Bismuth nanoparticle (Bi NPs) have wide-ranging applications in both biomedical imaging and nanotechnology. These applications are hampered by the absence of a sized-controlled, uniform synthesis using conventional laboratory techniques. The current synthesis of such particles is arduous and requires specialized techniques and equipment, specifically air-free techniques, which are not readily available to all. By using air stable and cost effective reactants, we hope to develop and characterize a synthesis that is reliable, aerobic, free of lithium based organic bases, and potentially offers size control between 30 nm and 70 nm. This synthesis would produce high quality Bi NPs that are available to a wider range of researchers.

The appearance of plastic in the world’s oceans has increased since the early 1970s. As these plastics get older, they begin to break down and erode, which causes microscopic fibers of these plastics to end up in the ecosystem. These microplastics end up in the world’s oceans through runoff and other freshwater inputs. The main issue with these plastics is that they can be toxic to marine organisms. This study can bring awareness to this problem that the world is facing. A localized study in Possession Sound to monitor these microplastics incorporates two components: visual surveys from plankton tows and a targeted sieving of seawater. I hypothesize that there will be higher concentrations of microplastics in areas that are closer to the Snohomish River. The data that is being looked at is from 2013 plankton counts to present day, as well as data that I have collected by pumping surface seawater through three different sieves (with mesh sizes of 60 microns, 150 microns, and 250 microns) for three minutes. With the data collected by plankton counts, the most microplastics were found at a station called Buoy (an area just outside of the Snohomish River) with 42 counted, and the least plastics that were found was at North Jetty (an area that is adjacent to the mouth of the Snohomish River) with one plastic fiber counted. My hypothesis was not supported because there was only one fiber that was found at the mouth of the river, while 42 fibers were counted were counted further away from the river.

Advancements in the design of robotic prostheses have provided promising new prototypes as well as highlighted the need for continued development in myographic sensors essential for fine control of prosthetic limbs and other assistive devices. Force myography (FMG) sensor matrices have the potential to improve upon the granularity of motion control present in current non-invasive robotic prostheses. FMG utilizes pressure sensors to detect flexion and extension of muscle fibers, requires no complex signal processing, and has the potential for improved accuracy and robustness over other myographic interfaces, which have known issues of data clarity. In this study, FMG was investigated through sensor matrix design and data collection. A matrix of force sensors was constructed for use in human-computer interaction, using the open-source prototyping platform Arduino for data collection and mechanism control. The sensors were constructed from pressure-sensing material (Velostat) layered between conductive foil, arrayed in a grid, and housed in an adjustable sleeve. Data collection was focused on identifying force patterns which correspond to the eight classes of motion critical to a robust transradial prosthesis. Preliminary prototyping and data analysis indicate that simple, widely-available materials provide sufficient pressure sensitivity for prosthetic applications when compared to more common, prohibitively expensive sensors. The matrix returns pressure data regardless of radial position on a user’s arm, such that pattern recognition algorithms will likely be sufficient to translate user intent into action. This approach will provide a framework for further development of low-cost prosthetic control alternatives, of particular interest for developing countries with limited funds for healthcare.

The acoustic properties of trumpets have been studied thoroughly, but little to no previous work has been done regarding the acoustics of trumpet mutes. Harmon mutes have a distinctively "buzzy" sound when they are used in performance and it is the opinion of a large number of trumpet players who use Harmon mutes that they should be dented before they are used in performance. Modifications were made to several Harmon mutes by either denting them or drilling holes in them. An impedance head was used to measure the resonant frequencies of the trumpet and mute to see what effect these modifications had on the acoustics of the system. It was found that Harmon mutes have similar behavior to Helmholtz resonators, but significant deviations from this behavior were observed. If Harmon mutes are better understood, then a more informed decision as to whether or not they should be dented can be made.

Alkanes are the major constituents of natural gas and petroleum. The development of catalysts for the efficient and cost-effective catalytic conversion of alkanes to functionalized organics is highly desirable. Previously, Nishiyama and coworkers found that the Ir^III complex (^dmPhebox)Ir(OAc)₂(OH₂) (Phebox = 3,5-dimethylphenyl-2,6-bis(oxazolinyl)) could activate the C-H bonds of n-octane at high temperatures under nitrogen. Goldberg and coworkers found upon increasing the temperature, β-H elimination could be promoted, giving isomers of n-octene. Reactions of similar Ir complexes with alcohols, ketones and other organic substrates will be presented.

Stimuli-responsive hydrogels represent a promising class of inks for use in 3D-bioprinting. These materials respond to external stimuli such as heat, light, pH, and pressure, and researchers have utilized these different stimuli responses to develop inks for direct-write 3D printing. The objective of our work is to understand how the composition of poly(alkyl glycidyl ethers)--more specifically, allyl-, ethyl-, isopropyl, n-propyl-, and methyl-glycidyl ether homopolymers--affects their thermal response in aqueous solutions. UV-Vis Spectroscopy was used to determine the lower critical solution temperature (LCST) of each homopolymer, varying the molecular weight, concentration and polymer composition to demonstrate poly(alkyl glycidyl ethers) versatility as a platform for synthesis of stimuli-responsive hydrogels. We determined that the identity of the alkyl group significantly affected the temperature-response of the respective hydrogel. These studies will guide our work toward developing temperature and shear-responsive inks for 3D-bioprinting. In particular, these materials can be used to develop printed hydrogel lattices for whole-cell catalysis to produce medicinal compounds.

Exposure to ionizing radiation is the longest-established environmental cause of human breast cancer in both women and men. Evidence indicates that HER2-positive breast cancers are more likely to spread and are more resistant to anticancer-treatments. The goal of our study is to investigate genetic variations that are introduced during breast carcinogenesis. As a progressive stages of breast carcinogenesis cell model, normal human breast stem cells were sequentially transformed with SV40 large T antigen, x-ray radiation, and HER2 (ERBB2) overexpression to generate non-tumorigenic/immortalized cells, weakly tumorigenic cells, and highly tumorigenic cells, respectively. We sequenced 82 genes that are frequently mutated in breast cancer using next generation sequencing. We identified 26 radiation-specific mutations in exonic or intronic splicing regions that are not present in immortalized cells, but are present only in weakly tumorigenic cells. Among the 26 mutations, a single nucleotide deletion in RECQL gene, which is involved in DNA repair, is highly mutated (43%). Mutations induced by the overexpression of HER2 were identified by analyzing mutations present in highly tumorigenic cells but not in weakly tumorigenic cells. Two HER2 overexpression-specific mutations PRSS1 p.K225R and RET p.F683Y were identified with the mutation RET p.F683Y present at a high prevalence of 54%. The RECQL deletion induced by radiation and the two nonsynonymous mutations PRSS1 p.K225R and RET p.F683Y induced by HER2 overexpression have not been previously reported by others. Studies are underway to further characterize genetic variations that are induced by SV40-T Ag, radiation, and HER2 overexpression in breast carcinogenesis. The identified mutations that are specific to these oncogenic treatments aid in characterizing molecular subtypes of breast cancers and provide a foundation of identifying potential target mutations that can serve as biomarkers for the sequential stages of breast carcinogenesis.

Each year bee specialists report detection of larger numbers of bee colonies suffering from colony collapse disorder. One of the suggested causes is the increasing use of neonicotinoid pesticides in farming agriculture. To study the effects these pesticides have on the environment, it is necessary to establish a method to quantify the amount of imidacloprid in an environmental sample. Previously, we found that off-line C-18 cartridges were suitable for enriching imidacloprid from complex matrices and for preparing samples for analysis in GC-MS (gas chromatography-mass spectrometry). Our previous work primarily focused on achieving a reliable calibration curve by enhancing signal-to-noise in the GC-MS. To further evaluate our findings, a spiked environmental sample was subjected to C-18 extraction and analyzed. This method resulted in a limit of detection as low as 160 micromolar, which is similar to the 120 to 320 micromolar range for limits of detection achieved by other research groups using more complex extractions. This is important to the future study of imidacloprid because it allows us to progress towards the analysis of environmentally relevant concentrations of imidacloprid in soil, plant and honey samples.

Since the 20th century, the concentration of atmospheric carbon dioxide has increased dramatically, inducing several environmental issues. Our research seeks to reduce this excess carbon dioxide in our atmosphere by using a chemical method to capture and utilize carbon dioxide. In our research, we have developed a bimetallic complex coordinated by a multidentate carbene-phosphine ligand. We hypothesize that the carbon dioxide molecule will be cooperatively reduced by the two metal centers. Using nuclear magnetic resonance and crystallography techniques we have been able to analyze our complex's molecular structure. So far, we have concluded that this complex successfully reduces a molecule with a shape and stability similar to that of carbon dioxide. Moving forward in our research, this bimetallic complex should behave similarly with the bonds of carbon dioxide and thus represents a potential method of reducing the amounts of carbon dioxide in our atmosphere. 

Cancer stem cells (CSCs) are small populations found within tumors and appear to be more resistant to anti-cancer treatments. Although evidence supports that CSCs play a major role in initiation and in the clinical outcome of cancers, the roles of breast CSCs in metastasis and invasiveness are not fully understood. Moreover, the association of the invasiveness of breast CSCs with genomic variations is unclear. The overall goal of this study is to investigate the migration abilities and genomic expression changes in breast CSCs. Human breast CSCs and immortalized/non-tumorigenic cells were derived from the same parental breast normal stem cells. To recapitulate tumor microenvironment, the cells were cultured on nanotopography-defined extracellular matrix (ECM)-mimetic platforms. We have employed a live cell imaging microscopy to measure migration at the single cell level as an indicator of invasiveness in breast tumorigenic and immortalized cells. Our preliminary results indicate that breast CSCs migrate significantly faster than immortalized cells on ECM mimics. Interestingly, we only find this difference under ECM-mimetic culture conditions, but not under flat control surfaces lacking ECM structures. This suggests that the invasive abilities of breast CSCs are lost when they are cultured in vitro on flat control surfaces without ECM structures. We will perform RNA sequencing to identify differences between the transcriptomes of breast CSCs and immortalized cells, and between cells cultured on ECM-mimics and substrates without ECM-mimics. The current study will characterize transcriptome profiling in breast CSCs and identify the genes that are differentially expressed between breast CSCs and immortalized cells. This will have implications in classifying molecular subtypes of breast cancers and in identifying potential therapeutic targets to regulate specific gene expressions for the inhibition of breast CSC invasiveness.

The blood-brain barrier is a very selective membrane that works to protect the brain from most pathogens; however, this means that it is a difficult therapeutic target for drug targeting. Cutting-edge nanotechnology research is being performed by Dr. Elizabeth Nance on targeted drug delivery mechanisms to combat brain tumors such as glioblastomas, neuromas etc. Nance’s group has found and supported good drug release results with poly-ethylene glycol coating (PEG) for which they hope to perform controlled and targeted release. Currently, there is a lack of molecular level understanding of the drug-polymer interactions that dictate controlled release. Better computational models are needed to understand release kinetics and facilitate rational design of biomaterials to control the release or retention of a particular drug or enzyme. In the present work, molecular dynamics (MD) simulations were used with GROMACS software to study glutamate diffusion in water to validate our method and set a baseline for more advanced systems. Diffusion constants calculated with the Einstein equation based on glutamate diffusion in MD were consistent with experimental values of ∼0.75 μm2/msec. We are currently extending this approach to probe glutamate diffusion in the presence of biocompatible polymers such as PLGA (poly(lactic-co-glycolic acid)) and PEG. Then, we compared and contrasted the diffusion constants to make sure they were on the same order of magnitude as glutamate. We also studied the system kinetics to discover the polymeric matrix that allows the system to get closest to a diffusion-limited rate. We will relay this to Dr. Nance to modify her experiments to lead to improved drug delivery systems that help fight off brain cancers.

Protein therapeutics such as monoclonal antibodies (mAbs) are gaining importance and number in the drug development pipeline and in the clinic. Most standard techniques used to characterize these drugs require dilution into aqueous buffers, which do not accurately resemble biological conditions. We hypothesize that the heterogeneous and crowded environment in serum affects the binding and self-aggregation behaviors of mAbs. We directly measured binding affinities and self-aggregation of mAbs in fetal bovine serum and phosphate-buffered saline using fluorescence correlation spectroscopy (FCS). FCS detects individual fluorescent particles diffusing across a small (~1fL) confocal volume. The diffusion time is directly proportional to hydrodynamic radius. We determined binding affinity between Alexa488-labeled streptavidin and anti-streptavidin mAbs based on the difference in diffusion time between the free antigen and the larger antigen-mAb complex. We found that binding with an anti-streptavidin IgG2 was ~3-fold tighter in neat serum versus buffer, while affinity of anti-streptavidin IgG1 did not change. We tested the effects of viscosity, excluded volume, and specific interactions with serum components through experiments in solutions of sucrose, polymeric crowders, and serum components such as albumin and γ- globulins, respectively. Viscosity, crowding, and specific serum components may contribute differently to the difference in IgG2 binding affinity. We also used FCS to measure self-aggregation of a labeled IgG1 in serum and buffer and found that aggregation occurred faster and formed larger complexes in serum at 45°C. These results demonstrate that antibody behavior is different in serum than in buffer conditions. Advancing our understanding of how mAbs and related molecules behave in physiological conditions such as serum will accelerate the development of safe, effective protein therapeutics and thereby improve our ability to treat diseases such as cancer and autoimmune disorders.

Proteins serve very specific and powerful functions in the cell by maintaining a specific shape through chemical interactions in the protein sequence. Under stress conditions however, these chemical interactions holding the protein together may break apart, leading to the protein’s loss of function and exposure of hydrophobic patches that can interact with other proteins to form toxic aggregates. Heat shock proteins (sHSPs) act as safeguards under conditions of stress and protein misfolding as molecular chaperones that interact with exposed hydrophobic patches to stabilize unfolded proteins. The importance of sHSPs in the cell is exemplified through various disease associated mutations of sHSPs. sHSPs are a large and diverse family of proteins distinctive in their ability to form multimers of various sizes, which correlates with varying ability to prevent aggregation. However, little is known about how different structural elements of sHSPs influence function. The Klevit lab aims to better understand the molecular interactions of human sHSPs with themselves, how these sHSPs are activated in cellular conditions, and how they recognize and bind unfolded protein. My work focuses on the characterization of the structure and function of the human sHSP, HSPB8. Specifically, I am studying the effects of HSPB8 binding to zinc, the cellular levels of which vary in response to stress and regulate the function of many other proteins. By comparing zinc bound and non-zinc bound protein samples using various biophysical techniques such as NMR, circular dichroism and fluorescence, I aim to elucidate the effects of zinc binding on structural and functional elements of HSPB8.

The obligate human pathogen, Mycobacterium tuberculosis (Mtb), is the primary causative agent of the infectious disease tuberculosis (TB) and ranks as one of the world’s deadliest pathogens; infecting about 1.5 billion people worldwide. Mtb evades the host immune response via intracellular persistence in alveolar macrophages, where it faces chemical stresses such as a drop in pH, various hydrolytic enzymes, increased toxic ion concentration, and highly reactive oxygen (ROI) and nitrogen intermediates (RNI). Modification of mycobacterial proteins by the prokaryotic ubiquitin-like protein (Pup) and their subsequent proteasomal degradation plays a critical role in Mtb survival in this harsh environment. Thus enzymes associated with protein pupylation may be valuable targets in anti-tubercular therapy. This knowledge is exploited in a rational approach to drug design wherein known structural information about the sole Pup ligase, PafA, is used to develop specifically targeted inhibitors. These inhibitors take the form of stabilized short α-helical peptides, which mimic the hydrophobic and electrostatic interactions in Pup essential for binding to PafA. Production of these compounds was achieved using both automated and manual solid-phase peptide synthesis, as well as 'Click' chemistry to modify cyclized variants. Quantitation of inhibitor performance was accomplished by densitometric analysis of SDS-PAGE results from in vitro pupylation assays with purified proteins. Here I report my work on the successful design, synthesis, and testing of helical peptides which display significant activity as PafA inhibitors.

The meeting of the fresh water from the Snohomish River and the salt water from Possession Sound creates a unique habitat that is important for many species that make up the biodiversity within this ecosystem. The purpose of this investigation was to observe seasonal variation trends from the pH, chlorophyll, dissolved oxygen (DO) and temperature data at the Deep site in the Possession Sound in Everett, Washington. The study was conducted using a Yellow Springs Instrument 85 and Exo Sonde to collect temperature, dissolved oxygen, pH, and chlorophyll data between 0-3 meters in comparison to 7-10 meters in the the water column. It was hypothesized that during the spring and summer months, there will be a higher chlorophyll concentration due to photosynthesis causing an increase in pH and DO throughout the water column. We also predicted during fall and winter months, there will be a decrease in photosynthesis causing a decrease in chlorophyll, pH, and DO. The hypothesis was not supported. There was an inverse relationship between temperature and dissolved oxygen; as temperature was increasing during the summer months, dissolved oxygen was decreasing. When the temperature was decreasing during the Fall and Winter months, there was a increase in dissolved oxygen.

36.7 million people are living with HIV worldwide, with most in low- to middle-income countries. Among many clinical interventions for controlling HIV, Antiretroviral Therapy (ART) has shown success in reducing HIV transmission. However, the effectiveness of these ART is undermined by rising drug-resistant HIV. The lab-based Oligonucleotide Ligation Assay (OLA) can specifically detect HIV drug resistance as an alternative to expensive complete-genome sequencing, but its reliance on moderately expensive lab equipment prevents its universal access. Our lab, in collaboration with the Frenkel Lab (Seattle Children’s Research Institute) and the Lai Lab (UW Bioengineering), is developing a simplified OLA-based HIV drug resistance test. In the OLA, PCR amplification of the HIV Pol region is necessary before subsequent detection of Single Nucleotide Polymorphisms (SNPs) conferring HIV drug resistance. Current amplification relies on a thermal cycler, a >$2.5k instrument not widely available in low resource settings. Isothermal Helicase Dependent Amplification (HDA) eliminates need for a thermal cycler. HDA has been used to amplify the conserved Gag and Env regions of HIV, however it has never been used for amplification of the Pol region, a non-conserved region containing mutations that interfere with DNA annealing in isothermal amplification. We have designed and validated three sets of primers that successfully amplified three regions in the HIV Pol gene covering six common SNPs that confer HIV drug resistance against 1st-line ART. These primer sets each could detect down to 10 copies of HIV Pol DNA per reaction.  We are currently working on a multiplexed HDA assay that simultaneously amplifies three regions in a single tube to reduce cost and consolidate workflow. Implementation of HDA will allow OLA to be further simplified for use in low resource settings by allowing accurate diagnoses of HIV-infected individuals, controlling transmission rates, and reducing the evolution of drug-resistant HIV within entire populations.

Expansion Microscopy (ExM) is a revolutionary fluorescence microscopy technique that enables super-resolution imaging of fixed biological specimens on ordinary microscopes. By chemically incorporating the specimens into a hydrogel polymer matrix, enzymatically digesting the proteins that hold the structures together, and physically expanding the hydrogel complex, this technique yields expansion of fourfold along each x/y/z axis and a spatial resolution of ~70nm. The original ExM approach utilized special tri-functional antibodies containing a DNA-dye-linker complex to attach the fluorescent dyes to the expandable polymer. Previously, our group has developed an improved and simplified ExM protocol, which is compatible with conventional antibodies and fluorescent proteins, by using only commonly available reagents, yielding increased fluorophore retention for cultured cells and tissue specimens. In this project, we extend this new approach of ExM to enable ultrahigh resolution imaging of Drosophila melanogaster tissues which had previously been incompatible with expansion. To do so, we adapted our existing ExM protocol to Drosophila embryos, larval brains, and larval and adult body walls by utilizing an enzyme system that can efficiently digest the tough outer cuticle of the organism. We assessed the uniformity of expansion achieved by our new procedure using correlative pre-expansion and post-expansion imaging and found the distortions to be minimal (<2%). With our new ExM approach, we were able to resolve features of presynaptic active zone structure and study the interactions between the epidermis and dendrites of somatosensory neurons in Drosophila. This work should open up this powerful super-resolution microscopy method to a large community of researchers who utilize Drosophila as an important model system in biology.

Human pluripotent stem cells (hPSCs) are defined by their ability to self-renew, proliferate, and differentiate into the three germ layers. Controversy remains as to the role of canonical Wnt/β-catenin signaling (β-catenin dependent) in hPSC maintenance and mesoderm formation. Wnt/β-catenin signaling has shown to be important in the context of cellular homeostasis and embryonic development, and improper signaling can cause growth of cancerous tumors. By determining the role of this signaling pathway in hPSCs, we aim to improve our overall understanding of the molecular processes behind hPSC directed differentiations and early embryonic development. We assessed the role of Wnt/β-catenin signaling in hPSCs by generating a CRISPR/Cas9 mediated knockout (KO) of the signal transducer β-catenin. Expression levels of protein biomarkers of pluripotency (SSEA4 and OCT4) were evaluated and no difference was noted between wild type (WT) and β-catenin KO hPSCs. Additionally, the relative level of cell proliferation between WT and β-catenin KO cells was analyzed by quantification of EDU incorporation using flow cytometry, and no difference was noted between the two cell types. After confirming β-catenin KO hPSCs can self-renew and proliferate, we assessed the ability of β-catenin KO cells to differentiate to form mesoderm in vitro. In order to further demonstrate the level of pluripotency of the KO cells, we also attempted to differentiate the KO cells towards ectoderm. Analysis of the effectiveness of both the mesoderm and the ectoderm differentiations show that while KO cells cannot effectively form mesoderm, they are still able to form ectoderm. In this study, we demonstrate that hPSCs lacking β-catenin can still proliferate, fail to form mesoderm, but can still form ectoderm. Thus, β-catenin may not be required for maintaining the proliferative characteristics of hPSCs, but is required for formation of the mesoderm germ layer, and is therefore an important component of early embryonic development.

Currently, only photo- or thermochromic filaments exist. We are the first group to synthesize a professional grade mechanochromic filament for extrusion 3D printing. The mechanochromic unit used in this poly(ε-caprolactone) (PCL) polymer-based filament is spiropyran. By shining UV light, or stretching the filament, the spiropyran activates and undergoes a reversible isomerization from the yellow-colored closed spiropyran form to its purple-colored open merocyanine counterpart. This filament could have a huge range of uses. For example, it could be used in safety and sports equipment where high impact, potentially concussive forces would result in activation of the polymer, and notify the wearer to seek medical attention. It could also be used as a coating that allows earthquake assessment of steel beams, as well as a diagnostic tool in orthotics and wearable prosthetics to determine if they need to be exchanged. We made this filament by sending our precipitated polymer to Functionalized Incorporated, who integrated it into a blend with more PCL to create over 25 kilograms of filament. We are now testing the filament on our extrusion 3D printers. Future work will focus on blending the spiropyran-PCL polymer with a softer, more elastomeric filament such as Ninjaflex to achieve lower strain activation that is also reversible, allowing for repeated use of a single printed object. Once we have our first samples, we will analyze the mechanical properties of the blends, their changes in strain of activation, and the potential for multiple cycles of activation. We intend to commercialize this filament, and make the material available to other research groups to study the material mechanics and mechanochemical properties. This is the only mechanochromic filament in existence, and its potential uses within these customizable print materials are numerous and important.

The goal of this project is to analyze and compare the carbonyl (C=O) stretch of ethylene carbonate before and after deuteration. This molecule is commonly used as a primary component of electrolytes in batteries, and exhibits a strong C=O stretching mode. The C=O mode is a sensitive reporter to an electric field through a process known as the Stark effect. A shift in peak position is associated with a quadratic Stark effect, while a widening of the lineshape is representative of a linear Stark effect. Many times, a combination of the linear and quadratic Stark effect manifest in analysis of the lineshape. Unfortunately, the absorption peak of C=O stretching is split into a doublet due to the presence of a fermi resonant mode, causing a significant misrepresentation of the C=O lineshape. This would result in a poor estimate of an electric field associated with a linear and/or quadratic Stark effect. Substituting the hydrogen atoms on ethylene carbonate for deuterium results in a reduction of the previously mentioned fermi resonant effect, enabling a better interpretation of C=O lineshape for the analysis of Stark effects. Recently, we have observed a shift in the C=O peak position of carbonate-based electrolytes at the interface silicon nanoparticle-based electrodes. It is our hypothesis that this change in vibrational peak position is due to the previously mentioned Stark effect, a measurable electric field at the nanoparticle interface. This is significant because one could correlate the magnitude of this hypothetical interfacial electric field with the performance of a battery containing Si nanoparticles. To better estimate the linear and quadratic Stark effect contributing to the magnitude of this interfacial electric field in future studies, we have characterized the lineshape of deuterated vs. non-deuterated ethylene carbonate, as well as attempted to synthesize the material in both forms using inorganic catalysts.

Fundamental understanding of how effector T-cells are transformed into quiescent memory T-cells is vital for developing immunotherapeutic treatments for autoimmune diseases and cancer, and for the development of vaccines against diseases such as AIDS, tuberculosis and malaria. Previous research on conversion of memory precursor effector cells (MPECs) into memory T cells has found evidence of Treg cell involvement in the process. Treg cells are regulatory helper T cells which selectively inhibit effector T cells to prevent and suppress overly strong immune responses and autoimmune responses. Research has also shown that MPECs produce their own IL-2, a cytokine involved in immune tolerance and activation. Based on these data, we hypothesize that direct interaction between MPECs and Treg cells, possibly via MPEC-produced IL-2, is required for conversion of MPECs into memory T-cells. We also addressed colocalization of Treg cells with short-lived effector cells (SLECs). SLECs are IL-2 non-producing effector cell counterparts of MPECs and do not form memory. Potential MPEC or SLEC interaction with Treg cells was visualized through immunofluorescence microscopy of the secondary lymphoid organs (spleen and lymph nodes) of experimental mice injected with lymphocytic choriomeningitis virus (LCMV). To bypass the low precursor physiological frequencies of MPECs we employed the P14 LCMV GP33-specific TCR transgenic mouse system, which is a well-regarded model system for studying memory differentiation. To test the role of IL-2 in cell-cell interactions, we used IL-2 agonist and antagonist antibodies. A detailed understanding of how Treg cells are involved in the process of MPEC to memory T cell conversion is key to developing novel immunotherapeutic treatments.