Genomic engineering has a wealth of possible applications, from curing genetic disease to curbing the spread of malaria. Homing endonucleases (HEs), which cut DNA at long, specific DNA target sites, are useful for genomic engineering because of their high site specificity. However, to use HEs for engineering, we must be able to modify their target site. Though this can be done with current computational and experimental protein engineering techniques, some HEs are resistant to those approaches. Therefore, we are working to develop a general methodology to modify the site specificity of HEs, based on a new synthetic biology technique called phage-assisted continuous evolution (PACE). PACE is a continuous evolution system in which the evolution of a desired biomolecular activity is linked to the reproductive cycle of phage particles containing the evolving gene(s), and should allow us to develop proteins or nucleic acids with almost any desired activity or specificity. We have built an automated system to keep the growing host cells and phage at a constant cell density and temperature, which is required for PACE. We are also working to design a mechanism to link HE DNA binding activity to phage reproduction by engineering plasmids in the host cells and phage. In the future, we will be refining the hardware set-up and protocol even further, and looking for ways to modularize the PACE system.

Indole-3-acetic acid (IAA), or auxin, is a plant growth hormone that is vital to the regulation of growth and development in plants. Although auxin’s function in plants is to control growth, it potentially regulates any gene in other organisms, making it a powerful regulatory tool. By investigating the synthesis of auxin, it can be generated in organisms besides plants. The goal of this project is to take the first step towards that reality through the synthesis of auxin in Saccharomyces cerevisiae (baker’s yeast). Due to the thorough characterization of S. cerevisiae, the organism is an optimal choice because the mechanisms involved with the manipulation of its gene networks are well understood. This project integrated the tryptophan-2-monooxyngenase (iaaM) and indole-3-acetamide hydrolase (iaaH) genes from Agrobacterium tumefaciens into the yeast genome. The enzymes produced from these genes convert tryptophan into auxin. In an attempt to reproduce this synthesis pathway, the two genes were amplified and integrated into the yeast genome through the use of the Gateway® cloning system and integrating vectors. Checking for successful auxin synthesis utilizes the auxin signaling pathway that was built in S. cerevisiae by the Klavins Lab. This system involves interactions between two proteins, an AFB (auxin signaling F-box) and an Aux/IAA (a transcriptional regulator of auxin responsive genes). The presence of auxin triggers the degradation of an Aux/IAA. By fluorescently tagging an Aux/IAA, the expression of the protein was observed. A decrease in fluorescence indicates the production of auxin. Successful auxin synthesis in yeast creates further possibilities for establishing regulatory systems based on the use of auxin in non-plant cells.

In a population of organisms, traits that increase fitness, or the ability to survive and reproduce, are passed on to progeny and become more prominent in the gene pool. It has been similarly shown that E. coli with a phenotype that increases or decreases fitness given an environmental pressure can be tuned to correspondingly decrease or increase fitness through random mutagenesis and selection of desired mutants. However, this process does not focus mutations and does not confer the maximum fitness benefit or cost per mutation. In order to isolate mutation location and effect, and increase the rate at which evolution occurs, I am applying a tool called Simple Sequence Repeats (SSRs). SSRs are repeats of short nucleotide sequences (AAA…, ATATAT…, etc.) that have been shown to mutate primarily in the form of insertions or deletions of an SSR and have a rate of mutation four to five orders of magnitude higher than non-repeat sequences. Most importantly, it has been shown that when SSRs are inserted between the ribosome binding site (RBS) and start of a gene, every insertion or deletion has a discrete, predictable effect on the level of expression for that gene. We have inserted SSR spacers in front of lacZ, lacY, and lacI in the lac system, and will show that we can rapidly optimize the expression of the genes. This could serve as a significant tool for tuning other, novel behaviors in bacteria.

Over the last decade, neurofeedback has become increasingly popular as a potential treatment option for various neurodevelopmental and neurological disorders. As a completely noninvasive procedure, electroencephalographic (EEG) neurofeedback uses auditory or visual methods to provide an individual with information derived from scalp-electrode recordings of his or her own brain activity. The feedback information may relate to the spatial distribution of activation across the brain or the energy distribution across specific frequency bands. To date, no studies have been undertaken to investigate the potential use of EEG neurofeedback in the clinical management of stuttering. Stuttering is a high-prevalence (1% of the population) speech disorder that often develops into a chronic, life-long problem with severe social implications. The ultimate causes are unknown, but recent studies have revealed structural and functional neural deficits that impact sensorimotor functioning. The present project therefore aims to test the feasibility of developing EEG neurofeedback procedures that may reduce the severity of stuttering in adult participants. Initial testing with 19 data channels and BCI2000 software focuses on reliably discriminating between rest and speech or nonspeech motor activity and designing conceptually relevant feedback displays that represent the spatial or energy distribution of the recorded brain signals.  If successful, this research could have important clinical implications in the treatment of stuttering.

This project’s goal is producing microporous scaffold for tissue growth. These scaffolds are made out of crosslinked 2-hydroxyethyl methacrylate (HEMA), a hydrogel. They are formed by polymerizing the HEMA around polymethyl methacrylate (PMMA) beads that are of a constrained and predetermined size. The PMMA is washed away and we are left with a porous scaffold of precise pore size. These scaffolds are used to encourage tissue growth and reduce inflammation and the foreign body reaction. The purpose of my research is to investigate blood vessel penetration into scaffolds by producing the largest sized scaffolds I can using novel techniques. We are currently limited to tissue scaffolds of a maximum thickness of 1 mm using our current techniques. With a larger thickness our scaffolds could be used for many more applications that are presently unavailable with a thin scaffold. Our methods are varied and include creating multiple mold designs, polymerizing techniques, and chemical washing techniques. Once I can create these larger scaffolds we will test them in vivo to evaluate the foreign body reaction as well as angiogenesis in the region. There is a large range of possible future applications for these scaffolds. They could be tuned to encourage many different types of tissue growth.

Tuning the gene expression of biomolecular pathways is an area of intense interest and value to synthetic biologists. Through the use of a mixture controlled turbidostat (MCT), we have been able to streamline, and in many cases enable the processes involved in this area of study. A turbidostat is a continuous culture bioreactor that maintains constant culture volume and cell density while diluting with fresh media to keep cells in an exponential growth phase. With the addition of mixture control, the turbidostat can vary the chemical environment within the culture vessel without altering cell density levels. Tuning gene expression in Escherichia coli is a simple matter when using a MCT. Currently, an area of global interest is the industrial production of butanol with E. coli. The first step towards this is enabling E. coli to live in rich butanol environments. Because butanol is naturally toxic to E. coli, an increase in gene expressions of the biomolecular pathways responsible for detoxification of butanol is required. By placing laboratory grade E. coli into our MCT, the concentration of butanol can be increased over time while the cell density remains constant. This will allow fitness increasing mutations within the biomolecular pathways responsible for detoxification of butanol to prevail over many generations, with the hope of eventually converging to a final population of E. coli that subsists in highly concentrated butanol environments. Through increasing E. coli’s resistance to butanol, we hope to illustrate the usefulness of MCTs in tuning gene expression of biomolecular pathways.

Diffuse Large B-Cell Lymphoma (DLBCL) is one of the most common types of non-Hodgkin's Lymphoma with a 50.4% five-year survival rate.  Previous work has shown that some patients with aggressive DLBCL have increased expression of the transcription factor NF-kß, which can alter normal growth and function of B-lymphocytes.  Interestingly, a protein upstream of NF-kß in the B-Cell Receptor Pathway called PKCß is found to be highly activated in many DLBCL cases, suggesting that PKCß overexpression may be altering the lifecycle of those with DLBCL.  Therefore, PKCß may be a significant new target for therapy.  We designed a transgenic mouse expressing a dominant active form of PKCß expressed only in B-cells (Rosa26 DAPKCß XCD19-Cre).  With this model, we measured differences in B-cell  germinal center development and in antibody responses between WT and DA PKCß-Cre mice immunized with T-dependent antigen NP-CGG.  We hypothesized that DA PKCß-Cre mice may have an increased response compared to WT because of its dominant active form of PKCß, which leads to increased NF-kß and hyper-activation of the responding B-cells.  Thus, we used ELISA and ELISPOT assays to quantify the expressed antibodies and immunohistochemistry to analyze the architecture of the spleen.  Immunohistochemistry has shown that the immunized DA PKCß-Cre mice have slightly larger germinal centers than WT mice, although not statistically significant.  Immunized DA PKCß-Cre mice show no difference in serum antibody levels by ELISA, but have slightly increased numbers of anitbody producing cells by ELISPOT.  Overall, the data suggests that over expression of PKCß in B-cells results in a hyper-proliferative phenotype, but does not strongly alter the antibody response to the T-dependent antigen NP-CGG.  Since we saw a difference in mice immunized with Pertussis, which stimulates the Toll-like Receptor (TLR) Pathway, we will look at different responses of TLR ligands in vivo and in vitro.

The human body is roughly two-thirds water, much of which interacts with intracellular hydrophilic surfaces, materials that exhibit water-attracting properties. Near any hydrophilic surface such as biological tissue, a zone of water extending a few hundred micrometers from the surface called the “exclusion zone (EZ)” exhibits exclusive and stunning properties far different from those in normal, bulk water. For instance, EZ water does not exist as a solid, liquid, or gas but instead forms a unique, ordered liquid crystalline state that is solute-free, highly organized, and electrically polarized. When EZs are exposed to light, their sizes increase as they absorb and store energy. Cells could utilize this potential energy through growth or other cellular activities. However, it is hypothesized that EZs absorb not only light energy, but also other energy sources such as acoustic energy. To measure the effects of sound on EZs, sheets of hydrated Nafion, a hydrophilic polymer that generates EZs, were submerged in water and exposed to various frequencies and intensities of audible sound produced from an underwater speaker. Microspheres, small spherical particles made from synthetic material, were used to visualize the EZs. Because EZs exclude solutes, the EZs were represented by the areas void of microspheres. During and after exposure to sound, the EZ sizes were monitored over time. Recent results showed that during exposure to multiple audible frequencies (1-8 kHz), the sizes of the EZs decreased transiently. After the sound was removed, in some cases, the size recovered to its original value. These results may suggest enormous biological implications. Everyday sounds produced in our environment could have a large impact on cellular behavior.

Mesanchymal stem cells (MSC) can differentiate into a variety of cell lineages, including adipocytes, chondrocytes, and osteoblasts. This gives them great potential for use in regenerative medicine. To harness this potential, we must understand the effects of the various stimuli that can be applied to cells in in vitro culture. We produced an ECM mimetic nano-grooved cell culture substrate via capillary force lithography. An agarose diffusive microfluidic device was used to establish an SDF-1 α gradient across the nanogrooved substrate. Bone marrow derived mesanchymal stem cells (BM-MSC) were seeded on the substrate, and live cell imaging was carried out over a few days’ time. Using the time-lapse images, the differentiation, proliferation, motility, and elongation of the BM-MSC were analyzed and quantified. Understanding the synergistic effects of topography and bio molecular gradients will prove helpful in future efforts to create MSC regenerative medicines.

 A Plasma Pencil Atmospheric Mass Spectrometer (PPAMS) is a new surface analysis device which allows mass spectrometry (MS) to be conducted on samples under normal atmospheric conditions. This enables surface analysis on samples that could previously not be analyzed under the current standard vacuum-based techniques, broadening the field of surface analysis. However, characterization of this method of surface analysis needs to be done in order to maximize the benefits of the PPAMS device and to determine the extent to which the traditional vacuum-based methods can be replaced. This process of characterization involves determining the ionization rate of the plasma pencil probe, performing various experiments while changing parameters (i.e. sample concentration, thickness, and gas type), and comparing results to established methods. Our current investigation is focused on the determination of the ionization rate of the PPAMS device. Initial experiments were performed on polyurethane-based samples dissolved in dimethyl acetamide and spun-coated onto a gold-coated silicon wafer. These samples were then probed by the PPAMS over controlled time intervals. A multivariate statistical method, Principal Components Analysis (PCA), was used to evaluate whether the MS signals obtained varied as a function of exposure time. We hypothesized that the signal would resemble our bare gold background once all of the polyurethane had been ionized. Instead, the MS signal was found to stay constant over a 15 minute exposure time implying a longer exposure time is needed. Current work is focused on evaluating the effect of PPAMS exposure to polymer thickness as a secondary means of evaluating ionization. Future work will center on investigating the changes to the ionization rate under different experimental conditions. The results of our device characterization will provide valuable insight into the limits of the PPAMS device that will enable improved experimental design.

Ultrasound (US) and microbubbles (MBs), as tools for gene transfection, have promising implications for gene therapy. Through permeation, US facilitates the diffusion of a gene or drug from the capillaries into the cell. Binding of the gene or drug to the MBs can further enhance the delivery efficiency, and when exposed to US, the MBs are ultimately cavitated once they have reached their target. Our goal is to create MBs that will enhance US-mediated transfection. Concerning MB production, we have shown that our prepared MBs are more concentrated, stable, and consistent than those of the leading manufacturers, via flow cytometry and Coulter Counter analysis. In in vitro experiments, we have shown that US, by itself, increases transfection efficiency (plasmid concentration in cells) of pGL3 and pGFP in human embryonic kidney 293 cells. 293 cells are transfected very readily and consistently and are therefore useful as a way to accurately quantify gene expression from transfection. Combined with MBs, transfection efficiency is further improved upon. US parameters (intensity, exposure time, frequency) and MB characteristics (concentration, size, distribution, stability) are interdependent, and thus the optimization of each parameter is important. With regards to US parameters, exposure times have been varied from 1 to 5 minutes, at intensities ranging from 0.5 to 2 watts, and the optimal condition (cells transfected minus cells killed) has been found to be 2 watts per square centimeter for 3 minutes. These conditions have been combined to quantify 293 cell gene expression. It has been shown that MBs enhance transfection rates by up to 25 times when compared to those with just US. These results have important implications in gene therapy as a means of non-invasive, localized, and efficient gene delivery.

 Myocardial infarction, heart failure, and congenitive heart disease are leading causes of morbidity and mortality worldwide. However, current cell therapies remain inadequate in addressing this clear medical need. One of the primary obstacles limiting the success of cell therapies is that stem cell derived cardiomyocytes (SCDCM’s) tend to be functionally immature, providing only a small fraction of the force generation of their matured counterparts. Thus, the work here focuses on developing a growth platform which incorporates topographical and mechanical properties in order to rapidly and thoroughly mature SCDCM’s. The field of tissue engineering is becoming increasingly aware of the roll that the extracellular matrix (ECM) plays in driving cellular differentiation and behavior, and the importance this has on the design of biomimetic scaffolds and growth platforms. A study of the native cardiac environment has revealed that the presence of aligned nanoscale matrix fibers plays a key role in establishing organization within the myocardium. This insight has allowed us to construct polymeric scaffolds which mimic the in vivo ECM, greatly improving cardiac culture techniques. There exists an optimal feature size and configuration at which nanogrooved topography is successful in promoting anisotropic alignment and elongation of cardiomyocytes, an increased action potential conduction velocity, and the substantial expression of cell-cell coupling proteins. Our current focus rests on understanding the way in which mechanical and chemical variations to the growth substrate can affect cell morphology and contractile properties when manipulated in conjunction with nanoscale topographical stimulation. This research will provide a strong foothold towards the development of a cardiac patch, which, implanted directly onto the infarct surface of the heart, could reduce scar tissue formation, promote healing and angiogenesis, and provide crucial contractile and mechanical strength.

In recent years, increasing numbers of single amino acid mutations in cardiac troponin C (cTnC) have been identified in patients with cardiomyopathies. The troponin protein complex regulates cardiac muscle, as Ca2+ binding to cTnC initiates contraction. Previous studies of the mutations associated with hypertrophic cardiomyopathy (HCM) patients show increased Ca2+ sensitivity and of those with dilated cardiomyopathy (DCM) patients, decreased Ca2+ sensitivity. However, it is still unclear how a change in Ca2+ signaling might precipitate the disease, and unknown why a myofilament protein variant affects the Ca2+ transient, which is regulated by calcium handling proteins. This project focuses on the I61Q cTnC variant, which previous studies have shown decreases fraction shortening (produce weaker contractions) and Ca2+ binding of myofilaments. Determining how or if abundance and activity of sarcoplasmic reticulum (SR) proteins vary will shed light on the mechanism behind the change in Ca2+ transient. All cardiomyocytes were isolated from healthy adult rats and transfected by adenoviruses containing the I61Q gene. Transduced cardiomyocytes were cultured for 48 hours. Then, Western blotting determined abundance of Ca2+ handling proteins in the SR, such as phospholamban and SERCA-2.  SDS-Page gels were used to determine protein phosphorylation. Using imaging software to analyze the bands in the gel will result in a quantitative comparison of protein abundance and phosphorylation. The difference in protein expression and regulation provide insights for understanding the mechanism behind the change in Ca2+ sensitivity, and potentially allow I61Q to be delivered through gene therapy in patients with HCM to restore normal Ca2+ sensitivity. Past contractile measurements were performed at 25°C, but since the heart normally operates at 37°C, future experiments will be performed at this temperature for transduced cardiomyocytes. Contractions will be induced by electrical stimulation so that cell lengths, rates of sarcomere contraction/relaxation, and Ca2+ transients can be measured simultaneously using IonOptix video microscopy.

Vascular calcification (VC) is an indicator of atherosclerotic plaque burden and is a strong, independent predictor for future cardiovascular complications, significantly increasing the risk for cardiovascular morbidity and mortality. Type II diabetes (T2D), the 7th leading cause of death in the US, is associated with increased incidence of VC clinically, as well as in our recent animal study. Although VC was considered a degenerative process involving passive accumulation of calcium-phosphate salts associated with tissue necrosis, numerous studies have indicated that VC is an active, cell-mediated process that resembles embryonic bone formation and remodeling. Smooth muscle cells (SMCs) have been implicated in giving rise to bone- and cartilage-like cells in calcifying blood vessels by undergoing lineage reprogramming in response to local environmental cues. We investigated the association of S100A11, a calcium binding protein, with a multi-ligand receptor, the receptor for advanced glycation end products (RAGE), as a possible signaling mechanism involved in the lineage reprogramming of SMCs under T2D settings. We used a genetic fate mapping strategy to trace SM-derived cells in the model T2D mice that developed vascular calcification. Immunofluorescent dual staining for S100A11 and RAGE, along with immunohistochemical staining for Runx2/Cbfa1, an early osteochondrogenic marker, was performed on aortic sections of the T2D mice. The results revealed that S100A11-RAGE signaling is most likely to be involved in cartilage lesion development. This is supported by co-localization of S100A11-RAGE markers within cells of chondrocyte morphology in cartilaginous and calcific areas of blood vessels. Interestingly, almost all S100A11-RAGE positive cells were derived from SMCs that have been labeled by specific transgenes. These results highlight the significance of S100A11-RAGE signaling in VC and possibly provide for the development of preventative or therapeutic targets against T2D-related VC.

Cell transplantation has been shown to improve cardiac function following myocardial infarction (MI). The mechanisms behind improved function are not well-understood, but are correlated with changes in myofilament protein phosphorylation, which likely affect Ca2+- and length-dependent (preload) changes in force. This project was designed to test the hypotheses that 1) responsiveness to ventricular filling (Frank-Starling relationship) will decrease following MI, and 2) neonatal rat cardiomyocyte (NRC) transplantation after infarction will ameliorate this loss of function. One week after induction of MI, fluorescently-labeled NRCs were injected into the area of infarction of some of the animals. Sham-operated rats served as control. After 4-6 weeks, cardiac performance for all groups was preliminarily assessed using echocardiography. Then, whole working heart preparations were used measure cardiac power output, testing the heart’s ability to follow the Frank-Starling relationship. NRC-injected hearts exhibited higher average power and response to ventricular filling than sham (media-only) injected hearts, indicating that NRC-engraftment significantly ameliorates MI-induced decreases in preload responsiveness. Force and Ca2+ sensitivity (pCa50) of demembranated myocardial strips from these hearts were then assessed. NRC-injected hearts strips had a sarcomere length associated change in pCa50 that was similar to uninfarcted hearts (0.07 ± .02 vs. 0.10 ± .03, respectively), which was significantly improved as compared to strips from untreated, infarcted animals (0.0 ± .03). These results indicate that loss of preload dependence following MI is, at least in part, due to altered myofilament function and this is reversed following NRC engraftment. Current work involves investigating myofilament phosphorylation profiles to assess the mechanism of myocardial function changes. Preliminary results show decreased phosphorylation levels of cardiac troponin T (cTnT) in NRC-injected hearts compared to sham-operated and infarcted hearts. These changes in phosphorylation may be associated with the changes in force production following cell therapy.

Despite a surge in research efforts over the past two decades, scientific understanding of the mechanisms underlying motor learning remains limited. Although many procedures have been designed to assess an individual’s ability to update motor commands in an altered environment, few attempts have been made to design procedures that assess, and possibly improve, an individual’s ability to learn a completely novel mapping of motor commands and sensory consequences. This is unfortunate because the latter approach has potential clinical applications: various brain disorders cause sensorimotor deficits, and learning alternative movement strategies may aid recovery. Whereas the above studies focused on hand or arm movements, our own research focus is on the speech motor system. The present project aims to design protocols to test and quantify the ability of the speech motor system to learn neural representations of a novel link between motor commands and sensory results. We make use of 3D electromagnetic motion capture technology that tracks 3 position coordinates and 2 angles for up to 9 sensors on the tongue, lips, and jaw; thus for a maximum of 45 dimensions. These 45 dimensions are mapped onto 2 dimensions of movement for a cursor displayed on a computer monitor. As subjects are instructed to move the cursor, their performance (i.e., learning of the novel mapping) is quantified. We will also test individuals from a clinical population (such as those with motor speech disorders or stuttering) and analyze their performance over time to design the most optimal techniques and procedures for quantifying the speech sensorimotor system’s ability to learn the complex relationship between a high-dimensional movement space and a 2D visual space.

Minimally invasive surgical (MIS) techniques, such as laparoscopic surgery (performed through small incisions using specialized tools), are an integral part of modern medicine and the future of surgery. Technologically advanced tools such as MIS robots are promising to replace or augment existing MIS manual tools. MIS robots improve on manual surgery, as they can incorporate increasingly sophisticated features such as force feedback for surgeons, automatic identification of diseased tissues, and partial automation of surgical tasks. These features can reduce patient pain, recovery time and thus healthcare costs by eliminating unintended damage to tissue and providing surgeons with tools to perform surgery more effectively. Each of these new features either requires or would greatly benefit from an accurate mathematical description of the dynamic mechanical properties for the tissues with which surgeons interact. Dynamic tissue property quantification requires that we apply a range of forces to the tissue and measure the resulting displacements. We have designed, built, and calibrated a device capable of collecting the necessary data to obtain an equation relating applied force to tissue displacement for any tissue of interest. We will process the data obtained from this device using an advanced signal processing and system identification algorithm (unscented Kalman filtering), which will evaluate the fit to the data recursively over time. This will allow us to maintain accurate results as the tissue properties change over time. This setup allows us to collect and analyze data from tissues including (but not limited to) in vivo abdominal organs in pigs. In addition to being an important study that has not been performed in the literature, the real-time collection and analysis of this data could have profound implications in partially automating surgery.

The Paul Yager Lab in the University of Washington’s Bioengineering Department has been researching the dynamics of fluids on two dimensional paper networks of nitrocellulose. Knowledge of this science will allow for the development of distributed diagnostics, akin to pregnancy tests, that can be used in remote settings without access to electricity or medical supplies. By testing the storage and flow of various proteins and carbohydrates on nitrocellulose, we are developing lateral flow enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of various diseases. My research involves the control of proteins once they are already on the nitrocellulose membrane. By printing proteins and carbohydrates in various conformations onto the nitrocellulose, we were able to test the effectiveness of our methods for delaying and mixing protein flow. We showed that you can delay protein flow by printing a wall of sugar around the protein that needs to dissolve before the protein can flow with the liquid. We tested the flow patterns of two consecutive patterns of proteins and found that we can predict the time and efficiency with which they arrive at the downstream mixing site. We also developed ways to effectively load up to 1µl of solution onto the nitrocellulose strips. This research will be useful in the eventual development of a working ELISA, when combined with other results from researchers in the lab.

A key issue in synthetic biology is the evolutionary robustness of synthetic circuits. Creation of robust circuits requires the development of design criteria to extend evolutionary timescales, and insight into the relative evolutionary fitness of different circuit designs. Experimentally, this research requires the use of multiple cell culture runs that are time-consuming and laborious. To accelerate this work, development of a high-throughput, automated, and parallel mini-chemostat and turbidostat device is proposed. While the development of nutrient-replenishing chemostat devices dates back to 1950, and the usage of such devices is well-documented, even modern chemostats are not well-suited to evolutionary experiments. Current designs face significant issues of equipment fouling, specialized complexity, and prohibitive cost. Presently there is a lack of cost-effective options for highly parallel milliliter-scale chemostat devices which can be applied to problems in synthetic biology. The proposed device is small and modular, utilizing multiple standard petri dishes for parallel culture experiments. The design features integrated media refreshment and temperature control mechanisms. Visible and fluorescent direct-view, real-time imaging systems are included. The device is constructed of readily-available parts to maintain low cost. Parts are modular such that used petri dishes can be easily removed, disposed of, and replaced. The design is scalable such that numerous devices can be stacked or tiled for parallel experiments. At present, design and fabrication of the device hardware have been completed, and the device has been verified functional for an operational period of at least 24 hours. Directed evolution experiments to measure of loss-of-functionality mutations in engineered E. coli will be conducted to assess live-culture performance of the device. An open-source software package and electronic microcontroller system are being developed to allow for automated real-time measurement and customization of growth and measurement parameters.

Paper-based diagnostic assays have become popular for their use in the detection of both pregnancy and infectious disease. The majority of lateral flow tests are run using a paper-like membrane called nitrocellulose on which liquid can flow using capillary action until it reaches the spot or line containing capture reagents. They provide an inexpensive, user-friendly alternative to expensive lab-based analysis. However, commercial paper-based assays typically only run single step processes that can lead to inaccurate results. Our goal is to create more sophisticated assays that can perform multi-step processes for higher performance using paper networks. In order to achieve this goal, we need to create paper fluidic valves to regulate fluid flow in these devices. For a simple shut-off valve, we came up with the idea of placing a sugar cube in a gap between two strips of paper so it would act as a temporary bridge and allow a certain amount of fluid to flow through until the sugar dissolved. This would enable specific volumes of fluid to flow through different channels -- something normally regulated by trained personnel operating pipettes in the lab -- with an untrained user merely having to add excess fluid to a single well. Dissolvable sugar bridges can serve as a method for manipulating fluid/reagent volumes within paper-based devices. We've been able to demonstrate and characterize their operation, including tunability using parameters such as geometry and composition. We've also demonstrated the utility of dissolvable bridges in the important context of automated delivery of different volumes of the same reagent (e.g. water) from a common source to multiple locations in an assay for simple device loading and activation. Sugar bridges have the potential to help bring advanced testing using paper networks to limited-resource settings.

Aminoglycoside antibiotics such as kanamycin and neomycin have been known to cause severe hearing loss in some patients. The Rubel Lab in the UW Department of Otolaryngology has recently reported a molecule, termed PROTO1, which has been shown to protect cochlear hair cells from aminoglycoside induced degradation. However, due to the poor vascularization of the cochlea, it is difficult to deliver ototherapeutic drugs systemically, and thus other methods of delivery must be explored. This study reports on the development of a PROTO1-loaded hydrogel based on the amphiphilic triblock copolymer poly(ethylene oxide)-poly[(r)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO). In this study, two cross linking mechanisms were explored. The first uses the oligosaccharide α-cyclodextrin to thread α-cyclodextrin onto the terminal PEO chains of the micelles resulting in a supramolecular structure termed Pluronic/aCD pseudorotaxanes. Hydrogels formulated in this manner showed controlled linear release of PROTO1 over 25 days but are difficult to manipulate and requires sonication and heating to achieve gelation. An ideal hydrogel would be able to undergo in situ gelation, in which the hydrogel components remain liquid prior to being introduced to the active site, upon which they immediately cross-link to form a hydrogel. In this work, we synthesize materials for an alternative approach that is based on crosslinked cyclodextrin-modified polymers. The synthesis and characterization of the polymer components are reported.

A reversible tissue adhesive is a surface that is capable of sticking to tissues repeatedly without losing adhesive ability, similar to how geckos' feet can adhere to smooth and rough dry surfaces. This project seeks to combine the reversible adhesion exhibited by geckos and the bacterium E. coli, a model for receptor-ligand tissue adhesives. This technology has the potential to benefit laparoscopic forceps designs, which are required to manipulate delicate tissues easily punctured or torn during a medical procedure. FimH is an adhesive protein expressed at the end of E. coli fimbriae that binds reversibly to mannose, a sugar expressed in human endothelial tissues. A FimH receptor-ligand adhesive has worked on small length scales (μm²), but adhesion fails at larger scales (cm²), possibly due to the propagation of a single crack at the adhesive interface. My project addresses this failure by 1) fabricating the geometry of the existing adhesive in PDMS allowing an interface size without damaging force instruments and 2) combining E. coli fimbriae with polymer molds of micron-sized pillar arrays that mimic the topology of gecko footpads and arrest crack propagation on dry surfaces. Spherical PDMS slices of increasing size (40μm – 300μm radii) were coated with mannose and brought down to fimbriated cell-culture plates. Results show that PDMS measures higher adhesion (226 nN) than the existing receptor-ligand adhesive (25 nN), indicating a successful increase in adhesive interface size. The PDMS surface can be used to measure and compare the adhesive capabilities of various micropillar array geometries coated with E. coli fimbriae. In conclusion, micropillar geometries that measure higher adhesion than plates coated with fimbraie have the potential to be developed into a tissue adhesive. Future studies may show reversible adhesion is achieved when fimbriated-micropillar substrates are brought in contact with cultured and dissected tissues.

 Microencapsulation of xenogeneic recombinant cells within Alginate Poly-L-lysine Alginate (APA) matrices is an alternative approach to drug delivery. The APA encapsulation matrix protects xenogeneic cells derived from individuals or species foreign to the host immune system without the need for immunosuppressive or genetic therapy. Furthermore, matrix permeability allows for the continuous delivery of secreted cellular therapeutics at the implantation site. Although this technology is well-established, the ability of these cell-loaded APA particles to secrete antigens activating specific immunity towards HIV over long periods of time has not been studied. My research is aimed at developing implantable, cell loaded microcapsules capable of producing immunostimulatory molecules preventing the transmission of HIV in the genital mucosa. Using cellular viability and quantitative protein assays, my work will examine the long-term stability and protein production of human 293T cells expressing fluorescent HIV pseudovirions. The immunomodulatory effect of cell-loaded APA microcapsules will then be investigated using cell reporter assays. The results of this research may demonstrate the ability of cell-loaded microcapsules to deter HIV transmission over long periods of time by priming the immune system for HIV attack. We expect that this research could lead to studies of the ability of these particles to provide long term protection against HIV infection in animal models. The results of this work may also be applicable for developing vaccination strategies for other sexually transmitted infections.

Industry and academia often use approaches from systems biology in therapeutic protein production. Maximizing the yield of therapeutics from a cell culture is an important need. Current computational approaches use flux balance analysis (FBA) to predict outcomes given certain inputs with the goal of increased protein production. FBA is a method for tracking fluxes within a biological system and studying these systems quantitatively. While commonly implemented within the biotechnology industry, few studies quantitatively validate the results of FBA. This project attempts to conduct a thorough in silico comparison between fluxes determined through FBA and the steady-state fluxes via kinetic simulation. I developed a method for constructing realistic but synthetic networks. Each network features randomly-generated reactions, including a biomass function around which the reaction rates are optimized. The optimization of the model’s kinetic parameters is crucial, for FBA assumes such when determining its solution of fluxes. A genetic algorithm is then applied to select the combination of rate constants that optimize the biomass function. The fittest “organism” determined through genetic algorithms and in silico evolution and the resulting steady-state fluxes will then be statistically compared against the solution of FBA. This assessment of FBA using kinetic simulations could give the scientific community a clearer view of this methodology and its capabilities. Further experimentation and analysis of the findings could direct other groups to where exactly FBA succeeds and where it falls short, thus pointing out the areas that require additional attention. In addition, the construction of synthetic, randomly-generated metabolic networks is novel. The applications of these networks could expand beyond the intentions of this project. It could provide others with a useful tool for tutorial building and a limitless supply of artificial networks for testing and validating other methods or applications.

Human immunodeficiency virus (HIV) has claimed more than 25 million lives worldwide, and a further 33.2 million are currently living with the disease. Current treatment for HIV involves highly active antiretroviral therapy (HAART), which commonly involves the oral administration of a combination of small-molecule drugs that target HIV at different stages in the viral replication cycle. Oral administration requires larger doses of drug due to loss during metabolism, which can lead to greater costs and increased toxicity for patients. To avoid these issues, a lipid-encapsulated hydrogel nanoparticle was synthesized as a mucosal drug delivery vehicle for small-molecule antiretroviral drugs. The nanoparticles, comprising a crosslinked polyacrylamide core surrounded by a phosphatidylcholine bilayer, were synthesized via photopolymerization and characterized using dynamic light scattering for size, zeta potential, and morphology. They were then synthesized encapsulating zidovudine or tenofovir, which are reverse transcriptase inhibitors, or maraviroc, an entry inhibitor, and the encapsulation efficiency and in vitro release kinetics were measured using high-pressure liquid chromatography. In vitro cellular uptake of the nanoparticles was measured using bone marrow dendritic cells (BMDCs). The nanoparticles showed a tenfold increase in the encapsulation efficiency of all three drugs and a slower release profile as compared to liposomes encapsulating the same drugs, indicating that this system allows for HAART dose reduction. The nanoparticles were also taken up preferentially by BMDCs as compared to drug-loaded liposomes and soluble drug, further indicating greater targeted delivery of drugs to infected cells and suggesting a future use for the delivery vehicle as an HIV vaccine.

Aging is a process that causes decline in normal biological function, eventually leading to disease and death. Aging is influenced by a number of factors, including stress-response pathways such as the hypoxic response. Over-expression of the hypoxic response pathway regulator, Hypoxia-inducible factor (HIF-1), increases lifespan in the nematode Caenorhabditis Elegans. Under atmospheric oxygen, HIF-1 is targeted for degradation by the Von Hippel Lindau (VHL-1) tumor suppressor protein, whose deletion increases nematode lifespan through increased HIF-1 activity. Since knocking out VHL-1 in mammals is known to cause disease, it is important to better understand the hypoxic pathway in nematodes to develop targeted therapies for age-related diseases in humans. The goal of this research is to determine the tissues and genes downstream of HIF-1 that modulate aging in C. elegans. To determine tissue specificity, we have developed vectors designed to reintroduce VHL-1 into VHL-1 knockout worms under the control of tissue specific promoters. Lifespan and other age-related parameters will be measured in these transgenic animals and compared to that of control groups. To further test tissue specificity, we are also developing vectors to overexpress a mutated non-degradable form of HIF-1 in specific tissues in wild-type worms, essentially the reverse of the reintroduction of VHL-1. To test for genes downstream of HIF-1 important for longevity, we have performed a screen and identified several genes that are important for HIF-1-induced lifespan extension. Our experiments will examine whether C. elegans lifespan is increased by the over-expression of these proteins, which would then suggest further characterization and a search for conserved proteins. Together, the results from these experiments will provide important information on how we might utilize the hypoxic response pathway to improve human health.

With more than 5 million new infections each year, the HIV pandemic presents a growing problem to which a cure has thus far been elusive. High rates of mutation and variability across HIV strains complicate antibody production via the secondary immune response. HIV surface protein shedding (gp120 and/or gp41) via membrane-proximal external region (MPER) specific antibodies has been shown to neutralize viroid infectivity1, however the conserved MPER of gp41 remains protected by surface proteins, and denatures when removed from its lipid environment. We have developed a vaccine system using lipid bilayer-polymer core virus-like nanoparticles (VLPs) that allows for the delivery of MPER in a native conformation. These VLPs have been controlled for size, polydispersity, and surface characteristics to allow intercalation and presentation of MPER in a hydrophobic state. We plan to characterize cellular uptake and cytotoxicity characteristics of these VLPs using in vitro assays. We will evaluate secondary immune response in a mouse model, examining antibody titers post inoculation. This vaccine platform may prove to be a robust technique for the delivery of hydrophobic antigen moieties, readily uptaken and producing a strong antibody response with low cytotoxicity.

The purpose of this research is to gain insight into prosthetic sock thickness trends of multiple sock stacks. For amputee patients, stacked sock combinations are necessary to accommodate volume change of the residual limb in the short and long term. The thickness of these stacks is quantified by the total number of ply. For example, a 1-Ply sock and a 3-Ply sock make a 4-Ply “stack.” However, with this method of measurement, total sock thickness depends on how many socks are worn. The focus of this test is to evaluate existing standards by comparing the thickness of multiple socks to a single sock of the same total ply. The results from this research should provide useful information to prosthetists fitting patients with prosthetic limbs. As the residual limb matures after amputation and swelling decreases, there is a reduction in volume and a change in shape. From the first prosthesis, the practitioner optimizes the fit with the expectation of the residual limb size decreasing. The practitioner advises the patient to add socks to take up space when the residual limb feels loose in the socket. By adding socks, the patient can maintain a proper fit, and the practitioner can track limb change and socket modifications so as to appropriately time fabrication of a new socket. The present research brings quantitative information about sock thickness, both for single socks and for stacks of socks, to clinical care. This quantification should help the practitioner determine just how much larger the socket is than the patient’s residual limb, and should help guide design of a new socket. This research collected in the present study may also facilitate documentation to justify the need for a new socket.

The control of infectious diseases in low-resource settings continues to be impacted by the limited availability of low-cost, easy to use diagnostics. However, even in developing nations, smart phones are common and can be used as "instruments" to control diagnostic devices. Here, we aim to use a smart phone to input a sound wave into a microfluidic card and measure a sound wave coming out of the card to determine whether a disease is present in the sample. This can be done by creating a network of fluid channels that can be modeled as an electrical circuit containing resistors, inductors, and capacitors (RLC circuit). When the sound wave is input to the system, it causes the fluid to oscillate and has a resonant frequency that is dependent on the characteristics of the system (the resistance, inductance, and capacitance). A chemical reaction of an analyte—the chemical that indicates a disease is present—causes a change in the fluid resistance, fluid inductance, or fluid capacitance (e.g. the creation of a bubble would change the capacitance). This change in R, L, or C shifts the resonant frequency of the fluid circuit, allowing the presence of the analyte to be recognized. This project seeks to integrate a means of detection (like a microphone) into the diagnostic card to measure the analyte-dependent resonant frequency. Completion of this would take the system one step closer to bringing simple and effective diagnostic platforms to developing nations.

Hydrocephalus is a medical condition affecting one in every 500 to 1,000 live births in which an excessive amount of cerebrospinal fluid (CSF) accumulates in the brain ventricles. Accumulation of CSF can occur if the pathway of CSF drainage is blocked or if too much CSF is being secreted. If CSF buildup is unchecked, intracranial pressure (ICP) will increase and lead to intracranial hematoma, cerebral edema, or brain herniation.Hydrocephalus is commonly treated by an implanted CSF shunt which is small catheter with one way pressure or flow controlling valve which diverts CSF from the brain ventricles to the heart or peritoneal cavity thus lowering ICP.However, 40% of the existing shunts fail in one year and 95% of the shunts fail in five years. Valve obstruction accounts for 50% of shunt failures. The high obstruction rate is due to the fact that existing CSF shunt valves suffer from complicated internal regulating mechanisms that are prone to obstruction. To have a significant impact on reducing the number of failures due to valve obstruction, a novel shunt valve design is necessary. Thus, the primary goal of our design is to reduce the valve failure due to obstruction. Our approach is to replace the internal regulating valve with a simple regulating mechanism. The key features of the valve designs are that there is no contact between CSF and regulating components and a simple flow path.This CSF shunt valve can maintain ICP within a physiological range by regulating the rate of CSF drainage according to ICP.

HIV has infected over 30 million people worldwide and has resulted in the loss of millions in healthcare costs. HIV vaccines have the potential to prevent infection and drastically reduce infection rates. A potential strategy for vaccine development is to elicit a mucosal immune response, since sexual transmission of the virus is the leading cause of new infections. A successful functioning vaccine would deliver an HIV antigen, such as the highly conserved regions of the HIV membrane surface protein gp41, and an adjuvant, such as the synthetic organic molecule R848, through mucosal and cellular barriers. Here, a delivery vehicle for a complex HIV vaccine is synthesized by using a single emulsion method to formulate a polymeric nanoparticle, consisting of a biodegradable polymer core surrounded by a lipid layer. Previous research has shown that a lipid shell increases serum stability and immunocompatibility in vivo. First, design parameters, such as solvent and surfactant properties, were modified to yield consistently monodisperse populations of nanoparticles with controlled size and surface chemistries. Different methods for encapsulating the adjuvant into the polymer core were also explored. The encapsulation efficiency and drug loading of the adjuvant, as measured using liquid chromatography, was less than 1%. We delivered an HIV antigen by intercalating it within the lipid layer surrounding the polymeric core. Encapsulation efficiency of the antigen was measured with a proof-of-concept peptide attached to a fluorophore. We used ELISA and cell culture studies to test for the controlled release and immunogenicity of the immunogens. Finally, a mouse model will be constructed to test the particles in vivo. This project demonstrates a design for creating nanoparticles with controlled characteristics for eliciting a comprehensive immune response against HIV.

Millions of people in developing countries die from infectious diseases (e.g. malaria and dengue), yet many of these deaths can be prevented if given the tools for accurate diagnosis. However, current diagnostic capabilities with the required clinical sensitivity, a measure of the number of people with the disease who are correctly diagnosed, are confined to laboratory settings due to cost, electrical, and personnel requirements. Thus there is a medical need for diagnostic tools with the required level of clinical sensitivity for use in low resource settings. The current standard test performed in low resource settings is a lateral flow test, like the common pregnancy test, which incorporates gold labeled molecules that allow for visual detection of antigen, e.g. disease specific protein, levels in a sample. In previous work, I helped design a prototype paper-based malarial diagnostic test that utilized a gold enhancement solution via a metal catalytic reaction to amplify the gold labeled molecules with a four-fold increase in sensitivity. The goal of my project is to increase sensitivity further by characterizing the effect of gold label particle size on the performance of gold and silver enhancement solutions. The gold label acts as the catalyst for the metal catalytic reactions. Smaller gold particles have the potential to bind at higher densities in the detection region, which could increase the catalytic surface area for the metal catalytic reaction and potentially lead to greater improvement in sensitivity. For my project I will redesign the current prototype malarial diagnostic test to incorporate the most promising signal amplification reagents, and the sensitivity of the device will be characterized.

Hydrogel tissue scaffolds have emerged as a useful tool for extensively mimicking and reproducing the cues of the natural extracellular matrix (ECM) of native cell environments. Polyethylene glycol (PEG) is a synthetic compound that is often used as a hydrogel as it is nontoxic and exhibits strong mechanical properties. PEG, however, does not allow cell adhesion and cannot be degraded by cells. To resolve this issue, the incorporation of denatured collagen, or gelatin, was used. This process was completed via addition of methacrylated gelatin (GelMA) to photocrosslinkable PEG hydrogels. The addition of the polysaccharide hyaluronic acid (HA), which is an integral part of the ECM, is in part a novel approach to further provide chemical cues to activate specific receptors (i.e. CD44) within cells. Moreover, cells have also been known to respond to physical stimuli such as changes in the topographical environment. We have developed a technique to produce a nanotopographic pattern on hydrogel scaffolds via the use of a nanopattern mold. Here, we propose the novel integration of the chemical signals produced from the hydrogel in conjunction with a nanopattern for directing cell migration and proliferation in vitro. In this experiment, Wnt1-Cre derived dental pulp stem cells (DPSC) were cultured on these scaffolds. Cellular regulation was reflected in changes in morphology and migration due to exposure to the combined chemical and physical signals of the tissue construct. Cells cultivated on patterned scaffolds showed significant alignment along the pattern compared to flat controls. In addition, cells on HA-PEG-GelMA hydrogels exhibited aggregation over time to form spheroids that persisted for the duration of the experiment. These findings suggest that the microenvironment of HA-PEG-GelMA hydrogels plays a significant role in regulation of the spatial arrangement of cells.

Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. Last summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. We constructed a strain of Escherichia coli that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. Using this method, we were able to produce on average 160 mg of alkanes per liter. Also, we identified a protease with gluten-degradation potential, and then reengineered it to have 100 times increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. Finally, to enable next-generation cloning of standard biological parts, BioBrick vectors optimized for Gibson assembly, an emerging DNA cloning technique, were constructed and used to construct the Magnetosome Toolkit: genes for biofabrication of magnetic particles.

Opiates are considered an effective treatment of severe pain for a long time. However, repeated exposure to opiates induces analgesic tolerance, which has been correlated with an increase in opiate associated deaths due to respiratory depression. This project aims to compare and contrast tolerance to opiate-induced analgesia and tolerance to opiate-induced respiratory depression. We hypothesized that the rate of development of the former tolerance phenomenon occurred faster than that of the later tolerance phenomenon. In such a case, patients may be more likely to develop respiratory depression as the dose of opiate is escalated in an attempt to provide continued analgesia. To date, animal tests have been conducted to look at effects on nociception and ventilation produced by various doses of opiates in opiate naive and opiate experienced rats. The nociception testing was conducted through paw withdraw experiments, where the time for rats to move their paws under a hot light indicated analgesic effects, and the ventilation testing was conducted using whole body plethysmography, where ventilation signals of rats were conveniently and accurately measured and recorded. In the ventilation testing, rats’ responses to different ambient carbon dioxide concentrations up to 5% were measured. From the collected data, a potential therapeutic window for morphine was found to be between 5 mg/kg to 15 mg/kg, under which no obvious respiratory depression was observed while above which severe respiratory depression happened and almost no respiratory response to carbon dioxide was noticed. Future results may indicate the relative large discrepancy between the magnitudes of tolerance to opiate-induced analgesia and that of tolerance to opiate-induced respiratory depression. This may further indicate the different mechanisms mediating these two opiate-induced tolerance phenomena, a better understanding of which may allow treatments that make opiates safer for patients with chronic pain.

The Community Action Research and Empowerment Project (CARE), launched in September of 2010, is a participatory action research project where elders and youth work together to assess the strengths and needs of the Vietnamese community in Seattle, and develop a long-term plan to address those needs and advance the community. Data from 16 focus groups, 85 interviews, and 316 surveys were collected. We learned that the Vietnamese community members have a broad range of concerns, such as youth and education, housing, jobs, health and social services. During Phase 2 (2011-2012), a strategic action plan will be created with extensive input from community members to address the issues identified—thereby creating a shared vision for the Vietnamese community.

South Africa’s KwaZulu-Natal province currently has the highest HIV prevalence in the world at approximately 30%. The University of Washington’s International Clinical Research Center is currently conducting a pilot program for a new HIV testing method, Home-Based Counseling and Testing for HIV (HBCT), to address the HIV epidemic. A mathematical model of heterosexual HIV transmission in KwaZulu-Natal was created to evaluate the long-term consequences of HBCT on HIV prevalence. This model simulates a population of susceptible and infected individuals with varying sexual activity levels and therefore, risk. The model also divides the population among twelve age cohorts and the infected individuals are further divided into three HIV stages and a final AIDS stage, as defined by the World Health Organization. The model was created in MATLAB and validated with HIV prevalence data for KwaZulu-Natal from the Joint United Nations Programme on HIV/AIDS. Preliminary results from the model indicate that HBCT will diagnose more cases of HIV than other testing methods and, with successful linkage to care, will decrease HIV prevalence in the future. While the possibility of HIV elimination by HBCT is yet to be determined, preliminary results indicate that HBCT is effective at linking infected individuals with care, including antiretroviral therapy. This work has the potential to guide health policy in South Africa, and given the newfound effectiveness of pre-exposure prophylaxis and treatment as prevention, HBCT provides a promising future for controlling the HIV epidemic.

The acid-catalyzed dehydration of 4-methylcyclohexanol yields a mixture of several isomeric methylcyclohexene products. The major product of this elimination reaction, 4-methylcyclohexene, is produced through the E2 mechanism. However, mechanisms for the formation of the minor methylcyclohexene products are not predicted by the traditional textbook explanations of carbocation rearrangements. 1-methylcyclohexene is thought to be produced by means of an E1 mechanism in which rearrangement of a carbocation intermediate takes place. This experiment was designed to classify the hydride shifts by observing the products of this reaction using 1,2-²H-4-methylcyclohexanol. The deuterated starting materials for this reaction were prepared in our lab. Gas chromatography-mass spectroscopy and nuclear magnetic resonance analysis were used in the characterization of the products and for the classification of the pair of 1,2 hydryde shifts in tendem and of the single 1,3 di-axial hydride shift, leading to the formation of the 1-methylcyclohexene product. This is useful to know when trying to predict the products of reactions in which intermolecular rearrangements occur during carbocation intermediates.

Drug targeting is highly advantageous because the ability to deliver drugs exclusively to specific cells or organs reduces both the dosage and toxicity effects associated with nonspecific administration. However, targeted delivery of therapeutics is still challenging due to lack of an efficient drug carrier design. Carbohydrate complexes, such as glycolipids and glycoproteins, serve as cellular markers and receptors universally found on the outer membranes of mammalian cells. These glycoconjugates distinguish different types of cells from one another and enable cellular recognition and adhesion. Both the immune system and foreign pathogens rely on these glycoconjugates to bind and enter host cells. For viruses and bacteria, adhesion to host receptors is the first step towards infection. Because of this broad physiological usage, carbohydrates must be highly diverse, with each carbohydrate corresponding to a different receptor. By mimicking nature’s mechanism of cellular adhesion, chemists are using carbohydrates to target specific cells for drug delivery. The designed glycopolymer, as a potential drug carrier, is composed of two main components: carbohydrate for targeting cells in cell-specific manner and pyridal-disulfide (PDS) groups for bioconjugation and fluorescent labeling. In the first step of the synthesis of the glycopolymer, three different glycomonomers (mannose, galactose and N-acetyl-glucosamine) have been synthesized and characterized in order to construct structurally well-defined glycopolymers. Each glycomonomer can be utilized for the synthesis of reversible addition-fragmentation chain transfer (RAFT) glycopolymers. This carbohydrate-based polymer construct has promising potential in targeted drug delivery for its reduction in toxicity and increase in drug efficacy.

Modern healthcare and education have allowed people to age more gracefully. Consequently, healthcare cost continues to rise and an emerging population of people has deteriorating visual perception. The need to perform routine ocular checkup to diagnose diseases at an early stage is necessary to provide effective treatment plans. Fourier Domain Optical Coherence Tomography (FD-OCT) imaging technique is an emerging low-cost non-invasive medical imaging technique that is capable of obtaining high spatial resolution images of biological tissues in vivo. Relative to other medical imaging modalities, OCT system requires the lowest financial cost and least regulatory issues because it can image the eye non-invasively. However, because OCT measures the interference pattern of light to acquire vital information of the tissue of interest, the light emitted to the sample arm must to have the same optical path length as the light emitted to the reference mirror. The process of adjusting the position of the sample to match the optical path length as the reference mirror for in vivo imaging of the human eye is a very operator-dependent and patient reliant process. To solve this problem, we designed an augment system that allows automatic alignment of the reference mirror and focusing lens in the OCT system so that the optimal imaging is always achieved without any manual interventions. We tested the designed system on the tissue phantoms and found that the system worked well toward our expectations. In future, we will integrate the system with a pre-clinical OCT system for in vivo imaging of human eyes for the diagnosis and treatment of ocular diseases, such as glaucoma.

Flight control in dynamic environments is a complicated task that many animals accomplish with ease. It requires rapid integration of sensory input as well as balancing long-term planning with reflexive behaviors. The giant hawk moth Manduca sexta is a useful model organism for studying flight control because its physiology is well understood and it is easy to rear in captivity and work with experimentally. In order to make flight control adjustments, Manduca constantly integrates information from slow-sensing visual sensors and fast-sensing mechanosensory organs. Both visual motion and mechanical rotation elicit strong abdominal reflexes in Manduca during tethered flight, however the exact role of the abdomen in flight is unclear. Previous work has suggested that abdominal reflexes help maintain flight stability, but it has not been shown that the abdomen is actively used for control. The goal of my research is to determine if Manduca can actively use their abdomen to stabilize the visual field. To answer this question, I have created a virtual flight arena composed of a projection system, magnetic tether, and IR sensor. The visual stimulus is projected onto an acrylic dome within which the moth is suspended by the tether. The experiment consists of a system where the moth controls the velocity of a horizontal bar displayed on the dome with its abdominal position. The bar has an initial velocity that the moth counteracts by moving its abdomen in order to stabilize the visual field. The precision of the moth’s abdominal control capabilities can then be tested by differing the initial velocities of the bar and varying the way that abdominal position controls the bar’s velocity. In addition to answering my research question, this project provides a framework to study the general multi-input multi-output problem in neural control.

Stroke is the degeneration of brain tissue caused by blockage of blood flow to the brain. It is one of the leading causes of adult disability and death worldwide. We used ultrasound elastography, a technique that measures local tissue deformation from ultrasound-induced shear wave propagation, to measure shear modulus, the ratio of shear stress to the shear strain, of brain tissue after ischemic infarction, loss of brain function due to low brain blood supply, to determine changes in individual hemisphere due to that injury. We imaged live mice that were 3 hours (hyperacute), 24 hours (acute) and 72 hours (subacute) after surgical occlusion of the middle cerebral artery, and measured the shear modulus of ipsilateral and contralateral brain hemispheres compared to corresponding values of control mice. The mice with hyperacute stroke showed statistically significant increase in shear modulus in both hemispheres compared with control animals. The mice with acute stroke showed a significantly decreased shear modulus in ipsilateral hemisphere and a significantly increased shear modulus in contralateral hemispheres compared to control mice. The mice with subacute stroke showed no significant difference in shear modulus compared to control mice. Significant differences were shown between ipsilateral and contralateral shear modulus values measured 24 hours and 72 hours after infarction. We hypothesize that the shear modulus differences among hyperacute, acute, and subacute stroke mice reflect the initial development of edema and reduction of cerebral blood flow in the ipsilateral hemisphere, and the initial reduction of blood flow then later development of edema in the contralateral hemisphere. Thus, ultrasound elastography may be a sensitive method to detect subtle changes in brain elasticity post stroke. Future work will include an attempt to remove the imaging artifact through the design of a new elastic imaging machine. This research will be a key step toward exploring the ultrasound parameters for imaging stroke, which will ultimately contribute to the development of a better imaging tool for potential stroke patients.

Gene therapy is the treatment of disease by inserting genes or altering them in a subject’s cells and tissues. The Pun lab develops cationic polymer-based delivery vehicles. Our agenda was to determine the intracellular fate of drug delivery vehicles carrying the therapeutic genes in order to better understand cellular barriers that limit efficient delivery. For this project, we applied two methods to study the intracellular trafficking of polyplexes (polymer-DNA complexes). The first approach is a quantitative method using subcellular fractionation to separate organelles and determine the distribution of polyplexes in various cellular compartments after internalization. We hypothesized that by using methods of centrifugation, organelle-specific enzyme assays, and radiolabeling, polyplex distribution could be quantitatively analyzed. We demonstrated successful separation and quantification of organelles using a combination of differential and density-gradient centrifugation. The polyplex distribution in each fraction was determined through radiolabeling. In this way, a quantitative “snapshot” was taken of the distribution of polyplexes inside the cell, and yielded information for the identification of potential bottlenecks in trafficking to the nucleus. Our second approach is to study the intracellular trafficking of drug delivery vehicles by time-lapse fluorescence imaging combined with single-particle tracking. This complementary approach reveals both the time-course and behavior of particles as they are trafficked within the cell. By fluorescently -labeling the polymer and applying the polyplexes to cells, we successfully observed their motion within a single cell. We further optimized the quantity of polyplexes delivered and the image capture techniques to robustly track polyplexes within a single cell. Our work therefore provides improved methods for determining the mechanism and efficacy of gene delivery systems and potentially any particle-based drug delivery system. For future work, we plan to investigate the process of unpackaging of the polyplexes inside cells.

Ultrasonic neuromodulation is the process of stimulating different neural pathways in the brain through the use of transcranially delivered ultrasound in order to induce clinically relevant emotional, cognitive, physical or behavioral reactions. My research goal is to find out if it is possible to add anatomical specificity to neuromodulation through the use of modulated focused ultrasound (mFU), where 'modulated' refers to adding complex temporal structure to the waveform to optimize neuromodulation. Since mFU has a small focus relative to the brain, the acoustic power from the ultrasound can be moved within the brain; previous experiments have used unfocused ultrasound which sends ultrasound through the entire brain. We hypothesize that we can map varying physical reactions through stimulation of different parts of the brain, with our initial emphasis on the motor cortex because stimulation there produces readily observable results. With lightly anesthetized mice as our subjects, and always delivering ultrasound through the skin and skull, we began neuromodulation with unfocused transducers to transmit our ultrasound. This critical step was done to determine the optimal intensity, waveform shape and carrier frequency of ultrasound that created a robust observable effect. Next we translated these ultrasound parameters to our mFU transducer. The current stage of the process includes moving through different parts of each hemisphere and recording the associated motor response such as leg movements and tail flicks. We have already seen a change in activity between activating the quadrant of the brain containing motor cortex relative to the opposite quadrant. Going forward, we intend to see if we could produce varied motor movements as we change the point of stimulation within the brain hemisphere containing the motor cortex, along with changes in applied intensity, and duration of stimulation. Clinical implications for neuromodulation research include retraining brain function after injury.

Cell labeling and tracking hold enormous potential to illuminate cellular pathway mechanisms. Because current labeling methods are either unsafe or unavailable for humans, a need exists for a safe and effective cell labeling technique suitable to track cells administered for cell therapy. The stable carbon isotope, C13, is safe for human use, incorporates into cellular DNA when taken up as C13 glucose in culture medium, and can be detected by GC mass spectrometry. We proposed to evaluate the use of C13 to label and track exogenous bone marrow derived macrophages (BMMCs) administered after myocardial infarct (MI). To confirm exogenous BMMCs can be detected in the heart, we injected green fluorescent protein (GFP) labeled BMMCs into the tail vein of mice three days following MI. Some heart sections were assessed for GFP positive cells by immunohistochemistry. Other hearts underwent dispersion and isolation of macrophages. Quantitation revealed the percentage of exogenously delivered C13 labeled GFP BMMCs in the cardiac macrophage population was 10 percent. Preliminary cell proliferation assay results suggest no considerable difference between the growth of BMMCs with or without C13 glucose. Finally, we cultured GFP BMMCs for ten days in 100% C13 glucose media for tail vein injections to mice following MI. Three days later, we excised the hearts to isolate cardiac macrophages. Cultured heart cells and macrophages were analyzed via GC mass spectrometer to quantify labeled DNA. Although we could detect C13 in in vitro labeled macrophages, increased C13 cellular label could not be detected in isolated macrophages. Unlabeled macrophages appropriately had no increase in label. Ultimately, while this study was unsuccessful in detecting a C13 cellular label in vivo with a GC mass spectrometer, in vitro successes suggest technique refinement could lead to a powerful and safe therapeutic stem cell label.

Alzheimer’s Disease (AD) is a costly, debilitating, and irreversible neurodegenerative disease that affects at least 15 million people worldwide. There is no accurate early diagnosis for AD. When symptoms are noticeable, it is often at the end stage when treatment is of little use. Current treatment of AD cannot cure the disease, and is only able to slow its progression. Thus, an early diagnosis may prevent people from experiencing late-stage AD because people can begin treatments early and could slow disease progression significantly. The goal of this project is to explore the feasibility of such a diagnostic approach through Optical Coherence Tomography (OCT) imaging of ex vivo human lens. Specifically, I am writing MATLAB code that can screen OCT images for supranuclear cataracts. Goldstein et al. have described unique supranuclear cataracts in the lens of post-mortem AD patients. I will also correlate OCT findings to histology of the lens and current known AD biomarkers for a more definitive confirmation of AD. If supranuclear cataracts are indeed unique to AD and OCT is able to detect supranuclear cataracts, a diagnosis for AD through in vivo OCT imaging of the lens is promising.

The field of gene therapy relies on vehicles that can introduce exogenous genetic material into target cells in order to affect the production of a desired therapeutic protein. Viruses have long been used as delivery vehicles, but concerns over immunogenicity have fueled efforts to develop alternative nonviral delivery vehicles. Current polycationic polymers used in gene delivery methods generally exhibit poor transfection efficiencies and also dose-dependent cell toxicity. This project seeks to address these parameters through the synthesis of a novel copolymer of 2-hydroxypropyl methacrylate (HPMA) and a methacryated oligoarginine peptide (MaAhxR10). In phase one, the HPMA-oligoarginine copolymer was synthesized and tested for basic functionality. Transfections of HeLa cell populations demonstrated that the transfection capabilities of this polymer were comparable to those of the oligolysine standard, but the toxicity was significantly enhanced. Phase two consisted of modifying the peptide with neutral amino acid residues (such as glycine) to reduce the charge density of the polycationic peptides. Further, a more efficient approach to preparing arginine peptides was developed by chemically guanidinylating lysine residues. This approach was used to generate a lysine and a homoarginine version of each polymer. The resulting copolymers were tested for transfection efficiency and cell toxicity in cultured mammalian cells. Copolymers made from the resulting ‘spaced’ peptides (MaAhx(GK)5 and MaAhx(GhR)5) demonstrated reduced cytotoxicity compared the MaAhxR10 and PEI standards, but were still comparable in transfection efficiency to the oligolysine standard. While the delivery vehicle has not performed as expected, the project may have revealed a relationship between charge density (and therefore accessibility of charge) and cytotoxicity. The next step will be to generate another panel of polymers, this time with even more spacing between positive charge residues. This will allow for the identification of a correlation between charge density and cytotoxicity.

The treatment of a disease is often based on that disease's progression. In cancer, such information is derived from indicators such as the location and extent of tumor within tissue. In addition to determining the presence of a disease, biopsies are often taken with the goal of finding these indicators. Unlike cell biopsy, samples from tissue biopsy preserve biological architecture, allowing these indicators to be more easily determined. For this project, Thin Needle Core Biopsy (TNCB) is used to obtain small cylindrical tissue samples 250µm in diameter and up to 2cm in length. Tissue samples are imaged with Optical Projection Tomography Microscopy (OPTM), which is currently used to generate high-resolution three-dimensional (3D) images of individual cells. Imaging through the tissue is facilitated by axial scanning through the optically cleared tissue; however, the microscope has limited lateral field of view and the TNCB specimen must be imaged in a series of 3D images. This project has three focuses. First, stitch a series of OPTM images using morphological operations, histogram manipulation, and phase correlation. Second, perform image reconstruction to create a single continuous 3D image of all cells within the biopsy. Third, present the 3D images to pathologists in a user-friendly interface. In addition to displaying the 3D image, the computer interface allows clinicians to view cross-sections of the 3D image in the x-, y- , and z-planes to simulate pathological tissue slices. The results allow pathologists to analyze the entire tissue sample, not just samples of cells, and have potential to reduce sources of error in making diagnoses.

All-atom molecular dynamics (MD) simulations are increasingly being used to predict protein motion and to represent the range of dynamic structures populated. MD simulations are now commonly done on nanosecond to microsecond timescales for improved sampling of biologically relevant processes. With the increasing size of simulation data, data visualization has become a challenge. In this research project, we address this problem by building an interactive 2D/3D rendering software based on Windows Forms and Windows Presentation Forms (WPF) within Microsoft’s .NET 4.0 Framework. The Protein Dashboard (PD) exists as several plugins for the in-house software program called DIVE (Data Intensive Visualization Engine). The Protein Dashboard offers 2D plots for rendering protein properties as well as 3D rendering for protein structures. Datapoints in both 2D and 3D plots are fully interactive. As plugins of DIVE, PD offers microscripting for both novice and advanced users. With microscripting, users can manipulate both 2D and 3D plots to examine data in various ways. All DIVE plugins have the capability of receiving and outputting data to one another. Thus, users can examine their data in different depth by connecting two different 2D plots and have the latter display the subset. The flexibility and interactivity of PD will allow researchers to explore MD simulation data much more efficiently and ultimately speed up scientific discovery.

Diabetes is a serious and prevalent disease affecting the lives of many people. Studies have shown that cardiovascular calcification, or calcium-phosphate mineral deposition in blood vessels, cardiac valves, and myocardium, is prominent in patients with Type II diabetes (T2D), accounting for the heightened cardiovascular morbidity and mortality in this population. Using a T2D mouse model, our laboratory has shown a positive correlation of hyperglycemia with cardiovascular calcification. A multi-ligand receptor, namely the receptor for advanced glycation-end products (RAGE), was high in cartilaginous and calcific areas of blood vessels and aortic valves and translocated to the cell membrane of the cells with osteochodrogenic (bone and cartilage precursor) and chondrocytic (cartilage) properties. To study whether RAGE is involved in the early signaling of osteochodrogenic differentiation and calcification of cardiac valves, we cultured bovine valve interstitial cells (BVICs) in procalcific medium containing normal and high glucose in a range of hyperglycemia. We found that high glucose increased calcification of BVICs by up to 3 folds. Expression levels of RAGE and its ligands are determined by reverse-transcription PCR and Western blot analysis. The biological activity of RAGE are blocked by a commercially available neutralizing antibody to see whether RAGE is required for the high glucose-associated BVIC calcification. Our results show a decrease in BVIC calcification when RAGE activity is blocked by a neutralizing antibody. For future studies, we will create a cell line with lower expression levels of RAGE via small RNA interference.

Heart disease is the leading cause of death in the US. Current therapies are limited, and most slow progression of the disease but do not address restoring function. Dr. Regnier’s laboratory is pursuing a novel heart disease treatment. The lab discovered that a nucleotide analog of ATP, 2-deoxy-ATP (dATP), which is produced in heart cells (cardiomyocytes), greatly improves cardiac performance by increasing myofilament contraction. We have established a transgenic mouse line that overexpresses ribonucleotide reductase (RR), the enzyme that converts ATP to dATP. In addition, we have developed viral vectors to overexpress RR specifically in the heart. Both methods of RR overexpression elevate intracellular dATP, improving contractile strength of cardiomyocytes and cardiac function. An added benefit is increased rate of relaxation by dATP, as is the decay of the calcium transient. The calcium transient behavior is primarily governed by proteins of the sarcoplasmic reticulum (SR), which is the storage site for intracellular calcium. I am studying what causes the improved rate of calcium decay. I measure calcium handling protein expression and activity modification via western blots, and measure contraction and relaxation of isolated cardiomyocytes. I am comparing healthy and infarcted hearts from transgenic and viral RR overexpression mice. Mechanical measurements demonstrate the frequency dependence of the amplitude and kinetics of contraction, relaxation, and the calcium transient. Western blots reveal if faster calcium transient decay is due to SR changes in relation to RR overexpression. The infarct hearts have loss of function due to the death of muscle tissue, but we predict that RR overexpression and subsequent increased concentration of dATP will increase myofilament calcium sensitivity, thus rescuing cardiac function and restoring damaged cells to regular contractile strength. RR gene therapy may be a breakthrough treatment of heart trauma if damaged cells are rescued and normal contraction is achieved.

Many soldiers acquire traumatic brain injuries (TBI), such as edema, hemorrhage, and traumatic axonal injury, from improvised explosive devices. Currently there are no adequate brain-imaging devices for use near the battle field. Patients with TBI may require a complete hemicraniectomy to examine the extent and location of the injury. This presents a great need for field-deployable imaging devices optimized for brain imaging with the spatial resolution of CT scans. Previous work has shown that intraoperative ultrasound, ultrasound used during surgery, can create images of brain-based tissue stiffness. For this project, rats have been given TBI using controlled cortical impact methods and their brains have been imaged using shear wave imaging to quantify the stiffness of brain tissue. The ultrasound images have shown patterns of brain stiffness that exhibit the extent and location of TBI. After the brain tissue stiffness for TBI was quantified using shear wave ultrasound, the data was used to optimize ultrasound machines for imaging moderate TBI. The Verasonics ultrasound device in the lab was optimized to image brain tissue by tracking the shear wave, generated by the transducer, as it propagates through brain tissue and correlating propagation speed with stiffness values. The images obtained from the ultrasound device were compared against histology. The success of this project would ultimately lead to increased quality of care for soldiers with TBI near the battlefield.

MRI is by far the most sensitive technique for screening high-risk women and assessing the extent of breast cancer. However, dynamic contrast-enhanced imaging (DCE-MRI), the standard breast MRI method, produces many false positives. As the American Cancer Society now recommends annual MRI screening for high-risk woman, estimated to affect up to 1.8 million women in the US, there is a quickly growing need to reduce the false-positive rate of breast MRI in order to reduce the number of unnecessary biopsies that could result. Our research explores the diagnostic potential of diffusion-weighted imaging (DWI), a MRI technique that depicts the diffusivity of water molecules in the tissue as a way to characterize the cellularity of breast lesions. Research has shown that DWI has the potential to increase the diagnostic accuracy of breast cancer, serving as a promising adjunct tool to DCE-MRI for reducing false positives. Our goal is to evaluate the ability of DWI for non-invasively discriminating benign and malignant breast lesions that were identified as suspicious on conventional breast MRI. Specifically, we will investigate image registration to improve alignment of DWI images, the effects of the contrast agent used in DCE-MRI on DWI measures, and the optimal diffusion sensitization of DWI in discriminating benign and malignant breast lesions. These characterizations of DWI can potentially help to optimize the acquisition and processing of DWI scans for future clinical implementation and help guide the appropriate use of DWI as an adjunct tool in radiological diagnosis of breast cancer.

Embryonic diapause or delayed implantation is an occurrence that effectively lengthens the gestation period of the embryo and allows offspring to be born into optimal conditions. In response to adverse environmental conditions, the mouse blastocyst enters a reversible state of cell quiescence, or extremely slowed development, resulting in delayed implantation. Our aim is to uncover the mechanisms that occur in diapause that allow stem cells to endure stressful environments. Theoretically we could generate “super” stem cells that retain pluripotency for longer periods and withstand harsh in vitro treatments. Gene expression profiling of the stem cell compartment of diapause embryos showed that the transcription factor Runx1 is upregulated and might be required to initiate diapause. Runx1 thus far has shown to induce a state of cell quiescence in haematopoietic stem cells (HSCs) and is responsible for haematopoiesis, the formation of blood cellular components, in early development1. Runx1 is also known as acute myeloid leukemia 1 protein (AML1) because mutations in it are the most common aberrations found in acute leukemia2. Additionally, we observed up-regulation of RUNX1 protein expression level in the primitive endoderm (PE) layer of the diapause embryos. We hypothesize that the increased expression of RUNX1 is required to maintain pluripotency of the stem cell compartments in the embryos. In vivo mouse models of Runx1 wild-type (+/+), heterozygous (+/-), and deficient (-/-) diapause embryos will be examined using confocal microscopy with antibodies against RUNX1, pluripotency marker (OCT4), and PE marker (GATA4), and the resulting morphologies of the Runx1 embryos will also be characterized.

Ubiquitin is a protein modifier that is essential for many eukaryotic cellular processes. Attachment of ubiquitin to substrate proteins (ubiquitination) occurs in a three-step cascade that involves a ubiquitin activase, ubiquitin conjugase, and ubiquitin ligase; it is the ligase that targets substrate proteins. One challenge for the ubiquitin field is identification of substrate cohorts for ubiquitin ligases. Although ubiquitination only occurs naturally in eukaryotic organisms, we have previously reconstituted the ubiqutination cascade in E. coli. Isolation of the ubiquitin pathway within a prokaryotic cell, such as E. coli, allows for the investigation and modification of individual components of the pathway and the measurement of immediate effects of these changes upon the overall process of ubiquitination. This project, which consists of the construction of a split protein assay, aims to address the abovementioned challenge by building on our previous work reconstituting ubiquitination in E. coli. The assay uses two fragments of the protein dihydrofolate reductase (DHFR) to detect the ubiquitination of proteins in E. coli, outside of the natural eukaryotic environment of ubiquitin. All of the essential components of the ubiquitination pathway, including individual fragments of DHFR fused to ubiquitin and a substrate protein of interest, are transformed into the bacterial cell. The assay tests for the covalent attachment of ubiquitin to the substrate protein through growth on selective medium, which is possible only through the combination of the separated fragments of DHFR. This reporter system allows for experimentation regarding the interaction and selectivity of the ubiquitin ligase with the substrate protein of interest. Thus, a functional reporter system will allow for the streamlined analysis and determination of the substrate cohort for a given ubiquitin ligase. Changes to the components of the system may include using different ubiquitin ligases and substrate proteins.