Cooperativity can be used to manipulate binding affinities of DNA biosensors – improving specificity without sacrificing sensitivity; examples include tentacle probes (TPs) and cooperative primers (CPs). This thesis body of work: (1) used TPs to develop a rapid, low-cost diagnostic for detecting the point mutation leading to Navajo Neurohepatopathy (NNH) and (2) used CPs to amplify a symmetric bowtie-barcoded origami with captured t-cell receptor (TCR) α and β mRNA of a single cell.

NNH (affecting 1-in-1600 Navajo babies) is a fatal genetic disorder often caused by 149G>A mutation and is characterized by brain damage and liver disease/failure. Phoenix Children’s Hospital currently uses gene sequencing to identify the 149G>A mutation. While this process is conclusive, there are limitations, as it requires both time (3-4 weeks) and money (>$700). Ultimately, these factors create barriers that can directly impact a patient’s quality of life. Assessment of the developed TP diagnostic, using genomic DNA derived from FFPE patient liver samples, suggests nearly 100% specificity and sensitivity while reducing cost to ~$250 (including cost of labor) and providing a diagnosis within 48 hours.

TCR specificity is dependent on V(D)J recombination as well as pairing of the αβ chains. Drs. Schoettle and Blattman have developed a solution in which a bowtie-barcoded origami strand nanostructure is transfected into individual cells of a heterogeneous cell population to capture and protect αβ mRNA. When PCR of the origami template is performed with Vα, X, Vβ, and Y primers, the α and β gene segments cannot be tied back to a barcode – and paired. Assessment of the developed CPs for PCR suggests correct individual amplification using (1) Va + Xcp and (2) Vβ + Ycp primers, whereas combination of all the primers (Va, Xcp, Vb, and Ycp) suggests hybridization of the Vα + Xcp and Vβ + Ycp products due to the origami target symmetry.

Prosthetic joint infection (PJI) is a devastating complication associated with total joint arthroplasty that results in high cost and patient morbidity. There are approximately 50,000 PJIs per year in the US, imposing a burden of about $5 billion on the healthcare system. PJI is especially difficult to treat because of the presence of bacteria in biofilm, often highly tolerant to antimicrobials. Treatment of PJI requires surgical debridement of infected tissues, and local, sustained delivery of antimicrobials at high concentrations to eradicate residual biofilm bacteria. However, the antimicrobial concentrations required to eradicate biofilm bacteria grown in vivo or on tissue surfaces have not been measured. In this study, an experimental rabbit femur infection model was established by introducing a variety of pathogens representative of those found in PJIs [Staphylococcus Aureus (ATCC 49230, ATCC BAA-1556, ATCC BAA-1680), Staphylococcus Epidermidis (ATCC 35984, ATCC 12228), Enterococcus Faecalis (ATCC 29212), Pseudomonas Aeruginosa (ATCC 27853), Escherichia Coli (ATCC 25922)]. Biofilms of the same pathogens were grown in vitro on biologic surfaces (bone and muscle). The ex vivo and in vitro tissue minimum biofilm eradication concentration (MBEC; the level required to eradicate biofilm bacteria) and minimum inhibitory concentration (MIC; the level required to inhibit planktonic, non-biofilm bacteria) were measured using microbiological susceptibility assays against tobramycin (TOB) and vancomycin (VANC) alone or in 1:1 weight combination of both (TOB+VANC) over three exposure durations (6 hour, 24 hour, 72 hour). MBECs for all treatment combinations (pathogen, antimicrobial used, exposure time, and tissue) were compared against the corresponding MIC values to compare the relative susceptibility increase due to biofilm formation. Our data showed median in vitro MBEC to be 100-1000 times greater than the median MIC demonstrating the administration of local antimicrobial doses at MIC level would not kill the persisting bacteria in biofilm. Also, administering dual agent (TOB+VANC) showed median MBEC values to be comparable or lower than the single agents (TOB or VANC)

Traumatic brain injury (TBI) is a significant public health concern in the U.S., where approximately 1.7 million Americans sustain a TBI annually, an estimated 52,000 of which lead to death. Almost half (43%) of all TBI patients report experiencing long-term cognitive and/or motor dysfunction. These long-term deficits are largely due to the expansive biochemical injury that underlies the mechanical injury traditionally associated with TBI. Despite this, there are currently no clinically available therapies that directly address these underlying pathologies. Preclinical studies have looked at stem cell transplantation as a means to mitigate the effects of the biochemical injury with moderate success; however, transplants suffer very low retention and engraftment rates (2-4%). Therefore, transplants need better tools to dynamically respond to the injury microenvironment.

One approach to develop new tools for stem cell transplants may be to look towards the endogenous repair response for inspiration. Specifically, activated cell types surrounding the injury secrete the chemokine stromal cell-derived factor-1α (SDF-1α), which has been shown to play a critical role in recruiting endogenous neural progenitor/stem cells (NPSCs) to the site of injury. Therefore, it was hypothesized that improving NPSC response to SDF-1α may be a viable mechanism for improving NPSC transplant retention and migration into the surrounding host tissue. To this end, work presented here has 1. identified critical extracellular signals that mediate the NPSC response to SDF-1α, 2. incorporated these findings into the development of a transplantation platform that increases NPSC responsiveness to SDF-1α and 3. observed increased NPSC responsiveness to local exogenous SDF-1α signaling following transplantation within our novel system. Future work will include studies investigating NSPC response to endogenous, injury-induced SDF-1α and the application of this work to understanding differences between stem cell sources and their implications in cell therapies.

Controlled release formulations for local, in vivo drug delivery are of growing interest to device manufacturers, research scientists, and clinicians; however, most research characterizing controlled release formulations occurs in vitro because the spatial and temporal distribution of drug delivery is difficult to measure in vivo. In this work, in vivo magnetic resonance imaging (MRI) of local drug delivery is performed to visualize and quantify the time resolved distribution of MRI contrast agents. I find it is possible to visualize contrast agent distributions in near real time from local delivery vehicles using MRI. Three dimensional T1 maps are processed to produce in vivo concentration maps of contrast agent for individual animal models. The method for obtaining concentration maps is analyzed to estimate errors introduced at various steps in the process. The method is used to evaluate different controlled release vehicles, vehicle placement, and type of surgical wound in rabbits as a model for antimicrobial delivery to orthopaedic infection sites. I are able to see differences between all these factors; however, all images show that contrast agent remains fairly local to the wound site and do not distribute to tissues far from the implant in therapeutic concentrations. I also produce a mathematical model that investigates important mechanisms in the transport of antimicrobials in a wound environment. It is determined from both the images and the mathematical model that antimicrobial distribution in an orthopaedic wounds is dependent on both diffusive and convective mechanisms. Furthermore, I began development of MRI visible therapeutic agents to examine active drug distributions. I hypothesize that this work can be developed into a non-invasive, patient specific, clinical tool to evaluate the success of interventional procedures using local drug delivery vehicles.

Changes to the microenvironment of the endothelium can produce significant changes in the response of endothelial cells to stimuli. Human Aortic Endothelial Cells (HAECs) are tested in vitro for their fluid shear stress response when their substrates, and the solute concentrations of the fluids to which they are exposed, are modulated, and for their nitric oxide expression when they are exposed to hyperglycemic conditions. ImageJ is used to quantify either the degree of cellular alignment and elongation with the direction of flow, or the relative NO expression using the fluorochrome DAF-2. First, the results of Brower, et.al. are replicated: HAECs under normal glucose (4mM) conditions align and elongate with flow (p<<0.05), while high glucose (30.5mM) conditions negate this effect (p<<0.05) and is likely the result of Advanced Glycation End-products (AGEs). Then, in this study it is found that substitution of fibronectin for gelatin substrates does not impair flow (p<<0.05), indicating that fibronectin likely does not participate in the initiation of vascular lesions. High palmitic acid also does not prevent HAEC shear response (p<<0.05), which is consistent with Brower's predictions that AGEs are responsible for impaired elongation and alignment. NO production is significantly increased (p<<0.025) in HAECs cultured 24 hours under high glucose (30.5mM) conditions compared with normal glucose (4mM) conditions, indicating the presence of inducible nitric oxide as part of an inflammatory response. Aminoguanidine (5mM) added to high glucose concentrations reduces, but does not eliminate NO production (p<<0.05), likely due to insufficient concentration. Modulation of the endothelial microenvironment leads to pronounced changes in HAEC behavior with regards to NO production under hyperglycemic conditions. Diabetic model rat aortas are explanted and imaged for the purpose of detecting aortic endothelial cell alignment and elongation; improvements in this method are discussed. A microvessel chamber used with explanted human tissue is re-fit to reduce required volumes of solutions and allow more effective experimentation.