world of corporate power, ubiquitous computing, data extraction and financial capitalism that has intensified since its early diagnosis. However, dominant narratives of art and visual culture continue to theorize artistic production according to traditionally avant-garde categories of resistance, criticality, transgression and subversion. This presumes art to have an agency that is difficult to imagine in the current social situation. In this respect, the thesis in part constitutes a critical reflection on the pressures placed upon our existing models of art and visual culture - for example, and centrally, the idea of an ‘avant-garde’ - by current social and technological conditions. Building on these observations, the thesis proposes a new model of contemporary art and visual culture that has no agency: images under control that are formed, as epiphenomena, by technological apparatuses of Control; studying examples such as extreme sports stunts, internet memes, online trolls, bad quality jpegs and impassive ‘artworks’. The purpose is to ask what value we can place on these emergent cultural forms, which seem to mirror, reflect and reiterate a pessimistic worldview deeply entrenched in the societies of Control