Annually, approximately 1.7 million people suffer a traumatic brain injury (TBI) in the United States. After initial insult, a TBI persists as a series of molecular and cellular events that lead to cognitive and motor deficits which have no treatment. In addition, the injured brain activates the regenerative niches of the adult brain presumably to reduce damage. The subventricular zone (SVZ) niche contains neural progenitor cells (NPCs) that generate astrocytes, oligodendrocyte, and neuroblasts. Following TBI, the injury microenvironment secretes signaling molecules like stromal cell derived factor-1a (SDF-1a). SDF-1a gradients from the injury contribute to the redirection of neuroblasts from the SVZ towards the lesion which may differentiate into neurons and integrate into existing circuitry. This repair mechanism is transient and does not lead to complete recovery of damaged tissue. Further, the mechanism by which SDF-1a gradients reach SVZ cells is not fully understood. To prolong NPC recruitment to the injured brain, exogenous SDF-1a delivery strategies have been employed. Increases in cell recruitment following stroke, spinal cord injury, and TBI have been demonstrated following SDF-1a delivery. Exogenous delivery of SDF-1a is limited by its 28-minute half-life and clearance from the injury microenvironment. Biomaterials-based delivery improves stability of molecules like SDF-1a and offer control of its release. This dissertation investigates SDF-1a delivery strategies for neural regeneration in three ways: 1) elucidating the mechanisms of spatiotemporal SDF-1a signaling across the brain, 2) developing a tunable biomaterials system for SDF-1a delivery to the brain, 3) investigating SDF-1a delivery on SVZ-derived cell migration following TBI. Using in vitro, in vivo, and in silico analyses, autocrine/paracrine signaling was necessary to produce SDF-1a gradients in the brain. Native cell types engaged in autocrine/paracrine signaling. A microfluidics device generated injectable hyaluronic-based microgels that released SDF-1a peptide via enzymatic cleavage. Microgels (±SDF-1a peptide) were injected 7 days post-TBI in a mouse model and evaluated for NPC migration 7 days later using immunohistochemistry. Initial staining suggested complex presence of astrocytes, NPCs, and neuroblasts throughout the frontoparietal cortex. Advancement of chemokine delivery was demonstrated by uncovering endogenous chemokine propagation in the brain, generating new approaches to maximize chemokine-based neural regeneration.

Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors.

Achieving effective drug concentrations within the central nervous system (CNS) remains one of the greatest challenges for the treatment of brain tumors. The presence of the blood-brain barrier and blood-spinal cord barrier severely restricts the blood-to-CNS entry of nearly all systemically administered therapeutics, often leading to the development of peripheral toxicities before a treatment benefit is observed. To circumvent systemic barriers, intrathecal (IT) injection of therapeutics directly into the cerebrospinal fluid (CSF) surrounding the brain and spinal cord has been used as an alternative administration route; however, its widespread translation to the clinic has been hindered by poor drug pharmacokinetics (PK), including rapid clearance, inadequate distribution, as well as toxicity. One strategy to overcome the limitations of free drug PK and improve drug efficacy is to encapsulate drug within nanoparticles (NP), which solubilize hydrophobic molecules for sustained release in physiological environments. In this thesis, we will develop NP delivery strategies for brain tumor therapy in two model systems: glioblastoma (GBM), the most common and deadly malignant primary brain tumor, and medulloblastoma, the most common pediatric brain tumor. In the first research chapter, we developed 120 nm poly(lactic acid-co-glycolic acid) NPs encapsulating the chemotherapy, camptothecin, for intravenous delivery to GBM. NP encapsulation of camptothecin was shown to reduce the drug’s toxicity and enable effective delivery to orthotopic GBM. To build off the success of intravenous NP, the second research chapter explored the utility of 100 nm PEGylated NPs for use with IT administration. Using in vivo imaging and ex vivo tissue slices, we found the NPs were rapidly transported by the convective forces of the CSF along the entire neuraxis and were retained for over 3 weeks. Based on their wide spread delivery and prolonged circulation, we examine the ability of the NPs to localize with tumor lesions in a leptomeningeal metastasis (LM) model of medulloblastoma. NPs administered to LM bearing mice were shown to penetrate into LM mets seeded within the meninges around the brain. These data show the potential to translate our success with intravenous NPs for GBM to improve IT chemotherapy delivery to LM.

Monitoring complex diseases and their comorbidities requires accurate and convenient measurements of multiple biomarkers. However, many state-of-the-art bioassays not only require complicated and time-consuming procedures, but also measure only one biomarker at a time. This noncomprehensive single-biomarker monitoring, as well as the cost and complexity of these bioassays advocate for a simple, rapid multi-marker sensing platform suitable for point-of-care or self-monitoring settings. To address this need, diabetes mellitus was selected as the example complex disease, with dry eye disease and cardiovascular disease as the example comorbidities. Seven vital biomarkers from these diseases were selected to investigate the platform technology: lactoferrin (Lfn), immunoglobulin E (IgE), insulin, glucose, lactate, low density lipoprotein (LDL), and high density lipoprotein (HDL). Using electrochemical techniques such as amperometry and electrochemical impedance spectroscopy (EIS), various single- and dual-marker sensing prototypes were studied. First, by focusing on the imaginary impedance of EIS, an analytical algorithm for the determination of optimal frequency and signal deconvolution was first developed. This algorithm helped overcome the challenge of signal overlapping in EIS multi-marker sensors, while providing a means to study the optimal frequency of a biomarker. The algorithm was then applied to develop various single- and dual-marker prototypes by exploring different kinds of molecular recognition elements (MRE) while studying the optimal frequencies of various biomarkers with respect to their biological properties. Throughout the exploration, 5 single-marker biosensors (glucose, lactate, insulin, IgE, and Lfn) and one dual-marker (LDL and HDL) biosensor were successfully developed. With the aid of nanoparticles and the engineering design of experiments, the zeta potential, conductivity, and molecular weight of a biomarker were found to be three example factors that contribute to a biomarker’s optimal frequency. The study platforms used in the study did not achieve dual-enzymatic marker biosensors (glucose and lactate) due to signal contamination from localized accumulation of reduced electron mediators on self-assembled monolayer. However, amperometric biosensors for glucose and lactate with disposable test strips and integrated samplers were successfully developed as a back-up solution to the multi-marker sensing platform. This work has resulted in twelve publications, five patents, and one submitted manuscripts at the time of submission.

Imaging analysis of local drug delivery is important because in both studies involving chemotherapy targeted toward glioblastoma and antimicrobial addressing infection, the drug concentration and distribution are unknown. There are a variety of studies focused on the local delivery of drug to a targeted location, but we are presenting a way of quantifying the concentration of the drug and the distribution of the drug during a period of time. This study aims to do that by utilizing Materialise Mimics to analyze the MRI images of local drug delivery in glioblastoma in canines and antimicrobial gel in rabbit femurs. The focus of the technique is to register the anatomy in T1-weighted spin echo images to the drug delivery in T2 flow attenuated inversion recovery (FLAIR) images in order to see where the drug went and did not go relative to the anatomical part. Both studies focus on addressing effective volumes of drug to a designated anatomical area, in which the delivery can be difficult as it involves bypassing the blood brain barrier in the first study and achieving effective volumes while preventing toxicity to the kidneys in the second study. The goal of this project lies in determining the drug volumes and location for the specified duration and anatomical part.

Microvillus Inclusion disease is a fatal disease found in the Navajo population caused by a single nucleotide polymorphism. It is characterized by intractable diarrhea and is often fatal early in life.1 The current method of diagnosis is sending duodenal biopsies for histopathological examination and confirmatory testing through genomic sequencing. The purpose of this experiment was to create a more simple and cost-effective diagnostic method for detecting Microvillus Inclusion disease. Three methods were explored (RFLP2, ARMS3,4, and Tentacle Probes5,6) and two methods were tested to determine their ability and their efficiency in detecting the SNP that causes the disease.2 Tests using the RFLP2 method and synthetic DNA resulted in 9% false-positive rate and 11% false-negative rate in a blind trial for detecting both target (mutation present) and non-target (mutation absent) DNA when gel analyzing software was used to compare Rf values after gel electrophoresis. Using the ARMS method3, a nine-sample randomized test was run that ended up with 22% false-positive rate and 19% false-negative rate from a blind trial when using a gel analyzing software to determine presence of the SNP by band intensity. Disclaimer: No DNA from human patients was used in this study. Only synthetic DNA used.

Background: Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems.

Methods: We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe.

Results: Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA.

Conclusions: Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

MPV17-related hepatocerebral mitochondrial DNA depletion syndrome, previously known as Navajo Neurohepatopathy (NNH), is a rare genetic disease affecting Navajo children of the American Southwest. These children can suffer from several severe symptoms like brain damage and liver disease, and a diagnosis leads to death by age 10, on average. The only known effective therapy for NNH is a liver transplant. Currently, the disease is diagnosed through a lengthy and expensive process of gene sequencing, but oftentimes patients with the most severe forms of NNH deteriorate quickly; thus a rapid diagnostic would be beneficial to beginning the transplant process as early as possible. Here, Tentacle Probes, a novel technology to detect genetic mutations, were proposed to rapidly and accurately diagnose NNH. Because of Tentacle Probes' double binding site kinetics, they can detect mutations more accurately than other types of genetic probes. Probes specific to the NNH mutation were designed for use with a real-time polymerase chain reaction (PCR) detection platform. Initial synthetic DNA testing of Tentacle Trobes showed capable differentiation between mutated and non-mutated samples. However, experiments to validate those results at Phoenix Children's Hospital before moving to patient samples showed that test viability decreased over time. Efforts to diagnose the issues that led to decreased viability suggested four possible explanations that are as follows (in order of decreasing likelihood): first, undesired products from improper PCR primer design was supported by double bands in DNA gel electrophoresis; second, DNA may have degraded over time or due to repeated cycles of freezing and thawing stock solutions, and this was supported by smeared DNA gel electrophoresis; third, probe degradation, specifically of the fluorescent reporter, is possible; finally, contaminants that inhibit the PCR reaction may have been introduced. A combination of these factors may also have caused the change in assay viability. As a result of these most likely possibilities, new primers were designed and steps suggested to return viability to the assay. Thus, the various limitations and requirements for this Tentacle Probe diagnostic have been identified, and as assay development continues following the promising initial results achieved, we are confident that a rapid method if diagnosing NNH is on its way to help the children afflicted with this devastating disease receive timely access to treatment.

Adaptive thermogenesis is an innate mechanism that assists the body in controlling its core temperature that can be stimulated in two ways: cold and diet. When adaptive thermogenesis is stimulated through diet, the metabolic rate of the body should increase and the metabolic efficiency of the body should decrease. This activation should, theoretically, help to control weight gain. A protocol was developed to study four male Sprague-Dawley rats throughout a fourteen week period through the measurement of brown adipose tissue blood flow and brown adipose tissue, back, and abdomen temperatures to determine if diet induced thermogenesis existed and could be activated through norepinephrine. The sedative used to obtain blood flow measurements, ketamine, was discovered to induce a thermal response prior to the norepinephrine injection by mimicking the norepinephrine response in the sympathetic nervous system. This discovery altered the original protocol to exclude an injection of norepinephrine, as this injection would have no further thermal effect. It was found that ketamine sedation excited diet induced thermogenesis in periods of youth, low fat diet, and early high fat diet. The thermogenic capacity was found to be at a peak of 2.1 degrees Celsius during this time period. The data also suggested that the activation of diet induced thermogenesis decreased as the period of high fat diet increased, and by week 4 of the high fat diet, almost all evidence of diet induced thermogenesis was suppressed. This indicated that diet induced thermogenesis is time and diet dependent. Further investigation will need to be made to determine if prolonged high fat diet or age suppress diet induced thermogenesis.

Flipped classrooms invert the traditional teaching methods and deliver the lecture online outside of the classroom. An increase in technology accessibility is increasing the prevalence of this teaching technique in universities. In this study, we aim to address some of the uncertainties of a flipped classroom by implementing a new lecture format in Transport Phenomena. Transport Phenomena is a junior level biomedical engineering course originally flipped in Spring 2013. Since transitioning to a flipped classroom, students have been required to watch 75-minute lectures outside of class where the instructor covered key concepts and examples using paper and marker on a document camera. In class, students then worked in groups to solve problems with instructor and teaching assistant feedback. Students also completed self-graded homework with the opportunity to earn lost points back by discussing fundamental misconceptions. We are introducing re-formatted mini lectures that contain the same content broken down as well as example problems worked out in a tutorial technique instead of traditional solving method. The purpose of this study is to determine the effectiveness of newly created mini lectures with integrated questions and links in terms of student achievement and attitude [interest, utility, and "cost" (time, effort, and emotion)].