HIV remains a serious health challenge particularly in developing countries. This can be attributed in part to the fact that many common diagnostic procedures in high-income countries are too expensive and technically complex to be practical in low-income countries. While there are several strategies for diagnosing HIV, one promising method is using the detection of the p24 antigen. The p24 antigen is a viral protein that can be detected within two weeks after HIV infection. This is useful because it allows for the detection of HIV infection sooner than the commonly used HIV antibody detection tests and rivals the more technologically complex nucleic acid detection methods. Our goal is to develop this test using a paper-based lateral flow membrane and a technique called immune complex dissociation (ICD) to reduce cost and increase sensitivity. The laboratory p24 antigen test was adapted into a paper format using the same reagents as the laboratory test. By comparing the results of the respective methods, we found that the paper test performed well compared to the laboratory test. This performance indicates that there is value in using the paper format and in exploring methods to increase the sensitivity of the assay. Test sensitivity can be compromised by the presence of antibodies that form complexes with the p24 antigen. These complexes reduce the amount of antigen that is free for detection. ICD restores this sensitivity by dissociating the antigen from these complexes. We tested the ICD process, to show that it is compatible with the paper format and improves sensitivity, by running parallel tests with and without ICD to demonstrate the efficacy of that procedure. The completion of this project will bring the paper-based assay one step closer to making a previously expensive, technology intensive, and operationally complicated test, accessible to those in low-resource settings.

Growing evidence indicates rapid and sensitive diagnostics for infectious disease can alleviate the healthcare burden. Laboratory diagnostics are quality-oriented and highly sensitive, but they require professional training, high cost, and extensive time. Most importantly, they are not accessible in low-resource settings. Recently, research has been focused on transforming laboratory diagnostic into a “lab-on-a-chip” format, which could allow on-site screening for infectious disease with micro-scaled sample volume. These microfluidic devices are typically portable and disposable but still require a pump-based instrument to control fluid movement and timing. In addition, challenges also remain on how to dry and rehydrate the reagents on the device. Here we propose a solution by replacing the pumps with wicking as the driving force, and adding programmed wicking to automate assay steps. We print nanopatterns on the channel celling and/or floor to locally modulate the capillary flow rate, and we will also utilize dry reagents to minimize the user steps. The microfluidic channel and nanopatterns are fabricated by low-cost PDMS molding. The outcome will be a quantitative understanding of the tunability of the flow rate modulated by nanopatterns, and this knowledge can be applied to achieve sequential delivery.

Proteins that share similar 3D structures, or folds, can be grouped together into fold families, and evidence suggests that there may be common folding mechanisms that exist within these fold families. Finding these common folding mechanisms will shed light on the way in which folding is determined among proteins, which is a major question in biochemistry. Here I investigate the dynamics and unfolding pathways of 18 members selected from the sixth most populated fold family, the SH3-like barrel domain. The SH3-like barrel fold is an all-beta fold, constructed of beta strands only. Using molecular dynamic simulations at high temperatures, the unfolding pathways, including the characterization of transition states, can be studied for each of these proteins. A broad analysis of native and non-native contacts and secondary structure content will allow common folding patterns to be determined across the fold family. It is anticipated that a hierarchical loss of contacts between the beta-strand of the fold, with those between the turns being maintained longer will be observed. Here I report an initial analysis of the unfolding of these 18 members of the SH3-like barrel fold family and present the observed commonalities, as well as variances across the pathways.

It has been hypothesized that there is a correlation between the flexibility of amino acids in a protein and their conservation. Inflexible residues tend to be structurally important and thus less tolerant of change. By this theory, mutations at these positions are expected to typically be deleterious. The Dynameomics project is an ongoing effort by the Daggett Research Group to characterize protein folding space through molecular dynamics simulations of representatives of all known protein structures or folds. The representative domains are organized such that the lowest rank fold (rank 1) represents the highest populated fold – with each subsequent rank representing fewer structures. We have used a novel software module to calculate residue flexibility directly from the relevant molecular dynamics simulation files. The resulting data were used to test the hypothesized correlation between conservation and flexibility. Analysis of the entire dataset showed that hydrophobic residues and Cysteine were relatively conserved on average. Hydrophobic residues are generally found in the less flexible core of the protein while Cysteine residues form highly inflexible disulfide bonds. Thus, residues expected to be inflexible were conserved on average – providing support for the tested hypothesis, with the caveat that the standard deviations were large. To avoid these large variations, analysis of individual proteins was needed. Calculated correlation coefficients allowed for the quantification of how well each simulated structure agreed with the hypothesis. Work is now being done to characterize the difference between the various ranks of Dynameomics in terms of this conservation-flexibility relationship. We anticipate that a better understanding of evolution at the amino acid level of proteins will help us to better predict the impact of mutation. Combining this information with rotamer information can allow for the creation of tools useful in the field of protein design and engineering.

Calbindin D9k is a calcium binding protein that contains a helix-loop-helix motif, called EF-Hand. Members of the EF-Hand family are involved in calcium uptake and regulation of functions within the neuroendocrine and muscular systems. As calcium is involved in many cellular processes, such as muscle contraction and nucleotide metabolism, the function of these calcium-binding proteins is of great biological significance. Nuclear magnetic resonance spectroscopy (NMR) can provide a measure of protein dynamics, such as order parameters, that describe the residue-level mobility of a protein, including that of the backbone amide groups. These order parameters are often translated into conformational entropies, enabling an assessment of the entropic changes associated with unfolding or binding events. NMR studies that probe the stability and cooperative binding of calbindin D9k provide evidence that a temperature dependence of order parameters can identify regions of the protein that exhibit linear or nonlinear stability as a function of temperature. I will use molecular dynamics simulations to characterize the dynamics of Calbindin D9k and provide atomic level detail of the protein’s temperature dependent behavior over a temperature range of 3 to 63 Celsius. I will be comparing order parameters calculated from the simulations with experimentally derived order parameters for both the calcium loaded and unloaded states to determine the effects the calcium ions have on protein stability and conformation. Residues instrumental in calcium binding and structural stability are expected to exhibit smaller reductions in order parameters while non-structural regions should exhibit larger and more erratic reductions through increasing temperature. This study will further our knowledge of the dynamics of calcium binding proteins and offer an estimation of the free energy contribution that conformational entropy provides ligand binding. It may also be possible to extend these results to other calcium binding proteins known to act in disease and calcium deficiencies.

Vascular calcification greatly increases risk for heart disease, the leading cause of death in patients with diabetes and chronic kidney disease. Vascular calcification is an active process attributed to vascular smooth muscle cell (VSMC) mineralization. The sodium-dependent phosphate cotransporter, PiT-1, mediates VSMC calcification. The mechanisms by which PiT-1 causes calcification may include cellular matrix degradation, apoptosis, calcium phosphate vesicle release, and changes in gene expression. Of these mechanisms, my research focuses on the poorly understood signaling pathway by which PiT-1 induces VSMC calcification through up-regulating osteogenic (bone-forming) and chondrogenic (cartilage-forming) genes and down-regulating smooth muscle genes. These changes in gene expression make VSMCs more readily calcified in elevated phosphate. Recent literature suggests that VSMCs respond to elevated phosphate through PiT-1 with a novel cell signaling mechanism independent of phosphate uptake. My project concentrates on understanding the function of the PiT-1 protein's intracellular domain, which currently has no known function. I hypothesize that this section of PiT-1 is responsible for signaling in response to elevated phosphate, triggering a phosphorylation cascade that activates osteogenic transcription factors, such as Runx2, which initiate this osteogenic conversion. I am working toward integrating the gene for the intracellular domain of PiT-1 into the vascular smooth muscle cell genome to overexpress the truncated protein in cells. From my hypothesis that PiT-1 has a role in cell signaling, I predict that the overexpression of the intracellular domain of PiT-1 will reduce VSMC calcification by competing with the wild type PiT-1 protein for binding to proteins in the activation pathway, and inhibiting the signaling process. If my data support this hypothesis, I may engineer the intracellular domain of PiT-1 for therapeutic purposes.

Esophageal adenocarcinoma (EAC) is a type of cancer with the most rapidly increasing incidence rate in developed nations. EAC occurs as a result of the metaplasia-dysplasia-adenocarcinoma sequence initiated by Barrett’s esophagus. In this sequence of events, patients develop Barrett’s esophagus, wherein the acidic damage of gastroesophageal reflux disease (GERD) causes the esophageal epithelium to take on an intestinal epithelium phenotype. This metaplasia can then progress to Barrett’s esophagus with either low-grade or high-grade dysplasia. Patients that develop high-grade dysplasia (HGD) are at great risk for esophageal adenocarcinoma, and hence there is a need for higher specificity in the labeling of pre-cancerous tissues for endoscopic detection. Current imaging of dysplastic lesions is done through traditional endoscopy with the use of contrast agents for differential staining. However, current contrast agents have a low signal-to-noise ratio (SNR), and dysplastic lesions are often missed. Therefore, for early detection of esophageal adenocarcinoma, we are creating a contrast agent that increases SNR via fluorophore-labeled peptides that bind specifically to tissues with high-grade dysplasia. Peptides are ideal as targeting ligands as they can bind specifically to surface proteins on dysplastic cells and increase the likelihood of clinicians obtaining biopsies of pre-cancerous tissue. In the past year, we have isolated potential peptide sequences that bind immortalized cells lines generated from dysplastic tissues. A bacteriophage library was subjected to subtractive whole cell panning for isolation of phage expressing HGD-binding peptide sequences. The phages were added in vitro to cultured cell lines of normal esophageal tissue and non-dysplastic Barrett’s esophageal tissue to eliminate those that were not selective for dysplasia. After subtractive whole cell panning, left over phage were sequenced to determine the peptide sequences on them. Future work includes binding studies using flow cytometry to demonstrate that fluorophore-labeled peptides bind preferentially to dysplastic cell lines independent of the bacteriophage.

Low resource clinicians face several challenges in diagnosing disease at the point of care. One of the way diseases can be diagnosed is by DNA amplification, but first amplifiable DNA must be released from the cell. Staphylococcus Epidermidis is a bacterium that is very difficult to lyse. I examined three methods of S. Epidermidis cell lysis that are feasible to use in low resource settings by measuring the amount of amplifiable DNA recovered from the lysed cells. The three methods used were: a Bead Beating machine, the Omnilyse, and a boiling water bath. A bead beater is a mechanical method of lysing cells requiring electricity. An Omnilyse is a battery operated portable machine with glass beads to lyse the cells. The boiling water bath used was a beaker on a hot plate. All of these methods have comparable cost per samples value, and can be operated with relatively simple training. I used three to five intervals of time during which the S. Epidermidis cells underwent the different lysis techniques: 30, 60, 90, 120, and 360 seconds. I found the Omnilyse outperformed the bead beater and hot bath. It was 31.23 percent more efficient at extracting amplifiable DNA, making it the best of the options tested for point of care diagnosis. Future expansion of experiments could include testing the lysis methods with different bacteria to determine if the efficiency of recovery of amplifiable DNA is comparable to S. Epidermidis' efficiency.

Synthetic biology aims to engineer biological systems that perform complex tasks such as producing therapeutics and biofuels. Synthetic gene networks are developed that interact with an organism’s native genome to modify its behavior, and the design of synthetic gene networks is guided by computational models. However, the ability of computational models to accurately predict gene network behavior is limited by a poor understanding of how individual network components operate; and this often results in repeated rounds of experimental refinement. This work aims to improve the predictive power of computational models by characterizing the function of promoter sequences. We developed methods to easily detect intracellular RNA levels with high accuracy using an RNA-based fluorescence reporter. The method is used to quantitatively measure RNA synthesis rates in E. coli. This technique may be used to enable high-throughput characterization of DNA promoter sequences for use in synthetic biology applications.

Proteins are functionally dependent on their 3-D structure. Many proteins share similar structural folds arising from either convergent evolution or a common ancestor. Understanding a protein’s folding pathway is a fundamental problem of biochemistry. The Daggett Lab created the Consensus Domain Dictionary to categorize common 3-D protein folds, termed fold families, based on various arrangements of secondary protein structure. Evidence suggests there may be common mechanisms in the folding and unfolding of members of a fold family. Here, we look for such characteristic mechanisms within 14 members of the Ubiquitin-like family, the 8th most commonly found protein fold. These proteins have a common core structure with sequence identities ranging from approximately 25% to 85%. Using Molecular Dynamics simulations to model the proteins denaturing at 498K, we compare transition states in the unfolding pathway, various atom-atom contact data, and the alpha-helical content amongst protein fold family members to analytically determine the existence of family-specific folding mechanisms. We anticipate that the early pathway will revolve around helix-turn-helix rotations or splays, sequential alpha-helical melting, and spatial separation of the four main beta sheets.

As our understanding of the genetic basis for the pathophysiology of many diseases has broadened, gene therapy, which is the delivery of genetic material into cells in order to supplement or alter defective genes, is being explored as a potential treatment option. Despite its promise, no gene therapies have been approved for clinical use due to the lack of safe and effective vectors. There are many obstacles that must be overcome in order to successfully transfect a cell with exogenous DNA. First, the vector-DNA complex must navigate through the extracellular environment and be uptaken into the target cell. Then, the genetic material must achieve endosomal escape, be released from the complex, and then translocate into the nucleus where it can be transcribed and translated into functional or therapeutic proteins. In the Pun Lab, we previously synthesized a block-statistical copolymer comprised of different hydrophilic and hydrophobic segments aimed at providing a plethora of functionalities to the formed polyplexes. Due to this polymer’s unique architecture, it exhibits transfection efficiencies higher than branched polyethyleneimine, the gold standard for nonviral gene delivery, but still fails to reach the same level of transfection efficiency as seen with viral vectors. In this project, two additional modifications will be made to the copolymer and a library of well-defined polyplexes with varying formulations and structures will be synthesized and evaluated in vitro and in vivo. These modifications, guanidinylation and conjugation of a targeting peptide, aim to increase uptake into cells through electrostatic interactions and increase localization to the target cell type. From these studies, the ideal method to incorporate both modifications into one system to improve transfection efficiency and the structure-function effects on transfection efficiency will be evaluated.

HIV/AIDS is a pandemic that affects over 33 million people globally, and it is estimated that the HIV virus has claimed over 22 million lives. It has been shown that HIV therapeutics are most efficacious when delivered in combinations that target multiple points of the HIV life cycle. Small molecule therapeutics must remain within a narrow range of concentrations in the blood and tissues known as the therapeutic window to be effective. With traditional drugs the patient will spend a large amount of time out of the therapeutic window as concentrations of drug rise, peak, and then fall as the drug is metabolized, leading to inefficient treatment. Therefore to maximize efficacy it is necessary to develop technologies that are able to stabilize the serum concentration of multiple small molecule drugs simultaneously. Controlled combination delivery of antiretroviral agents promises to vastly improve the quality of care for HIV positive patients. However modern biomaterial platforms have fallen short of this goal. I propose the use of an adapted lipid-based, nanoscale biomaterial platform (ICMV) for the controlled and simultaneous delivery of multiple small molecule antiretrovirals as well as an antiviral protein. I further propose future work for the evaluation and optimization of the ICMV platform for sustained drug release, in hopes of developing a long acting HIV treatment. The ICMV system will significantly improve antiviral drug regimes, as well as the scope of deliverable medications. Controlled release platforms also play an essential role in maintaining systemic drug concentrations within the therapeutic window. These advances in therapeutic technology will lead to significant improvements in the treatment and prevention of HIV, which will directly impact millions of human lives.

Low intensity non-thermal non-ionizing electromagnetic fields (EMF) in the ranges of extremely-low to low frequency (< 200 kHz) have been shown to kill cancer cells in various studies. The external electromagnetic field cannot be concentrated to focus enough energy in the tumor site to be effective. We are designing an EMF-emitting microchip that can easily be surgically implanted into a tumor. The microchip emits electromagnetic fields when it is activated by an ‘activator’ outside of the body. Since the chip is in close proximity to cancer cells within the tumor, the cells would be exposed to a very high local field to achieve cytotoxic effect. A prototype of microchip (cylindrical, ~12 mm in length, ~2 mm in diameter) will be tested with Molt-4 cells (human leukemia). Molt-4 cells will be incubated for 24 hours at 37^oC in a humid atmosphere of 5% CO₂/and 95% air. After initial cell count, one ml of cells each will be put in four microfuge tubes. A microchip will be placed in two of the tubes. Two tubes (one containing a microchip and other not) will be exposed using an activator. The other two (with and without microchip) will be sham exposed, i.e., they will be subjected to the same experiment procedures as the ‘exposed’ samples except that the activator will not be turned on. During exposure, cells will be maintained in a water bath at 37^oC. Viable cell count will be performed at 1, 24 and 48 hours after exposure. Our preliminary results using the above-mentioned protocol are encouraging. We anticipate to show that a microchip can be used to kill cancer cells and produce data to proceed to in vivo (experiment within a living organism) study.

Sexually transmitted infections are a major health concern, both in the developing and developed world. Despite the fact that vaccines have been developed and are under development for many STIs, there is currently no efficient method of topical delivery for these vaccines. The barrier posed by the vaginal epithelium and the difficultly of achieving intracellular delivery hinder their ability to elicit immunity. The optimal topical drug delivery system would ideally transport vaccines through the epithelium and into immune system cells. There is a naturally-occurring pathway in the vaginal epithelium in which the FcRn receptor, which is present on epithelial cell surfaces, binds and bidirectionally translocates IgG antibodies across the epithelium. We hypothesize that a nanoparticle decorated with IgG could be transported by the same mechanism. Loading the nanoparticle with the desired vaccine would then enable delivery to cells populating the vaginal submucosal tissue, providing the topical delivery that is desired. Several methods of conjugating fluorescent polystyrene nanoparticles to IgG have been investigated. I have selected two methods to pursue. The first of these is a passive adsorption method, in which the antibody was allowed to stick to the nanoparticle surface through hydrophobic interactions. The second of these employed commonly used (thiol-maleimide) conjugation chemistry. Now that these methods have been identified as potentially viable, I will assay them to determine if the IgG protein can still bind FcRn while conjugated to the nanoparticle. If this yields positive results, I will move on to animal models to determine the biodistribution of the particles. This will allow me to see if the IgG modification has increased the particles’ ability to reach immune system tissues. If this project’s aim is successful, it may become as useful system for the delivery of vaccines against a variety of diseases.

Current medical diagnostic gold standard, the Enzyme-Linked Immune-Sorbent Assay (ELISA), requires additional labels and amplification to determine the existence of interested molecules. Such process is extremely time consuming and expensive, and thus a fast and low-cost alternative is in demand. Our project aims to develop a rapid and affordable diagnostic biosensor platform that can be administered in the Point-Of-Care (POC) setting. In our silicon photonic sensor chip, we exploit a unique aspect of light when it is confined into a waveguide much smaller than its own wavelength. Since different molecules have different refraction index, the portion of light travels outside the waveguide, called evanescent field, is capable of sensing molecules binding on the sensor surface. While the design and fabrication of these biosensing devices can be done quickly and affordably, characterizing and experimenting the devices remains challenging because of the complexity of testing submicron-scale components. Chips require careful alignment of the optical I/O fibers with micron-tolerances. Microfluidics must be integrated to deliver reagents to specific regions of the chip. Real-time processing of optical spectra is needed to facilitate user operation. Hence we have customized a low-cost test bench to perform experiments and characterizations on the silicon photonic biosensors. I have designed the software, which employs industrial standard Model-View-Controller (MVC) architecture, for instruments integration, experiments orchestration as well as data acquisition and analysis. This software has greatly enhanced the efficiency of the biosensor platform development and future improvement is foreseeable because of the ongoing progress of full automation of experiment preparation. I have characterized multiple sensor devices through optical data acquisition, analysis, model fitting and key parameters’ estimation this software provides. Characterization results show great promise on the sensing capability of the platform and we will soon proceed to biomolecules (e.g. protein), and even cell (e.g. cancer cell) sensing.

The majority of the world’s population resides in the developing world where infectious diseases are the leading cause of mortality and morbidity. Due to the lack of affordable and accurate diagnostic tests in low-resource settings, proper treatment of patients with infectious diseases such as malaria, HIV, and tuberculosis is greatly limited. As a result, there is an urgent need for point of care (POC) diagnostic tools targeted for low-resource settings that are inexpensive, rapid, portable, accurate, and require little to no training to operate. Paper-based diagnostic tests are portable and low-cost, and have shown promise for POC diagnostics appropriate for the developing world. The Yager Laboratory is currently developing a paper-based POC diagnostic platform for methicillin-resistant Staphylococcus aureus (MRSA) that utilizes an isothermal strand displacement amplification (iSDA) technique. The amplification method operates at a constant temperature and uses a mutant restriction endonuclease that cleaves only one strand of DNA, which is then displaced by a strand displacing DNA polymerase. While the incorporation of iSDA in the POC platform has been promising, it has been challenging to optimize performance in the paper matrix without a full understanding of its operation. The purpose of this investigation is to develop a kinetic model for iSDA. The model currently considers the major steps associated with the amplification technique and incorporates kinetic rate constants that have been selected from other published studies. Next, simulation results for various conditions will be compared to experimental results to validate the developed model. A successful kinetic model will provide a better understanding of the iSDA method and should enable optimizing the amplification technique. Ultimately, the information produced from this project could be used to improve paper-based POC devices that utilize iSDA technology.

Porous membrane-based lateral flow assays have become commonplace in the diagnosis of a wide range of conditions. Attributes of these assays vary greatly depending upon the application. Many commercially available lateral flow tests (LFTs) employ a 'sandwich' format, where the target molecule is effectively sandwiched between a surface anchored antibody and a labeled antibody. Because small molecules lack multiple epitopes (antibody binding sites), they cannot support the binding of two antibodies simultaneously, and thus cannot be detected using a sandwich format. Competition format assays offer a solution to this problem, but fall short in two key areas: they generally rely on a counterintuitive, inverted user read-out, where a darker signal translates to lower concentration, and are not compatible with chemical signal amplification. My work seeks to improve upon current competition methods by investigating assay formats that generate a colorimetric signal proportional to the concentration of small molecule analyte in a sample. Amplification chemistries have been utilized to improve the limit of detection for small molecules in this new format. Potential future applications of a robust small molecule assay include: rapid detection of cortisol to diagnose post-traumatic stress disorder or Cushing's disease, and field-use testing for active drug components like Δ9 THC in marijuana. Such an assay would have tremendous utility at the point-of-care, and would provide an efficient and effective alternative to expensive lab-based testing.

Myocardial infarction remains the leading cause of death and disability in developed nations. While current therapeutics are able to slow the progression of heart disease, there are no viable treatment options to repair damaged myocardium. As such, cardiac stem cell therapy has received much attention for its promise to repair damaged heart tissue. However, there currently is no ideal stem cell source for cardiac stem cell therapy. Cardiovascular progenitors (CVPs) are stem cells that can differentiate into cardiomyocytes, endothelial cells, and vascular smooth muscle cells, but CVP cell fate determination is not well understood. It is well known that stem cells respond to the physical, geometric, and chemical cues provided by the microenvironment. Herein, we report the development of a platform that allows specific design and control over the in vitro stem cell microenvironment. Polyurethane acrylate (PUA) is a synthetic polymer with tunable rigidity and can be used to form nanopatterned substrates through the use of capillary-force lithography (CFL). Bifunctional PUA-binding peptides that contain the RGD peptide sequence are used for presentation of cell adhesion sites. We report precise control over the physical rigidity, nanotopographical dimensions, and RGD peptide presentation. We anticipate that our platform will be able determine the optimal conditions required for directing CVP cell fate. Thus, by determining the conditions responsible for CVP cell fate determination, CVPs could be a viable cell source for cardiac stem cell therapies.

The increasing rates of drug-resistant bacterial infections is a concern to the healthcare system globally. Anti-adhesive therapy has emerged as a possible alternative therapy, focusing on preventing biofilm formation that is associated with increased drug resistance and disease. The study of anti-adhesive therapy is underway, but the basic transport mechanisms are still unclear. Adhesion depends on the bacterial concentration near the surface, as these bacteria are closest to any adhesive present. Flow chambers are commonly used to study bacterial adhesion and mechanics, but the relative concentration distribution within the chamber is often overlooked. Bacterial concentration without adhesion was found to depend on time – the concentration rises until reaching a peak level. After reaching an equilibrium level, the concentration deviates from the inflow concentration differently at separate locations along the chamber. Measured at three separate points, the peak concentration is highest towards the end of the flow chamber. Bacterial concentration remains space and time dependent without any adhesive protein in the chamber. With adhesion however, the concentration differs across the entire adhesive surface, and also dramatically over a small area near the inflow where adhesion first begins. Bacterial concentration of spotted and even surfaces will be affected by these settling and transport mechanisms - using different adhesives along the chamber increases this variability. Researching anti-adhesive therapy and dynamic adhesion requires the analysis of adhesion strength to different glycans, compounds on the outside of proteins, under different flow rates. The adhesion to each glycan can then be measured relatively and compared. Our data suggests that this data could be severely impacted by the placement of these glycans, in addition to the strength of their adhesion, a factor that has not been previously considered.

Blood flow plays a very important role in keeping cells and tissues alive. Many studies have been focusing on the basics of coagulation after injury. However, little is known about how blood perfusion responds to injury. Precise and real-time monitoring of the skin microvasculature before, during and after injury is required in order to understand this research question. Most of the previously reported imaging systems have either low spatial resolution or slow image acquisition, therefore fail to capture the transient microvasculature changes. Laser speckle contrast imaging (LSCI) is a fast technique that utilizes the backscattering of light to distinguish moving objects, such as red blood cells (RBC), from stationary objects, such as the surrounding tissue. We hypothesize that combining LSCI with localized injury to the skin can provide detailed information on the acute perfusion response after a blood vessel is occluded. In this study, a mouse ear model was used and laser speckle images were acquired continuously covering before, during and after injury. By separating the high frequency signal and the low frequency signal from the acquired images, contrast between dynamic and static speckles can be generated. We found that the downstream blood vessels were occluded immediately after injury. Flow velocity changes in nearby collaterals were observed. In summary, LSCI can be used to image skin microvasculature with high resolution and fast speed. It can also provide direct visualization on how blood perfusion responds after injury, which may provide novel insights to the field of wound healing.

Vascular calcification, the accumulation of calcium-phosphate salts in blood vessels, is a common complication associated with atherosclerosis and type II diabetes mellitus (T2DM). We have shown that reprogramming of vascular smooth muscle cells (SMCs) into osteochondrogenic cells is important in T2DM vascular calcification, and endothelin-1 (ET1) and its type A receptor (ETA) were found in the reprogrammed SMCs. Along with the in vitro study that showed a substantial inhibition of the high glucose-induced SMC calcification upon ETA antagonism, we hypothesize that signaling through ET1 and ETA may play an important role in ectopic calcification of atherosclerotic blood vessels of T2DM. To test this hypothesis, we fed low-density-lipoprotein receptor knockout (LDLr-/-) mice with a customized diet for 18-24 weeks to induce T2DM, atherosclerosis, and vascular calcification. Upon 5 weeks diet fed, LDLr-/- mice developed hyperglycemia with no detectable atherosclerosis and vascular cartilaginous metaplasia and calcification. Half of these mice were then treated with an ETA antagonist, atrasentan, to inhibit ET1-ETA signaling. Mice were terminated at 18 weeks under the diet for histological analysis and 24 weeks under the diet for quantification of vascular calcification. We found that atrasentan significantly reduced fasted blood glucose levels of the T2DM mice but not affecting their body weight. Surprisingly, quantitative analysis showed no difference of calcium amounts in aortic arches between the treated and untreated groups. We are currently in the process of analyzing the lesion sizes of atherosclerosis, cartilaginous metaplasia and calcification of 18-week diet-fed mice via morphometric analysis to determine whether atrasentan affects vascular calcification at an earlier time point and whether vascular calcification is correlated with atherosclerotic lesion size. Finally, our finding of ETA antagonism in the control of hyperglycemia provides a new therapeutic role of atrasentan in improving T2DM, which may be beneficial in alleviating the hyperglycemia-related complication.

Hypertrophic cardiomyopathy (HCM) is a heritable disease in which the heart undergoes pathological remodeling and thickening, leading to sudden cardiac death, especially in young adults. I am investigating how modifications to myofilament proteins, which are responsible for muscle contractions, contribute to HCM functional characteristics. My goal is to determine how the P82S mutation of cardiac troponin I (cTnI) alters the dynamic properties of cardiac troponin (cTn), such as the Ca²⁺ binding affinity to cTn and interactions between cardiac troponin C and cTnI (C-I interaction), as well as the overall effect of P82S on muscle activation and relaxation. I am testing the hypothesis that the C-I interaction serves as a critical point in contractile regulation, regulating systolic performance at basal activity and particularly during β-adrenergic stimulation. During β-adrenergic stimulation, the cardiac-specific N-terminus of cTnI is phosphorylated by protein kinase A (PKA), and the shifting protein conformation results in a cascade of conformational changes in regulatory proteins that promote myosin unbinding from actin, accelerating relaxation rates in cardiac muscle. This work combines state-of-the-art protein biochemistry, myofibril & cellular mechanics/kinetics and multi-scale computational modeling to study the behavior of P82S-cTnI with wild type (WT) cTnI. For each, recombinant troponin complexes containing WT-cTnI, P82S-cTnI, non-phosphorylatable cTnI (S23/24A), and phosphomimetic cTnI (S23/24D) are studied and compared. Initial results from the WT-cTnI variants show that with respect to the WT-cTnI, the S23/24A and S23/24D mutated troponin have decreased Ca²⁺ sensitivity to cTn, as well as the C-I affinity. Accompanying experiments are currently being done on the P82S variants. A better understanding of how protein structure and function is altered by mutations will bring the HAMM Lab closer to developing approaches to mitigate the impact of HCM-related mutations.

In 2012 there were an estimated 35.3 million people living with HIV globally and 2.3 million new infections. In Sub-Saharan Africa, the region of the world with the highest incidence of HIV, women are disproportionally infected with HIV. We hypothesize that the use of an electrospun polymer nanofiber vaginal drug delivery system encapsulating cyanovirin-N and etravirine can provide a method to combine two anti-HIV agents with different physiochemical properties and mechanisms of action into a signal microbicide product to improve prevention efficacy through improved potency and limiting the impact of drug resistance. Poly(vinyl) alcohol nanofibers encapsulating cyanovirin-N and poly(vinyl) pyrrolidone nanofibers encapsulating etravirine were electrospun. The cytoxicity and bioactivity of the nanofibers was determined in vitro in TZM-bl cells using the CellTitar blue assay and HIV-1 BaL assay, respectively. We found that our poly(vinyl) alcohol nanofibers encapsulating cyanovirin-N, poly(vinyl) pyrrolidone nanofibers encapsulating etravirine, and the composite nanofiber formulations showed limited cell cytotoxicity in vitro. Comparable inhibition rates of HIV infections in vitro to the free drug formulations were found, indicating that electrospinning does not impact the antiviral activity of cyanovirin-N and etravirine. Additionally, we found that the composite nanofiber formulations displayed enhanced potency compared to the single agent nanofibers, indicating a complimentary effect of combining cyanovirin-N and etravirine into a single microbicide. These results demonstrate the potential for the use of polymer nanofibers encapsulating cyanovirin-N and etravirine as a vaginal microbicide to prevent HIV infections.

Nucleic acid amplification tests (NAATs) performed in the laboratory are the gold standard for pathogen detection due to single copy sensitivity, and could be a useful tool for widespread diagnostics in a global health setting. Unfortunately these assays tend to be expensive and complex, but new developments in isothermal NAATs will allow for cheaper, simpler diagnostics to be possible. Another problem is that current NAATs require a preparation step of cell lysis to release DNA, which traditionally requires bench top equipment or harsh chemicals that can interfere with the assay. My research addresses this barrier in the diagnostic ability of NAATs through development of a low-cost, cell phone based point-of-care device capable of efficiently lysing cells in through mechanical agitation. By placing a sample tube with beads and a magnet into a coil of wire and using a portable audio device to play an audio signal that feeds power through said coil, the alternating magnetic field produced in the coil spins the magnet and agitates the beads. The spinning of the magnet also produces an alternating magnetic field of its own that can be used as real-time feedback. Thus far, experiment results have had moderate variability, potentially because they are carried out with a live model organism, Staphylococcus Epidermidis: a common and moderately difficult pathogen to lyse. Conventional NAATs are used to measure success of this new methodology, and results so far have shown up to 60% efficiency from the experimental device compared to a bench top standard. This device may also be capable of DNA shearing, where larger DNA strands are broken into smaller pieces, but more research is needed to confirm and quantify the process. Additional research will also look into developing a theoretical model for lysis, isolating potential sources of variability, and reducing negative control background noise.

Diagnosing infectious disease in the developing world continues to be a challenge due to the lack of infrastructure and resources needed to conduct state-of-the-art methods of nucleic acid detection. Surface enhanced Raman spectroscopy (SERS) is a potential alternative to current point-of-care (POC) nucleic acid tests because it could offer similar sensitivity. However, current SERS substrates that possess the reproducibility and stability needed for translation to POC applications are too expensive. Electrospinning gold nanorods (AuNR) into polymeric nanofibers was employed as a method to reduce the cost, improve reproducibility and lengthen shelf life of SERS substrates for POC applications. Electrospinning also offers a facile method of aligning anisotropic metallic nanoparticles, which can improve SERS sensitivity over non-aligned particles. AuNR size and dimension was varied alongside fiber diameter to optimize SERS enhancement. For the detection of target nucleic acid (or oligonucleotide), a sandwich-like design was used: Oligonucleotides complementary to the target strand were conjugated to SERS active fibers in a 96 well plate format, allowing for multiplex detection with different probes. After the sample was administered, probe oligonucleotides conjugated to a Raman reporter molecule were washed over the SERS substrate to complex with the bound target strand before Raman testing. Electrospinning produced homogenous fiber meshes with uniformly deposited AuNRs to create reproducible enhancement across the substrate. In addition, the SERS fibers showed prolonged shelf-life suggesting that the polymeric fibers offer stability for AuNRs. Successful conjugation of probe oligonucleotides to the polymer mesh is shown. Initial experiments showed presence of signature Raman spectra associated with the probe molecule, indicating specific binding of the target strand. However, additional experiments are necessary to improve sensitivity. Creation of this technology provides the initial framework that electrospun fibers can offer a mode of translation of SERS to sensitive detection of biologics for POC diagnostic applications.

Distal Arthrogryposis (DA) is an umbrella term for non progressive congenital skeletal muscle defect characterized by abnormal contractures of the limbs and the face. It occurs in 1 in every 3,000 live births and is inherited in an autosomal dominant manner. Currently, there are no treatments for the disease, and the patients usually go through regular physical therapy throughout their lifetime. Severe cases require surgical intervention and amputation. One of the more common syndromes is DA 2B, or Sheldon Hall syndrome, which is caused by mutations in two genes, TNNI2 and TNNT3. I am particularly interested in studying the R63H mutation that impacts the fast skeletal troponin T (TNNT 3) subunit. The troponin subunits are critical in regulating the interaction between actin and myosin myofilaments. Mutations in the TnT can alter the way the troponin subunit interacts with the tropomyosin and have large impacts on overall muscle mechanics. I hypothesize that this mutation increases the contractility and tension in mutated proteins. To verify the hypothesis, I will compare the wildtype to the mutated version of TNNT 3 by putting a fluorescent tag on the proteins and observe the differences in calcium sensitivity, ATPase, and maximum filamented actin velocity in a sliding filament assay known as the in vitro motility assay (IVM) to piece together information about how the mutation impacts the overall muscle mechanics. My project seeks to gather information about the mechanics of the mutated muscles. Putting all of the data together will enable me to identify causes in the mechanics behind abnormal contractions. Also, it will allow me to make connections between the genotype and the phenotype of the disease to better understand it on a molecular level, and thus open up doors for therapies.

Existing polymeric delivery systems rely heavily on non-covalent interactions between chains in order to provide reduced carrier toxicity and polymer clearance. A major side effect of this strategy is premature particle destabilization making them impractical for human treatment. Attempts to resolve this issue has included the creation of single molecule scaffolds but have suffered from a complex synthesis and inefficient degradation rates. In order to overcome these issues the design of a single molecule radical addition-fragmentation chain transfer (RAFT) polymerized system was undertaken which included a polyethylene glycol (PEG) stealth cornea and multifunctional brush architecture. PEG corneas are essential through their proven ability to increase biocompatibility. Therefore the design of polymerization conditions of a novel PEG macromonomer (Mn ~ 950Da) (O950) were conducted and resulted in the creation of polymers with a polydispersity index (PDI, a metric for determining the uniformity of a molecular size distribution) sub 1.1 at 80% conversion with lauryl methacrylate, a hydrophobic stabilizing monomer, across a degree of polymerization (DP, number of monomer repeats per polymer) from 25-100. The developed synthesis conditions were then adapted for conjugation functionalities to additional RAFT agents through a degradable ester bond. Further polymerizations were carried out from the added RAFT agents with a positively charged amine monomer to increase polymer solubility, producing a 34 armed brush polymer with a PDI of approximately 1.2 at 70% conversion. Further alterations added a pH responsive functionality for endosomal escape. Light scattering was employed to determine the particle size followed by hemolysis studies to determine the pH responsiveness. The result was a synthesis technique that allows the facile creation of multifunctionalized polymer multi-brush with accurate PDI and DP, a feat not seen throughout the literature.

 Despite decades of research, our best attempts at formulating a vaccine for the human immunodeficiency virus (HIV) have failed. Recent work in mucosal immunology and virology has highlighted the importance of the mucosal immune system in the successful infection of a host after mucosal exposure to HIV. A vaccine formulation that effectively induces humoral and cellular immune responses in the vaginal mucosa could clear local HIV populations before the virus is able to gain a foothold in the body. The proposed project will develop a novel vaginal mucosal HIV vaccine using therapeutic cell encapsulation. Briefly, a transgenic cell line producing HIV virus-like particles (VLPs) will be entrapped within a nanoporous polycaprolactone (PCL) capsule optimized to release VLPs. Release of the VLPs and viability of entrapped cells will be studied over long periods of time and controlled by an electrospun PCL membrane. Finally, the ability of the cell-based vaccine to interact with dendritic cells will be studied through the bone marrow dendritic cell uptake assay and the mixed lymphocyte reaction assay. This work would represent the first use of cell encapsulation technology to create a vaccine. If successful, this project could generate essential data that could be used to develop novel mucosal HIV vaccine for human testing.

Treatment for osteoarthritis is currently limited to pain management and surgical intervention, including arthroplasty. Long-term, these treatments fail as viable solutions as many metal implants erode bone and pain management does not address continued joint deterioration. To progress the treatment of arthritis, our group has begun to design an implantable scaffold to resurface arthritic joints. We have applied a nano architectural approach to designing a scaffold, which recapitulates the native extracellular matrix of cartilage tissue. To fabricate our scaffold we have developed a method to orient nano fibers in three-dimensional space within a hydrogel, thus allowing us to mimic the orientation of native collagen. Currently, this scaffold is undergoing mechanical and biochemical analysis to evaluate it’s potential for cartilage tissue formation. I have focused on mechanically characterizing our scaffold by determining the compression modulus across several architectural variables, and in comparison to native cartilage tissue. I have demonstrated reproducibility in our fabrication method by obtaining statistically independent groups based on varying nano fiber material, spacing and hydrogel concentration (p<.05). In comparison to native cartilage, I have identified a combination of materials and geometry (polylactic-co-glycolic acid nano fibers, Norland Optical Adhesive 76 polymer, and 200 micrometer nano fiber row spacing), which are equivalent to native cartilage tissue compression values (2-4 megapascals). I am currently following up mechanical characterization with live dead staining and confocal microscopy to evaluate the biocompatibility of the scaffold. In the near future, we hope to evaluate stem cell differentiation using mesenchymal stem cells (MSCs) by evaluating glycosaminoglycan and collagen type II concentrations. Thus far, our mechanical characterization and methodology for synthetically engineering a mechanical construct has demonstrated potential for therapeutic use in the treatment of arthritis and joint degradation.

Macrophages of our immune system can be broadly classified as pro-inflammatory M1 or anti-inflammatory M2 macrophages. Imbalance in the ratio of these two phenotypes has been associated with various chronic diseases, suggesting that targeting drugs to specific populations of macrophages may achieve therapeutic benefit. Phage display is one method of identifying peptides that can be used for targeting cell populations. In whole-cell peptide phage display, a library of bacteriophage, in which individual phage display a unique peptide sequence on a coat protein, is applied to target cells. Phages that preferentially bind target cells are isolated, and their peptide sequences can then be obtained by DNA sequencing. Targeting M1 macrophages has possible applications in treating arthritis, diabetes, and atherosclerosis. Recently, we identified a novel phage-bound peptide sequence (hM1pep), using phage display, that exhibited selective binding to M1 macrophages. I synthesized the peptide form of hM1pep and evaluated its selectivity of M1 macrophage binding in vitro using flow cytometry; however hM1pep showed insignificant binding. Tumor-associated macrophages (TAMs), a type of M2 macrophage, have been shown to facilitate tumor growth and are associated with decreased survival of cancer patients. Previously, we identified a peptide that preferentially bound to TAMs in mice. We demonstrated that peptide-mediated TAM elimination improved survival in mice, suggesting that an analogous strategy in humans could be a potential cancer therapy adjuvant. In this work, I attempted to identify human M1 and M2 macrophage binding peptides by panning against human M1 and M2 macrophages, while using M2 and M1 macrophages as a subtractive panning step, respectively. Targeting M2 macrophages has potential uses in treating diseases associated with increased tissue levels of M2 macrophages, including cancer, fibrosis, and asthma.

Muscle contraction results from interactions between thick and thin-filament proteins within the sarcomere of a muscle cell. The movement of tropomyosin (Tm) over actin on the thin filament exposes myosin binding sites and is a fundamental step in the regulation of muscle contraction. Single amino acid substitutions in Tm and regulatory mechanisms such as Tm phosphorylation can greatly impact muscle function. Tm monomers link end to end over actin, so shifting of one Tm monomer to expose myosin binding sites may cause strain in neighboring monomers via end-to-end interactions, resulting in cooperative behavior. The flexibility of Tm may play a role in modulating the strain induced in neighboring monomers, thus we chose to assess the flexibility of phosphorylation-mimicking and clinically relevant Tm mutants that are known to affect contractile function. We produced and purified recombinant Tm in E. Coli and imaged it using rotary-shadowed electron microscopy. The flexibility of single molecules was determined by skeletonizing Tm to determine its persistence length, a quantity from statistical mechanics that describes the curvature of rod-like objects. Skeletonization and analysis were performed manually and with a novel automated image analysis program. Molecular dynamics simulations were also performed with the clinically relevant Tm mutant to determine structural mechanisms that could lead to the altered contractile function observed in muscle containing mutant Tm. Preliminary results show decreased flexibility near the region of phosphorylation in phosphomimetic Tm compared to wildtype Tm. This result is expected because Tm phosphorylation has been shown to increase cooperative behavior and the phosphorylation site is located in the middle of the overlapping region of each Tm monomer.

Evaluation of cellular apoptosis and DNA damage at the single-cell level allows for highly sensitive measures pertaining to cell sample health. Apoptosis is an important constituent of the cell life cycle, and is relevant when considering tissue remodeling, cancer elimination, aging, and drug genotoxicity. Current techniques in apoptosis evaluation lack sensitivity, can be inaccurate, and require relatively expensive procedures, making them unideal for low-resource research where high accuracy is needed. The inexpensive and rapid alkaline halo assay (AHA) has recently come into more widespread use for studying DNA damage. Dr. Narendra Singh previously showed that the AHA can be used to measure apoptosis as well. The drawbacks to this assay include a labor intensive process of individually scoring thousands of cells, random variations in the height and placement of cells embedded in agarose gel, and the propensity for user error when taking measurements. Working in conjunction with researchers from Dr. Liyuan Ma’s laboratory at Worcester Polytechnic Institute, this project aims to address these shortfalls. The Ma group has developed a microfabrication technique to regularly pattern cells at uniform intervals and height for the AHA. In accordance, I am developing a program to automatically scan and find the level of apoptosis, necrosis, and DNA damage in a given sample. The program, image analysis algorithms, and GUI are written using MATLAB and its image analysis toolkit. Included in my work is the characterization of apoptotic, necrotic, and live cells by frequency spectrum analysis, and the creation of new metrics for DNA damage. The software is assessed in terms of processing speed, accuracy, and ease of use. Manual image scoring methods are used to evaluate algorithm sensitivity and false positive generation rate. In summary, we aim to reduce the cost and time required for research involving DNA damage and apoptosis.

Stem cell-based therapies provide new avenues to create cardiac tissues for in vitro screening and regenerative medicine applications. In order for stem cell-derived cardiomyocytes to effectively recapitulate the function of native tissue, these approaches must account for the mechanical properties of the cardiac tissue extracellular matrix (ECM). Nano-fabricated scaffolds that mimic the structural and mechanical properties of the myocardium may be an effective method of deriving clinically relevant stem cell-derived cardiac tissues. It is necessary to quantitatively understand how these nano-fabricated scaffolds affect the structure and function of the cells. Focal adhesions are specialized protein complexes that enable cells to sense mechanical forces and transduce signals from the ECM. We hypothesize that stem cell-derived cardiomyocytes will form larger focal adhesions on nano-fabricated scaffolds compared to traditional flat substrates, resulting in a more mature cardiac phenotype. We designed a computational method of quantifying these focal adhesions, which bears information about the stability and adhesion of the cardiomyocyte on its substrate. The method simultaneously measures shape and size of the cardiomyocyte, which indicates its state of cell maturity and ability to integrate with host tissue. We used confocal microscopy to obtain images of fluorescently labeled focal adhesions and cell membrane. Using ImageJ and MATLAB, fluorescent intensity was used to reconstruct 3D cell shape and measure expression of focal adhesions. Using this method of quantitative analysis, we can quantify both adhesion and maturation of cardiomyocytes, which will better allow us to characterize nano-fabricated scaffolds. The ability to quantify focal adhesions and cellular morphology will be useful in analyzing how cell-ECM interactions affect maturation, and ultimately aid the development of more effective stem cell-based methods for cardiac tissue engineering. This method can also be used to better understand the mechanisms involved in focal adhesion formation and the interplay between mechanical signaling and cell function.

 Over 90% of cancer-related death is caused by metastasis of invasive cells from one organ to another. Discovering more about this proliferative capability will be influential in creating new cancer therapies that will not only diagnose cancer, but may soon eventually fight it. Cancer metastasis involves migration of cells along bundles of extracellular matrix (ECM) fibers. In this context, understanding topotaxis through various kinase pathways which regulate cell migration is essential. An example of a kinase pathway is the PI3K pathway, one of the most mutated and well-studied pathways. When this pathway is over-expressed, proliferation of cancer cells is increased, while apoptosis of the cells is decreased. Additionally, cancer cells exhibit a more rigid nature suggesting that there are durotaxis involved for metastasis. Understanding these two mechanotaxes and how a particular oncogene influences the cellular response to these factors are vital in our quest to understand the process of cancer invasion. In our experiment, we studied two specific oncogenes in this pathway: PIK3CA and PTEN. Since PIK3A and PTEN are up-regulators and down-regulators of the PI3K pathway respectively, they will be an excellent model to investigate how PI3K is regulated by oncogenes. We hypothesize that cells not containing the down-regulated PTEN oncogene (knockout) and cells containing up-regulated PIK3CA oncogene (knock-in) will result in directional cell migration and increased migration speed and persistence time. To test this hypothesis, we utilized nano-scaled platforms of increased stiffness to simulate the ECM collagen fibers found on cancer micro-environments. Coupled with polymers such as polyurethane acrylate, polydimethyl siloxane, and poly(lactic-co-glycolic) acid, these platforms can vary the stiffness of cancer microenvironments, ranging from kiloPascals of normal tissue to gigaPascals of cancerous tissue. Thus, these platforms give us the opportunity to design a cancer tissue model to study effects of oncogenes on cancer invasion.

Thrombotic microangiopathy is featured by endothelial activation and microvascular thrombosis, and is associated with a group of life-threatening disorders, such as thrombotic thrombocytopenia purpura (TTP), sepsis, and other small vascular diseases. With systemic inflammation, the vessels are often found to be obstructed by platelet rich thrombi, leading to tissue ischemia and organ failure. Understanding the mechanism behind endothelial activation and induced thrombi formation during inflammation has important clinical implications and can lead to therapeutic development. Here, I developed a novel in vitro microvascular platform that mimics the in vivo vascular architecture and function. This platform is generated based on a biocompatible polymer, polydimethylsiloxane (PDMS), using a soft lithographic method. A vascular network is fabricated in the PDMS mold, which is subsequently sealed with a glass coverslip to create an enclosed network. The device is lined with an extracellular matrix using type I collagen for structural support. Optimization was made to provide consistent, high-throughput testing by controlling the diameter and integrity of the vessel lumen. Human umbilical vein endothelial cells (HUVECs) were seeded into this device and cultured for 7 days. To recapitulate the inflammation during diseases such as sepsis, lipopolysaccharide was used and perfused through the vessels to stimulate the endothelium. Granules or secretory vesicles carrying von Willebrand Factor (VWF) were observed to release its contents from the activated endothelium, forming strands of VWF along the direction of flow. On-going studies include examining changes in the transcription factors during endothelial cell activation, cleavage and self-association of VWF strands under flow, and drug screenings to prevent VWF secretion and strand formation. The study is expected to not only elucidate the fundamental mechanism of microvascular diseases, but to also provide a base platform to develop preventative or therapeutic medicine for those affected by these diseases.

Most bacterial infections are initiated when binding to receptors on the tissue surface via bacterial adhesive proteins. After adhesion, they form biofilms and become more resistant to the immune system and antibiotics. A novel approach to preventing infection is the use of anti-adhesive agents that inhibits bacterial adhesion to host tissues and thus blocks the infectious process. Therefore, understanding bacterial adhesion and physiology under different conditions is essential for the development of anti-adhesive drug therapies. This project involves studying the effects of pulsatile flow on Escherichia coli adhesion and testing the hypothesis that flow pulsatility enhances bacterial adhesion because bacteria are adapted to in vivo pulsatile flows caused by intestinal peristalsis, swallowing saliva, or beating of the heart. I used a programmable syringe pump to produce a pulsatile laminar flow composed of a low shear (e.g. 0.1 Pascal) and high shear (e.g. 1 Pascal) in a parallel plate flow chamber. The frequency and amplitude of this pulsatile flow was in the physiological range. Through a microscope with a digital camera, I recorded the number of E. coli that bound to the bottom plate of the flow chamber coated with the model receptor mannan. The preliminary results demonstrated that E. coli accumulation after 5 minutes under pulsatile flow was not significantly different from that under steady low shear but accumulation was more stationary with pulsatile flow. However, the difference in accumulation became significant when comparing with the steady high shear. These data suggest that E. coli initiate adhesion at low shear and they firmly maintain binding at high shear. Future work will require the adhesion assays of wide range of bacteria under various pulsatile flow conditions. This research will help us understand the bacterial physiology and advance the development of anti-adhesive drug therapies.

Today, approximately 13% of the world's population is affected by chronic kidney disease, with kidney transplantation being the only functional restorative treatment. However, the significant shortage of donor kidneys leaves 100,000 Americans with limited treatment options. My project seeks a solution to generate proper human kidney extracellular matrices through decellularization and subsequent recellularization to recreate functional kidney peritubular filtration unit for the application toward therapeutic development. The efficacies of cellular material removal using sodium dodecyl sulfate (SDS) and Triton X-100 were evaluated on porcine kidney sections. SDS solution showed better cellular removal and matrix preservation. To optimize organ decellularization protocol, decellularizations on organ porcine kidneys were performed using a pumping system that perfused SDS into the renal arteries of the kidneys. Decellularized organs retained intact microarchitecture and the essential extracellular matrix components such as laminin, fibronectin, and collagen IV. Future work will be focused on human kidney organ decellularization using the protocol previously developed and decellularized scaffolds repopulation, testing the cellularity and regenerated kidney functions. If successful, this project will not only lead to recreate human kidney peritubular filtration unit in vitro for toxicity testing and therapeutic development, but also provide insights about how the microenvironment controls the structure and function of the cells and tissues.

 In silicon photonics, light propagation along a narrow silicon waveguide at the nanoscale produces an electromagnetic field that extends beyond the waveguide surface. Microrings can “couple” light from an incident waveguide at certain wavelengths. Silicon microring resonators can detect changes in the effective refractive index at the sensor’s surface by producing minute shifts in the peak resonance frequency. For biosensing applications, this resonance frequency shift is utilized to quantify the binding of biological analytes to the microring surface. Combined with a microfluidic platform for directing chemical and biological reagents over the microring resonators, silicon photonics offer a promising method of determining the concentrations and binding activities of analytes of interest in biological assays. However, protein fouling- the non-specific accumulation of protein molecules on a surface- in blood samples presents a significant challenge in the translation of these assays toward clinical adoption. Ongoing research has aimed to significantly reduce non-specific adsorption by coating the chip surface with polymer chains, resulting in a hydrated surface which resists protein fouling. In this study, charged polymer layers were grown on the chip surface by atom-transfer radical polymerization (ATRP), producing a coating of polymer brushes of high density and uniform length. Microring sensors were then functionalized by conjugating biological capture elements using standard carbodiimide chemistry to the terminal carboxylic acid residues of a surface-grafted polymer. The polymerization reaction conditions were optimized to minimize the thickness of the polymer layer while maintaining the non-fouling properties of the surface as a whole. Non-fouling properties were observed in the residual resonance shift following plasma and buffer rinse flows via a microfluidic platform. By minimizing the surface polymer thickness, we increase the physical limit of analyte detection on the microring surface, thereby maximizing the sensitivity of the biosensor while reducing “biological noise” due to non-specific protein adsorption.

An inborn genetic disorder, phenylketonuria (PKU) is characterized by elevated phenylalanine (Phe) concentrations that interfere with neurodevelopment, leading to irreversible symptoms such as mental retardation and motor developmental delays if left undetected. Most countries, including the United States, implement PKU testing in newborn screening programs, in order to provide treatment to prevent the onset of irreversible damage. Current methods of detection include tandem mass spectrometry, high performance liquid chromatography, and fluorometric immunoassay. However, these methods require highly technical equipment and trained personnel that do not allow for the tests to be implemented in low resource settings. Thus there is a need for a fully disposable, easy to use, and cost-efficient device for the detection of Phe that can be implemented in areas that lack current Phe detection strategies. My project proposes to develop an enzymatic colorimetric assay for the detection of Phe in a simple paper-based disposable, such that an untrained user can perform and read the test results. Critical to this the incorporation of dry reagents on card. The assay requires a two-step reaction: 1) the conversion of nicotinamide adenine dinucleotide (NAD+) to its reduced form (NADH) in the presence of Phe and catalyzed by phenylalanine dehydrogenase (PheDH), and 2) the conversion of the tetrazolium salt, nitroblue tetrazolium (NBT), into a purple formazan dye due to the presence of NADH and the coenzyme phenazine methozulfate (PMS). Reagent drying protocols have been evaluated for PheDH, NAD+, NBT and PMS for short-term reagent stability. Preliminary data shows NBT and PMS should be stored separately, to avoid autoproduction, while PheDH should be stored with a low concentrated sugar. NAD+ stores well without added stabilizers.

Thermosensitive hydrogels have been actively investigated as a potential approach to preventing the recurrence of tumors after surgery. However, current hydrogels remain limited by their low drug retention rate and non-degradability, preventing their use in neural applications. Using a trifunctional dibromomaleimide-alkyne based cross linker, this project aims to synthesize a thermosensitive, biodegradable polyethylene glycol (PEG) hydrogel for the delivery of anti-tumor drugs. The crosslinker is synthesized through condensation of dibromomaleic anhydride with propargylamine and substitution of bromine groups with cysteamine. Our trifunctional crosslinker will provide the following properties: (a) crosslinking of two thermosensitive, PEG based polymers, (b) reducible to allow for triggered degradation and (c) site for biologic conjugation by click chemistry. With the site for biologic conjugation, we will be able to physically attach the drug to the gel and make drug release dependent on the more sustained gel degradation rather than passive diffusion. The liquid, thermosensitive gel will be able to fully fill the neural cavity after tumor removal and release a drug over a sustained period of time. Preliminary synthesis of crosslinker using cysteamine shows the fluorescent color indicative of substituted maleimide in addition to active amine reactivity. Initial crosslinking of thermosensitive polymers indicate an increase in hydrophillicity and swelling with increased temperature. Future experiments will include increasing length of crosslinker arms with PEG spacers for more effective linking and attaching a biomolecule to the alkyne group of the crosslinker. The toxicity and release profile of the crosslinked polymers will be characterized to retune the polymers to create a hydrogel with high localization and minimal toxicity.

 Antibodies play a key role in imaging, diagnostics and therapeutics. Antibodies are large molecules, often proteins, produced by our immune system that recognize and bind to specific targets. This project focuses on creating antibody-like recognition proteins that can be triggered to bind and release on command. We propose to create this activatable protein by mutating key regions on an existing protein that would act as a scaffold for the new switchable binding protein. FimH, found on the end of bacteria fimbria, is an ideal protein scaffold because it can be triggered to have an allosteric conformational change that affects binding affinity. To fully explore the different mutation possibilities, we propose to randomize 3 complementary determining region (CDR) loops that are crucial in binding to FimH’s original target. This “library” of proteins will then be expressed on bacteria, filtered to remove improperly folded proteins and screened against targets of interest in order to search for unique binders. The display library size is significant in our work because a larger library increases the probability that there could be more unique binders found while screening the library. We have created a display library of 1.07x10⁵ variants of CDR loop 2 randomized. I have focused on maintaining the diversity of the expressed library by optimizing the library transformation rates while randomizing CDR loops 1 and 3. Through this work, I have found an optimum ratio of cells to library DNA that will express a sufficient sample of the library at an affordable cost. We predict that this technology could improve the specificity of diagnostics and imaging by exacting control through triggering binding and unbinding of targets in these systems.

Most important biological interactions with the environment occur at interfaces such as a lipid cellular membrane. These interactions are often dynamic and show time-dependent changes in material properties (e.g, viscoelastic properties). A detailed understanding of biomechanical properties at molecular and subcellular level is vital for basic understanding of normal/abnormal biological processes. Indeed, abnormal changes in the mechanics of biological systems are often indicators of pathophysiological states. For example, amyloid beta peptide (Aβ), a protein present in neural plaques formed in Alzheimer’s disease and cholesterol are reported to alter cell membrane mechanical properties leading to neuronal degeneration. Atomic force microscopy (AFM) provides an ideal tool to image the structure and examine the mechanics in physiologically relevant medium. We have created a new cantilevered probe for AFM for the study of nano-to-microscale mechanics of biological interfaces. We used it to examine the role of Aβ1-42 insertion in DPPC lipid monolayers and its effect on the viscoelastic properties that underlie altered membrane fluidity. Real-time analysis of mechanical properties of the model membrane monolayer in the presence of Aβ1-42 shows a decrease in the viscosity of the monolayers over time, consistent with increased membrane fluidity. The finding is consistent with changes in membrane permeability due to Aβ-inserted pores.

The human brain is rapidly growing during the last trimester and there is evidence that premature birth is related to adverse neurological events. Magnetic resonance (MR) imaging of premature neonates and subsequent automated segmentation allows us to quantify subtle disruptions in cortical growth and folding in large population based studies. We use an age-specific automated tissue segmentation technique combined with an age-specific parcellation scheme to study regional patterns of tissue growth in a large study of brain development in premature neonates. We aimed to investigate the additional information provided by regional analysis of MR scans when determining risks for delayed neurocognitive outcomes. Our image analysis framework incorporates a spatiotemporal reference atlas that covers the age range of 27.43 to 46.43 gestational weeks (GW), and consisted of manually segmented brain tissues and 8 hemispheric lobe regions. The tissue atlas was used to automatically segment all 269 MR scans, age range from 27.29 to 47.00 GW, using a hybrid framework that combines an age-specific atlas-based EM segmentation with a patch-based prior. This tissue segmentation was then used to re-align the subject anatomy with an age-specific tissue template and the region parcellation of each subject was carried out by template probability propagation into the subject anatomy from the spatiotemporal parcellation model. Multiple linear regression models of the parcellation volumes, divided into gray matter and white matter tissues, showed a significant correlation between regional volume and Bayley’s Scale of Infant and Toddler Development scores, beyond gestational age at birth. Parietal and occipital lobes were clear markers for language and motor abilities, while the temporal lobe volumes were correlated with cognitive abilities. These findings support a more anatomically-specific analysis of brain structure after premature birth and may provide the possibility of earlier and more specific clinical diagnostics and timely developmental interventions.

Magnetic microbeads are a common tool used in biological assay purification, however, they don’t diffuse quickly nor do they have a high surface area, which results in low binding efficiency. Magnetic nanoparticles have more total surface area and can diffuse quickly, but cannot be separated magnetically as individual particles. The research I do uses magnetic nanoparticles (MNPs) coupled with pNIPAAm (p(N-isopropylacrylamide)) as a temperature-responsive reagent system to purify solutions for diagnostic tests in order to increase the sensitivity and specificity. This works by combining MNPs coated with pNIPAAm and free pNIPAAM-antibody conjugates. The antigen binds to the antibody and when heated to the cloud point temperature (around 37°C for pNIPAAm), the polymers aggregate, allowing the MNPs and the antibody conjugates to be magnetically separated together. The conjugates are synthesized using NHS chemistry and purified through HPLC. The separation efficiency of these conjugates is determined though ELISA and can reach near 100% and the total aggregation and capture time for this system is less than 4 minutes. This is useful because it can rapidly concentrate and purify samples to reduce false positives and allow for a lower limit of detection. This can be incorporated into a multitude of point-of-care tests to deliver earlier, faster, and more accurate results.

Cardiomyocytes are the optimum cell source to model and study heart diseases and to screen drugs for cardiotoxicity in vitro. Cardiomyocytes differentiated in vitro from human pluripotent stem cells, however, are structurally and functionally immature. It is necessary to accurately recapitulate the behaviors of cells in the native myocardium for pathogenesis and drug-screening assays. Therefore, before the application of human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs), we must address their maturation state. Physical cues presented to neonatal rat cardiomyocytes in a specific scale matching their native microenvironmental niche have been shown to profoundly regulate their structure and function, as characterized by elongated anisotropic morphology, well-organized contractile machinery, and faster action potential propagation. It is unknown, however, whether the same nanotopographical cues would have a similar positive influence on the maturation of hPSC-CMs. In this study, we tested the hypothesis that anisotropic nanopatterned substrates would enhance the maturation of hPSC-CMs, resulting in cardiomyocytes more suitable for in vitro cardiac tissue engineering applications. To test this hypothesis, we seeded hPSC-CMs differentiated from the matrix sandwich method on nanopatterns of various dimensions and compared their maturation with hPSC-CMs cultured on traditional flat substrates. To characterize maturation, the structural and functional phenotypes of hPSC-CMs were analyzed from their morphology/alignment, calcium handling properties, and gene expression. It was anticipated that, similar to neonatal rat ventricular cardiomyocytes, hPSC-CMs would exhibit a size-dependent change in their structure and function and that there would be an optimal substrate dimension size resulting in the most mature hPSC-CMs. The experimental result could yield a hPSC-CMs culturing protocol for deriving mature human cardiomyocytes for future drug screening assays and heart disease modeling. After optimizing substrate dimensionality, other culturing aspects such as chemical and mechanical cues and their temporal introduction to cells can be tested to further enhance the maturation of hPSC-CMs.

 Impaired systolic function resulting from acute injury, congenital defect, or aging can lead to cardiac complications and heart failure. Current therapies are unable to rescue cardiac function, but merely slow disease progression. We have demonstrated that when myosin uses 2-deoxy-ATP (dATP) as an energy substrate for contraction instead of ATP, cardiac contractility is enhanced by 20% or more. Viral-vector mediated overexpression of R1R2, the enzyme that catalyzes production of deoxy-nucleotides, increases intracellular dATP. In this study we determined the expression pattern of R1R2 in mice following systemic delivery of recombinant adeno-associated viral vectors pseudotyped with the type 6 capsid (rAAV6) carrying R1 and R2 subunits, and a cardiac specific gene regulatory cassette (cTnT455). Cardiac function was assessed in vivo via echocardiography, and ex vivo using Langendorf perfusion to test the hypothesis that elevating myocardial dATP by a gene therapy approach enhances cardiac function. We also performed metabolomics evaluation of both skeletal and cardiac muscle to correlate with changes in cardiac function. Our current data suggest that systemic delivery of this rAAV6 system results in myocardial specific overexpression of R1R2 and improves basal cardiac performance, which supports our previously published data for transgenic animals with long-term overexpression of R1R2. Metabolic analysis suggests that R1R2 overexpression in the heart alone alters the glucose metabolism of both skeletal and cardiac muscle, indicating improved basal cardiac performance can directly impact skeletal muscle metabolism. Preliminary studies in an aging model demonstrate that treatment with this rAAV6 vector increases oxidative metabolism in cardiac tissue and increases glycolysis in fast twitch skeletal muscle. These data indicate that vector-mediated overexpression of R1R2 improves basal cardiac function and impacts skeletal muscle metabolism, demonstrating potential to improve cardiac function of patients with systolic dysfunction. HL11197.

Affecting millions of people across the globe, the WHO deems myocardial infarctions (heart attacks) as the largest cause of death worldwide. The heart’s weak regenerative capacity leads to scarring of the dead tissue and a reduction in cardiac output that presents numerous health consequences. Tissue engineering is a promising outlook in restoring the structural integrity and contractile force of the damaged area. Recent findings have demonstrated that engineered heart tissues grown from stem cells can display significantly improved characteristics when subjected to mechanical stimuli. Inspired by these results, this project seeks to improve tissue performance even further by investigating a novel concept where stem cell-derived cardiac tissues are treated with the same mechanical profile experienced by native heart tissue. In order to simulate and implement these forces, tissues are grown and then anchored onto two posts that can vary in distance and stiffness. Upon contracting, post stiffness is increased providing a resistance to each beat. This resistance simulates the afterload stress that the ventricular wall contracts against to eject blood into the aorta. Next, the tissues are stretched by increasing the distance between each post. The stress produced by elongating the tissues simulates the preload stress exhibited by the ventricular wall as blood is pumped into the chamber prior to the next contraction. Finally, combining the stiffness (afterload) and stretching (preload) schemes allows the tissues to experience the entire force profile exhibited by the native heart. Simulating the cardiac mechanical environment gives the engineered tissues the stimulus to yield contractile forces and a cellular structure at par with their native counterparts. This investigation thus aims to advance regenerative medicine by bringing stem-cell derived cardiac tissues one step closer towards curing myocardial infarctions.

Recent pharmaceutical development that targets specific cell signaling pathways has been in the rise. However, many of the drugs can also induce off target effects especially in cardiovascular system. Current in vitro cardiotoxicity screening utilizes genetically modified cell lines, which do not characterize the biochemical and electrophysical parameters of human heart cells. The discrepancy of cell characteristics produces false negatives and false positives resulting in the release of potentially lethal drugs and the loss of valuable drugs, respectively. The project aims to improve the predictive value of the cardiotoxicity screening by creating an assay with well developed cardiac tissue composed of matured human pluripotent stem cell-derived cardiomyocytes. Made possible by well-established nanopatterning techniques, cardiomyocytes are cultured on a poly(dimethylsiloxane) thin film with topographical features layered with multiple functional substrates using spin coating and soft molding. The thin film with tunable dimensions is employed as a substrate upon which force generation of cardiomyocytes can be characterized. Under the poly(dimethylsiloxane) layer, poly(N-isopropylacrylamide), a thermal responsive polymer that transforms from a hydrophobic form to a hydrophilic form, is spin coated and utilized as a sacrificial layer that will release the thin film when the temperature is lowered. After the release of the thin film, measurement of the deformation of the thin film allows for the calculation of force generation and gives a direct representation of the effects of cardiotoxicity on the overall cardiovascular system functionality. Optimization of the thin film fabrication was completed and upon scanning electron microscopy observation, high fidelity of the nanopattern was observed. This biomaterial platform better mimics the native human heart tissue and has the ability to characterize the force generation of cardiomyocytes. Furthermore, this platform could be utilized as a drug-induced cardiotoxicity assay which could potentially benefit the pharmaceutical drug development.

Cardiovascular disease remains the leading cause of death worldwide. The heart is one of the most resistant organs to self-repair from injury, e.g. myocardial infarction. Stem cell therapies, and tissue engineering have had limited success in inducing heart tissue to regenerate owing to the failing incorporating morphological, electrical, mechanical cues that are important for physiological functionality of transplanted heart tissues. Pre-engineered functional tissue on scaffold prior to myocardial transplantation enables recapitulating topological, mechanical, electrical and biological cues of healthy myocardium extracellular matrix (ECM), while providing cites for adhesion and migration of cells. In this study, we investigated one of the potential novel materials, Graphene Oxide, for pre-engineered cardiac tissue maturation. We performed various analyses to confirm maturation of cardiac tissue on graphene oxide physiologically, morphologically, and functionally. Graphene Oxide showed positive effects on maturation of cardiac tissue. Neonatal Rat Ventricular Myocyte tissue showed anisotropic cooperation with contraction when it was on the material. Interestingly, Graphene Oxide substrates were reduced during cell culture, which was confirmed by Raman spectroscopy. However, mechanisms for the reduction are still need to be explored. We would like to continue our study on finding underlying mechanisms and role of Graphene Oxide on cellular redox reaction. We are cooperating this material with nanogrooved substrates to build a platform for cardiac tissue maturation for tissue implantation.

The brainstem's important role in the motor and sensory systems makes it a vital structure for studying pediatric development. However, the developing brain's rapid growth presents significant challenges when studying preterm infants. We investigated the regional differences of the brainstem between healthy and developmental delayed preterm infants. A spatiotemporal atlas was constructed from 28 training subject magnetic resonance (MR) scans and their brainstem manual tissue segmentations (whole brainstem) and parcellations (midbrain, pons, medulla) to capture a wide range of growth over time. The brainstem in all 269 MR scans in our database was automatically segmented using an age-specific template derived from the tissue-atlas training data to initiate an atlas-based Expectation-Maximization framework. We used tissue segmentations to re-register the age-specific tissue maps and derive an accurate non-rigid transformation from the atlas into the space of the subject scan. Template propagation was used to place an age-specific brainstem parcellation from the atlas into the space of the subject MRI. Multiple linear regression analysis was performed to model the relationship between regional brainstem volume calculated from the automated parcellations and specific Bayley Scales of Infant and Toddler development-Third Edition (BSID-III) cognitive, language, and motor composite scores at 18 months of age. We covaried with post-menstrual age at scan, age at birth, gender, and with and without supratentorial volume. Significant association existed for all brainstem volumes except for the pons and all BSID-III scores near birth age (mean=32.7+/-3.2 weeks) while there was nearly no significance for the same models in children near full-term age (mean=40.6+/-2.7 weeks). This supports the hypothesis that smaller total brainstem, midbrain, and medulla volumes measured shortly after preterm birth may be useful as a biomarker to predict poor developmental outcome at 18 months of age, while measurements later (near full term age) may be less useful as brain volume recovers.

A prosthetic liner is a sleeve that wraps around the residual limb of an amputee and couples the limb to a prosthetic socket.  The inner surface is an elastomeric gel that is in contact with the skin, and the outer surface is a fabric support.  Lower limb amputees must support their body weight on tissues which were not suited for high stresses, and the liner plays an important role in how ambulatory loads are distributed.  Quantitative data on the properties of liners is limited, so a clinical prosthetist's knowledge is confined to insight gained during practical experience.  The goal of this project is to create an online tool, the Prosthetic Liner Assistant (PLA), which will provide engineering data in a clinically accessible format.  Twenty new and ten used liners from six manufacturers were tested.  The material properties of the liners were quantified using six different material tests that measured the compressive elasticity, bulk elasticity, shear elasticity, tensile elasticity, coefficient of friction, and thermal conductivity.  Across all liners, gel material determined the shear elasticity and coefficient of friction, while the fabric backing had a strong influence over the tensile elasticity.  The gel and fabric backing contributed more equally to the free compression response of the liners.  As liners aged, the gels became softer while the fabrics became stiffer.  This was seen in decreased shear and compressive elasticity, and an increased tensile elasticity.  The PLA is published online at www.LinerAssist.org.  Differences between liners can be unclear, but the Prosthetic Liner Assistant will potentially allow a better fit for patients by quantifying the physical properties of liners, providing evidence for prosthetists to improve the quality of patient care.

While therapeutic cancer models offer potential for novel drug discovery and personalized medicine, current models seldom yield clinically-useful measures of drug efficacy because the physiology of tumor microenvironment is not accurately mimicked or fail to produce data in time to inform the best possible treatment. Thus, we have developed an approach that integrates live tumor slice culture with a 96-well plate-based microfluidic device that allows for the exposure of intact tumor slices to large numbers of drugs. The device integrates a porous membrane that allows for organotypic culture of tissue slices and contains open fluidic channels beneath the culture membrane (membrane acts as a roof) in which drugs can be delivered in parallel microfluidic streams through the membrane to the tissue slices. The addition of a 96-well plate creates a user-friendly interface in which reagents can be added with a standard pipette. Considering current advances in 3-D printing technology, we have used 3-D computer aided design (CAD) software to design a version of the proposed microfluidic device that can be printed using stereolithography, an additive manufacturing process where a photosensitive resin is solidified using a high resolution laser. This will allow us to assess the viability of this approach for the fabrication of complex microfluidic devices, and we are able to take advantage of the three dimensional design space to create a network of channels that exist on multiple planes. With this microfluidic platform, there is the potential to test up to 80 different drug compounds within an intact tumor tissue, which will ultimately be beneficial for cancer patients. 

The Fu lab, in collaboration with the Yager and Lutz labs, develops low cost disposable diagnostic assays, much like the common pregnancy strip, but capable of performing more complex sample processing. Our paper-based assays are capable of automatically sending multiple reagents over a detection zone in sequence. These assays have been developed to perform the detection of markers for diseases such as dengue fever and malaria. My project is to further expand the capability of these assays by providing methods to passively control the flow rate through the detection zone. By optimizing the flow rate for each reagent delivery step, the time to result of the assay can be reduced without sacrificing signal strength. Devices are designed in AUTOCAD and fabricated from nitrocellulose and cellulose using a CO2 laser system. Flow speed within a device is quantified using a fluid marking system, in conjunction with custom video tracking software composed in MATLAB. Our experiments demonstrate several preliminary designs, in which flow is modulated by modifying the geometry of the assay substrate. In one design, a steady flow rate eventually transitions to a secondary, slower steady flow. A second design performs the opposite, where a steady flow rate eventually transitions to a secondary faster steady flow. The third preliminary design incorporates both these results to have a slow steady flow, which eventually transitions to a faster steady flow, and then finally falls back to a slower steady flow again. These flow profiles are well tailored for common protocols currently in use, where fast flowing rinse steps alternate in between slow flowing reagent binding steps.

According to the World Health Organization, cardiovascular diseases are the leading cause of mortality worldwide. The low regenerative capacity of the heart renders it ineffective in healing itself after heart failure. Our lab has developed efficient methods to derive cardiomyocytes (cardiac muscle cells) from human embryonic stem cells (hESCs). However, it has become apparent that the cardiomyocytes derived using current differentiation methods exhibit immature morphological and functional properties. An important characteristic of mature cardiomyocytes is the presence of T-tubules, which are invaginations of the plasma membrane into the cell interior. These subcellular structures are critical for synchronized calcium release and contraction of mature cardiomyocytes. The protein caveolin-3 is a critical component of T-tubules, and thus its visualization can be used to assess the development of these important subcellular structures. I am currently evaluating the expression of caveolin-3 in hESC-derived cardiomyocytes using immunostaining techniques. For these studies, I expose the sample to anti-caveolin-3 as the primary antibody followed by a complementary secondary antibody conjugated to a fluorescent marker for visualizing the protein. Titration of several parameters, such as time of incubation and dilution of the antibodies allows for staining optimization. I have obtained detailed images of T-tubules aligning with striations in control heart tissue, but the staining is diffuse for the hESC-derived cardiomyocytes grown in standard 2D cultures. I am currently exploring whether this is a technical issue or the result of developmental immaturity. To assess the latter, I will stain hESC-derived cardiomyocytes that are more mature due to mechanical conditioning in 3D constructs. Optimization of this technique will facilitate our efforts to find novel agents that can accelerate the maturation of hESC cardiomyocytes. Finding ways to mature these cells will then provide a better platform for therapeutic applications such as drug screening, disease modeling and cardiac repair.

Myocardial infarction and resulting heart failure continues to be the leading cause of death worldwide. The heart does not respond effectively to injury through tissue regeneration because cardiomyocyte proliferation after differentiation occurs at a greatly diminished rate. The goal of this research is to study stem cell-derived cardiomyocytes to better understand the mechanisms and possible controls of cardiomyocyte proliferation. Achieving this goal would allow for the intitiation of cardiomyocyte proliferation, which would better direct new technologies aimed at regenerating cardiac tissue after injury. RUES2 human embryonic stem cells are differentiated into cardiomyocytes using a directed differentiation protocol that manipulates the TGFβ and Wnt signaling pathways, by the controlled addition of Activin A, BMP4, chiron, and Xav signaling factors. After twelve to fifteen days, cells are harvested and stained for cardiac troponin T (cTnT), which is a protein present in the sarcomere of cardiomyocytes. Flow cytometery is used to identify cardiomyocytes expressing cTnT and indicate the purity of the differentiated culture. Cardiomyocyte proliferation is measured by exposing cells to Bromodeoxyuridine (BrdU), which is a thymidine analog that is incorporated into synthesized DNA. A double immunohistochemistry stain for Beta Myosin Heavy Chain (β-MHC) and BrdU is used to indicate proliferating cardiomyocytes. RNA is isolated from cardiomyocyte cultures and analyzed using quantitative real time PCR. Specific sequences of RNA present in cells can indicate relative gene expression, and allow for genes of interest to be monitored through stages of differentiation to determine peak expression. Ongoing experiments are investigating the activity of the Notch signaling pathway during directed differentiation of cardiomyocytes and its relation to cardiomyocyte proliferation. Results from these experiments will better indicate time points for enhancing cardiomyocyte proliferation using Notch signaling and help further characterize methods for induced proliferation as a potential therapeutic after cardiac injury.

The development of drug-resistant HIV variants is an important cause of virologic failure. In order to choose effective regimens, assays for diagnosing resistance are essential. Pol gene mutations within the HIV retrovirus, which are associated with drug failure and phenotypic resistance, can be used as the assay targets. Current diagnostic assays include various genotypic and phenotypic assays, recombinant virus assays and other commercial assays. However, these assays are time consuming. To address this issue, we are applying engineering principles to convert the oligonucleotide ligation assay (OLA), developed by Frenkel et al., for point-of-care (POC) diagnostics, to significantly reduce the timeframe for diagnosing drug resistance, leading to regimen decisions. In order to detect the HIV pol gene using OLA, nucleic acid (NA) from bodily fluids need to be extracted and purified. Current extraction methods are time consuming, and require complex procedures, instrumentation, and expensive reagents unavailable in low-resource settings. We are developing novel approaches to capture NA without instrumentation through the use of stimuli-responsive polymers. These polymers are capable of responding to specific changes in their environments, such as temperature variations, resulting in a change of their physical properties; our polymer becomes cationic and aggregating. Magnetic nanoparticles (mNPs) can be utilized in conjunction with these polymers for biomolecule separation by applying a stimulus and a magnetic field. The new NA extraction strategy starts from complexing cationic stimuli-responsive polymers with NA, and then magnetically separating NA from solution via the co-aggregation with mNPs and heating (characterized using standard agarose gel protocol). This extraction method utilizes 2 reagents and takes about 20 minutes, which will reduce cost and enable POC diagnostics in low-resource settings. The extracted NA can then be amplified, tested for the presence of the mutant pol gene, and used to better diagnose HIV drug-resistance and determine patient regimens.