A versatile diffractive maskless lithography for single-shot and serial microfabrication.

We demonstrate a diffractive maskless lithographic system that is capable of rapidly performing both serial and single-shot micropatterning. Utilizing the diffractive properties of phase holograms displayed on a spatial light modulator, arbitrary intensity distributions were produced to form two and three dimensional micropatterns/structures in a variety of substrates. A straightforward graphical user interface was implemented to allow users to load templates and change patterning modes within the span of a few minutes. A minimum resolution of approximately 700 nm is demonstrated for both patterning modes, which compares favorably to the 232 nm resolution limit predicted by the Rayleigh criterion. The presented method is rapid and adaptable, allowing for the parallel fabrication of microstructures in photoresist as well as the fabrication of protein microstructures that retain functional activity.

Antisense Gene Silencing and Bacteriophages as Novel Disinfection Processes for Engineered Systems

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

Recent national trends in Salvia divinorum use and substance-use disorders among recent and former Salvia divinorum users compared with nonusers.

CONTEXT: Media and scientific reports have indicated an increase in recreational use of Salvia divinorum. Epidemiological data are lacking on the trends, prevalence, and correlates of S. divinorum use in large representative samples, as well as the extent of substance use and mental health problems among S. divinorum users. OBJECTIVE: To examine the national trend in prevalence of S. divinorum use and to identify sociodemographic, behavioral, mental health, and substance-use profiles of recent (past-year) and former users of S. divinorum. DESIGN: Analyses of public-use data files from the 2006-2008 United States National Surveys on Drug Use and Health (N = 166,453). SETTING: Noninstitutionalized individuals aged 12 years or older were interviewed in their places of residence. MAIN MEASURES: Substance use, S. divinorum, self-reported substance use disorders, criminality, depression, and mental health treatment were assessed by standardized survey questions administered by the audio computer-assisted self-interviewing method. RESULTS: Among survey respondents, lifetime prevalence of S. divinorum use had increased from 0.7% in 2006 to 1.3% in 2008 (an 83% increase). S. divinorum use was associated with ages 18-25 years, male gender, white or multiple race, residence of large metropolitan areas, arrests for criminal activities, and depression. S. divinorum use was particularly common among recent drug users, including users of lysergic acid diethylamide (53.7%), ecstasy (30.1%), heroin (24.2%), phencyclidine (22.4%), and cocaine (17.5%). Adjusted multinomial logistic analyses indicated polydrug use as the strongest determinant for recent and former S. divinorum use. An estimated 43.0% of past-year S. divinorum users and 28.9% of former S. divinorum users had an illicit or nonmedical drug-use disorder compared with 2.5% of nonusers. Adjusted logistic regression analyses showed that recent and former S. divinorum users had greater odds of having past-year depression and a substance-use disorder (alcohol or drugs) than past-year alcohol or drug users who did not use S. divinorum. CONCLUSION: S. divinorum use is prevalent among recent or active drug users who have used other hallucinogens or stimulants. The high prevalence of substance use disorders among recent S. divinorum users emphasizes the need to study health risks of drug interactions.

Phase Locked Loop and Modulo Games, The Dogma Loops; and “Finding Ibrida”

This dissertation consists of two independent musical compositions and an article detailing the process of the design and assembly of an electric guitar with particular emphasis on the carefully curated suite of embedded effects.

The first piece, 'Phase Locked Loop and Modulo Games' is scored for electric guitar and a single echo of equal volume less than a beat away. One could think of the piece as a 15 minute canon at the unison at the dotted eighth note (or at times the quarter or triplet-quarter), however the compositional motivation is more about weaving a composite texture between the guitar and its echo that is, while in theory extremely contrapuntal, in actuality is simply a single [superhuman] melodic line.

The second piece, 'The Dogma Loops' picks up a few compositional threads left by ‘Phase Locked Loop’ and weaves them into an entirely new tapestry. 'Phase Locked Loop' is motivated by the creation of a complex musical composite that is for the most part electronically transparent. 'The Dogma Loops' questions that same notion of composite electronic complexity by essentially asking a question: "what are the inputs to an interactive electronic system that create the most complex outputs via the simplest musical means possible?"

'The Dogma Loops' is scored for Electric Guitar (doubling on Ukulele), Violin and Violoncello. All of the principal instruments require an electronic pickup (except the Uke). The work is in three sections played attacca; [Automation Games], [Point of Origin] and [Cloning Vectors].

The third and final component of the document is the article 'Finding Ibrida.' This article details the process of the design and assembly of an electric guitar with integrated effects, while also providing the deeper context (conceptual and technical) which motivated the efforts and informed the challenges to hybridize the various technologies (tubes, transistors, digital effects and a microcontroller subsystem). The project was motivated by a desire for rigorous technical and hands-on engagement with analog signal processing as applied to the electric guitar. ‘Finding Ibrida’ explores sound, some myths and lore of guitar tech and the history of electric guitar distortion and its culture of sonic exploration.

UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.

The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extracellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40-binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.