VictorJuiz/thesis huggingface dataset:
Engineering cell access using nanostraws [electronic resource]

The study of biology is driven by our ability to perturb biological systems and observe the outcomes. At the cellular level, by predicting the presence of a biological interaction and perturbing elements of that interaction, we increase our understanding of how a cell functions. Interactions between biomolecules consist of binding events, enzymatic breakdown, metabolic synthesis, and many others, but one valuable distinction between interactions is the locale, which can be extracellular or intracellular. Extracellular signals allow cells to interface with their environments including other cells and soluble signals, but the majority of signaling events occur intracellularly. To protect the integrity of the cell, the cell membrane tightly regulates the passage of molecules, which provides a robust barrier between the extracellular and intracellular space. Therefore, perturbing intracellular elements of the cell effectively requires a method for accessing the cell without disruption. One strategy for passing material through the cell membrane is to use the cell's natural uptake mechanisms, thereby limiting the disruption to the cell. Unfortunately, methods which leverage endocytosis or other uptake mechanisms can do little to protect the cargo from the cell's inherent defense mechanisms. An alternative strategy is to physically deliver material into the cell by creating gaps in the cell membrane. Until recently, this was difficult because small nanometer scale gaps could not be reliably formed in cells. However, advances in fabrication have allowed nanometer scale objects to be reliably produced, which have demonstrated promise as a pathway to cell access. This thesis describes the development of a technique based on high aspect ratio hollow nanowires, called nanostraws, which we use to deliver material into cells. Compared to other nanowire techniques, nanostraws are capable of timed delivery, which provides a powerful, unique lever in engineering cell access. Beginning with fabrication, we discuss the types of nanostraw devices used for delivery and we characterize cell behavior on the nanostraws. Later, we apply the nanostraw devices to study a point of controversy in the field of nanowire delivery, the question of whether or not nanowires penetrate into cells. This study is complemented with studies of cell behavior or adhesion and their effects on delivery. We then use the nanostraws to observe the effects of perturbing the cell cytoskeleton on delivery and penetration. This thesis concludes with a discussion of the field of nanowire delivery at large, and future directions for nanowires and nanostraws in particular.

Keywords: Materials Science and Engineering.
Advisor: Melosh Nicholas A. primary advisor. Thesis advisor
Heilshorn Sarah advisor. Thesis advisor
Wang Shan
Engineering electron metabolism in cyanobacteria [electronic resource]

Photosynthesis offers tremendous promise for driving sustainable production of the chemicals our society requires. Recent years have seen many inspiring demonstrations of an array of biochemical pathways in photosynthetic hosts, but progress has been limited by a lack of foundational engineering capabilities. We have made advances along two avenues towards using photosynthesis for sustainable chemical production: metabolic engineering of a cyanobacterium to produce hydrogen from sunlight, and new procedures to make cyanobacteria easier to engineer. As a metabolite, hydrogen is unique. The pathway is rather simple, just one reaction away from a major product of photosynthetic electron transport. What is complex is the enzyme that catalyzes the hydrogen production reaction. We describe two projects to prepare a cyanobacterial host to express a complex foreign hydrogenase: upregulating the synthesis of iron-sulfur clusters required for electron transfer, and developing the ability to express proteins anaerobically. Both are ultimately connected to electron metabolism. Our work and that of others has been hindered by the relatively limited state-of-the-art of cyanobacterial engineering. Thus, we made parallel efforts to improve two fundamental operations in the strain engineering cycle: transformation with foreign DNA and cell lysis for intracellular analysis. This resulted in a simple procedure to reliably lyse cyanobacteria for high-throughput and small-sample applications.

Keywords: Bioengineering.
Advisor: Swartz James R. primary advisor. Thesis advisor
Bhaya Devaki
Bryant Zev David advisor. Thesis advisor
Covert Markus advisor. Thesis advisor
Engineering fluorescent proteins for improved FRET performance and four-color biosensing [electronic resource]

Engineered fluorescent proteins (FPs) have been widely used as biosensor components and protein tags. Improved and diversified variants continue to expand the FP toolbox, allowing us to re-design existing biosensors and develop new ones. In my thesis work, I present three new FPs and demonstrate the relevance of their improved properties in two applications. In the first example, I show that Clover and mRuby2, new green and red FPs engineered as a FRET (Förster Resonance Energy Transfer) pair, improve the performance of FRET-based reporters when substituted for traditional cyan FP (CFP) and yellow FP (YFP) pairs. Clover and mRuby2 improved photostability, FRET dynamic range, and emission ratio changes; and these improvements enhanced detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones. In the second example, I show that an engineered red FP, Maroon1, possessing a uniquely red-shifted excitation, enables the use of genetically encoded reporters in a fourth wavelength channel beyond the three common blue-cyan, green-yellow, and orange-red channels. In conjunction with the Fucci cell cycle reporter system, I used Maroon1 and the cyan FP, mTurquoise2, to label histone H1.0 and PCNA, respectively. Using this system, Fucci4, all four cell cycle stages can be visualized during live-cell imaging.

Keywords: Bioengineering.
Advisor: Lin Michael Z. primary advisor. Thesis advisor
Bryant Zev David advisor. Thesis advisor
Meyer Tobias advisor. Thesis advisor
Engineering in vitro photobiological hydrogen production [electronic resource]

Hydrogen is an important industrial chemical and attractive next-generation fuel. Current methods for its production rely on steam reformation of fossil fuels which liberates greater than 1% of the annual global carbon dioxide emissions; therefore, there is a need for sustainable hydrogen production processes. Nature has provided a biological catalyst for hydrogen production in [FeFe] hydrogenases. These complex enzymes catalyze the reaction which combines electrons and protons to generate hydrogen. Our long term goal is to integrate a non-native [FeFe] hydrogenase into the photosynthetic electron transport chain of a cyanobacterium to couple photosynthetic water-splitting with hydrogen production. One challenge to implementing this pathway is that heterologous hydrogenases interact inefficiently with the photosynthetic machinery and electrons will be lost to native, competing pathways in the cyanobacteria. Engineering the hydrogenase and other pathway components in vivo is challenging due to the limited tools available for genetic engineering these photosynthetic organisms. We developed an improved in vitro pathway using Photosystem I complexes isolated from a cyanobacterium to mimic in vivo electron flow to the hydrogenase or the main competing pathway, the production of NADPH. This system provides fine-tuned control over the reaction components and environment including concentrations of proteins in the pathways to study and control electron flux. We demonstrate the use of a non-native ferredoxin to facilitate electron transfer between the photosystem and hydrogenase. Finally, we will describe coupling our photosystem-pathway with a high-throughput hydrogen sensor to assess hydrogen production rates in microtiter plates. This approach can be extended to the high-throughput screening of in vitro synthesized enzyme or electron carrier libraries to develop components that support high photobiological hydrogen production rates.

Keywords: Bioengineering.
Advisor: Swartz James R. primary advisor. Thesis advisor
Cochran Jennifer R. advisor. Thesis advisor
Smolke Christina D. advisor. Thesis advisor
Engineering new capabilities into optical microscopes [electronic resource] : toward measuring the three-dimensional position and orientation of single molecules in living cells

The labeling of specific biological structures with single fluorescent molecules has ushered in a new era of imaging technology: super-resolution optical microscopy with resolution far beyond the diffraction limit down to some tens of nm. With the features of these exquisite tools in mind, this Dissertation discusses optical strategies for measuring the three-dimensional (3D) position and orientation of single molecules with nanoscale precision and several super-resolution imaging studies of structures in living cells. The concepts of single-molecule imaging, super-resolution microscopy, the engineering of optical point spread functions (PSFs), and quantitative analysis of single-molecule fluorescence images are introduced. The various computational methods and experimental apparatuses developed during the course of my graduate work are also discussed. Next, a new engineered point spread function, called the Corkscrew PSF, is shown for 3D imaging of point-like emitters. This PSF has been demonstrated to measure the location of nanoscale objects with 2-6 nm precision in 3D throughout a 3.2-micrometer depth range. Characterization and application of the Double-Helix (DH) PSF for super-resolution imaging of structures within mammalian and bacterial cells is discussed. The DH-PSF enables 3D single-molecule imaging within living cells with precisions of tens of nanometers throughout a ~2-micrometer depth range. Finally, the impact of single-molecule emission patterns and molecular orientation on optical imaging is treated, with particular emphasis on multiple strategies for improving the accuracy of super-resolution imaging. The DH microscope is shown to be well-suited for accurately and simultaneously measuring the 3D position and orientation of single molecules.

Keywords: Electrical Engineering.
Advisor: Moerner W. E. (William Esco)
Fan Jonathan Albert advisor. Thesis advisor
Solgaard Olav advisor. Thesis advisor
Engineering non-antibody scaffolds and growth factor ligands as protein therapeutics and diagnostics [electronic resource]

Protein therapeutics have emerged as an important class of pharmaceuticals, with monoclonal antibodies making up the largest group of protein drugs. While antibodies have many desirable characteristics, there are some applications in which they may be the best option. In this dissertation, we describe alternative classes of proteins that can complement the use of antibodies in the development of protein therapeutics and diagnostics. Cystine knots, or knottins, have highly desirable properties for use as molecular imaging agents due to their small size and stable structure. Previous approaches to engineering knottins have focused on a small subset of knottin scaffolds; in this work we extend those studies to engineering Agatoxin, a knottin from spider venom, for high affinity binding to tumor associated integrins. Growth factor ligand-receptor interactions modulate many cell signaling pathways and biological processes. The vascular endothelial growth factor (VEGF) and the platelet-derived growth factor (PDGF) mediate two important signaling pathways involved in angiogenesis. In this work, we used the structural similarity of PDGF and VEGF to rationally design a chimeric PDGF/VEGF ligand, creating a multi-specific antagonist of their receptors PDGFR and VEGFR. The PDGF/VEGF chimera was then engineered using combinatorial methods, including yeast surface display, for increased binding affinity to PDGFR and VEGFR. The resulting engineered protein variant strongly inhibited PDGFR and VEGFR signaling with in vitro cell models. Given the important roles of PDGFR and VEGFR in angiogenesis, the PDGF/VEGF chimera has potential application in anti-angiogenesis therapy. Collectively, the engineered proteins in this work demonstrate the promise of using non-antibody scaffolds and growth factor ligands for the development of protein therapeutics and diagnostics.

Keywords: Chemical Engineering.
Advisor: Cochran Jennifer R. primary advisor. Thesis advisor
Swartz James R. primary advisor. Thesis advisor
Khosla Chaitan
Engineering synthetic ligand-responsive RNA devices for controlling the cell cycle [electronic resource]

Synthetic biology aims to program cells by applying engineering principles to the design of genetic circuits to control and monitor cellular behavior. Programmed cells can harness existing dynamically coordinated biologically processes to rescue cellular malfunctions, control existing cellular morphology, and introduce useful novel functionality. Recent advances in the field are leading to genetic circuits of higher-order complexity in mammalian cells, which may ultimately be used to study and reprogram phenotypes that play a key role in human health and disease, such as those linked to the cell cycle. RNA-based control devices can provide specific and modular control of gene expression in response to endogenous or exogenous inputs in living cells. Specifically, we show that for a ribozyme-based device platform, ribozyme switches previously prototyped in yeast are able to regulate gene expression in a predictable (R-squared = 0.63--0.97), ligand-responsive manner in human HEK 293, HeLa, and U2-OS cell lines without any change to device sequence nor further optimization. We also show that these simple, general-purpose components (an RNA ribozyme coupled to an RNA aptamer) are a compact (~200 nt) and effective way to control a complex phenomenon such as arresting up to 80% of a population of mammalian cells in the G2/M phase of the cell cycle. Current methods for altering cell cycle progression can present a number of limitations, including exhibiting specificity towards particular cell types, being broadly disruptive of cellular processes, and being limited in capacity to extend to different networks. A genetically encoded system supporting control over cell cycle progression has the potential to address limitations with existing chemical approaches. Further, these switches are modular and readily adapted to arrest cells in the G0/1 phase of the cell cycle and in response to at least two different small molecule inputs. This work describes the first example of the application of synthetic RNA-based gene-control devices to the regulation of cell cycle progression in human cells. We further show that siRNAs can induce the arrest of > 90% of a population of cells in the G0/1 phase of the cell cycle. This indicates the potential to build miRNA-based devices to control the cell cycle that complement the ribozyme-based approaches. Thus, ligand-responsive RNAs represent a class of synthetic biology tools that are both genetically encoded and capable of regulating intracellular protein levels in response to user-specified molecular signals, and can be adapted for sophisticated control of complex processes in human cells.

Keywords: Bioengineering.
Advisor: Smolke Christina D. primary advisor. Thesis advisor
Skotheim Jan
Swartz James R. advisor. Thesis advisor
Entanglement [electronic resource] : a study in Neolithic resource exploitation in the Middle East

Although Neolithic communities in the Middle East have been the focus of much archaeological research, there has been little agreement concerning how these societies developed. This dissertation examines how Neolithic communities were shaped through acts of resource-dependency -- the constraints and limitations which emerge from relying on particular resources. It examines how certain communities came to depend on the use of specific local resources by studying how such resources were acquired and consumed. An evaluation of the long-term effects of this dependency shows that using particular resources not only promoted certain behaviors, but restricted the development of others. Ultimately, it is argued that such intimate entanglements between people and resources produced a cohesive force capable of influencing how the Neolithic developed. Understanding the processes which create these entanglements, and the resulting effects, allows us to more thoroughly address the relationship between economic practices and community development.

Keywords: Anthropology.
Advisor: Hodder Ian primary advisor. Thesis advisor
Meskell Lynn advisor. Thesis advisor
Shackley M. Steven advisor. Thesis advisor
Enzymatic activation of dioxygen at mononuclear and binuclear copper sites [electronic resource]

The activation of dioxygen at copper sites in metalloenzymes is essential for the biosynthesis of a number of natural products including neurotransmitters and melanin. In biological systems, these reactions occur at either a mononuclear or binuclear copper site. Central to the function of these enzymes is the geometric and electronic structure of the copper dioxygen species that is formed. For a number of copper enzymes, dioxygen is activated at a single reduce copper to form a mono-nuclear copper(II) superoxo species that is activated for H-atom abstraction. Due to challenges isolating this intermediate in the enzymatic systems, these studies will focus on the characterization of synthetic copper(II) superoxo and copper(II) hydroperoxo species. Results presented here indicate that the triplet ground state of the end-on copper(II) superoxide is activated for H-atom abstraction as well as proton coupled electron transfer. In contrast, the copper(II) hydroperoxo specie is not activate for this reaction but rather decays via homolytic Cu--O bond cleavage. In contrast to these mononuclear systems, dioxygen is activated by two electrons at a binuclear copper site to form a side-on (μ-η2∶η2) copper(II) peroxide intermediate in enzyme systems including Tyrosinase (Ty). Two other Cu2O2 core isomers have been observed in synthetic systems, the binuclear end-on (μ-1,2) peroxo and the binuclear copper(III) bis-μ-oxo structure. Previous studies on these synthetic systems have shown that the side-on peroxo core can be in equilibrium with the bis-μ-oxo isomer and that both are able to perform the biomimetic hydroxylation of phenolate. The studies reported here build upon this synthetic chemistry by characterizing a new end-on peroxo/bis-μ-oxo equilibrium. The observation of this interconversion suggests that the electrophilic reactivity (phenol oxygenation and sulfoxidation) observed for complexes that form end-on peroxo species results from an accessible bis-μ-oxo isomer that is responsible for the electrophilic reactivity. In addition, the coupled binuclear copper enzyme family is extended to include a new, fourth class of enzymes, the hydroxyanilinases. These enzymes have the same side-on peroxo active site and have similar reactivity to Ty while also possessing the unique ability to convert o-aminophenols to nitrosophenols. These studies provide insight into the function of mononuclear and binuclear copper enzymes and the dioxygen intermediates that make up their catalytic cycle.

Keywords: Chemistry.
Advisor: Solomon Edward I. primary advisor. Thesis advisor
Martinez Todd J. (Todd Joseph)
Stack T. (T. Daniel P.)
Epic and encounter [electronic resource] : form and culture in early modern narrative poetry

The prevailing reading of epic poetry is that it exists in a binary relationship with romance: epic, the argument goes, attempts to tell a linear and end-driven story, but is continually diverted by a romance impulse that acts upon it, derailing the narrative, fragmenting its structure, and prolonging its ending. In recent decades, this has taken on political dimension: European epics are seen as stories of victory attempting to marginalize or silence altogether the voices of the non-European vanquished that contradict the victors' vision of history, reflecting a real-life European colonialism. This dissertation offers an alternate model for reading the epic genre, arguing that epic tells not just the story of victory, but also of "encounter, " a register of indeterminacy and exploration that reflects Europe's greater awareness of and involvement in the world around it, rather than simply an incipient and in many cases largely imaginary colonialism. Reading epic in this way allows us to see as essential to the genre, rather than as subversive romance impulses, moments of ambiguity, conflicting perspectives, and both European and non-European visions of history as epic texts represent them. Likewise, it allows us to see different potential ways of organizing epic narrative, rather than attempting to separate out a "main" narrative from its supposedly dilatory and subversive elements. The dissertation begins by looking at the way that the Iliad and the Aeneid represent perspective and opposition, explore issues of ethnography and identity, and treat questions of cultural engagement and historical interpretation formally, by reorganizing and redistributing narrative structure and attention. The tools of ambiguity, multiplicity, and contradiction that these texts provide would have appealed to early modern writers whose rapidly changing and broadening world would have called them to engage in new forms of cultural exploration and analysis. Each of the dissertation's four chapters focuses on a European epic of the sixteenth and seventeenth centuries to see how it, diegetically and formally, deals with issues of "encounter": Luís Vaz de Camões's Os Lusíadas (1572); Torquato Tasso's Gerusalemme liberata (1581); Edmund Spenser's The Faerie Queene (1590 and 1596); and John Milton's Paradise Lost (1667 and 1674).

Keywords: English.
Advisor: Greene Roland
Orgel Stephen primary advisor. Thesis advisor
Summit Jennifer advisor. Thesis advisor
Equal in theory [electronic resource] : gender egalitarianism and inequality in new marriage

This dissertation is comprised of three distinct papers, each of which draws from in-depth interviews conducted with a sample of 31 egalitarian-minded heterosexual couples (62 individuals) and explores gender inequality in marriage. The first paper shows how childless women and men's career aspirations are differentially shaped by anticipations of parenthood, with women being more likely than men to assume the mental labor of worrying about future parenthood, and thus more likely than men to scale back on their career aspirations even before the act of having children. The second paper demonstrates how social power differentials between women and men influence the marital surnaming choices women make following marriage. I find that many women who would otherwise keep their birth-given name choose to change it to appease male partners. The third paper shows that while couples resist gendered expectations in marriage proposals privately, instead choosing more egalitarian proposal options, many still present stories to the public that conform to traditional gender expectations. Collectively the papers demonstrate the continued presence of gender inequality in heterosexual marriage, despite the rise of egalitarian attitudes among contemporary young adults.

Keywords: Sociology.
Advisor: Correll Shelley Joyce primary advisor. Thesis advisor
Jiménez Tomás R. (Tomás Roberto)
Rosenfeld Michael