DCE Theses and Dissertations

The hair follicle cycles between growth, regression, and rest phases. This cyclic activity is regulated by the activation and quiescence of hair follicle stem cells (HFSCs). Recent studies indicate that the crosstalk between HFSCs and the stem cell niche play an important role in regulating HF maintenance. Due to the complex architecture and diverse skin cell types consisting of the skin, it is often challenging to study how a specific cell type of interest influences the microenvironment of HFSC niche. To enhance our understanding of the relationship between HFSCs and their neighboring cells, it is necessary to develop an investigation tool which can target and manipulate specific skin cell types without disturbing the sensitive microenvironment. The goal of this study was to evaluate transduction patterns of AAV with different conditions, ranging from serotypes, promoters, dosage, and timing of injection. Given these findings, we established the system by which AAV vectors can show targeted transduction of specific cell types in the skin. We first prepared the AAVs incorporated with a reporter transgene and injected intradermally on the back skin of mice. Next, we evaluated their transduction efficiency via fluorescence microscopy and quantified the labeled cells. Immunohistochemistry with different skin cell markers was performed to identify which skin cell types were infected by our AAV candidates. As a result, the pattern of AAV transduction in skin varied depending on capsid serotype, promoter, and the timing of injection. The adult injection of AAV8-CAG-GFP showed the most widespread transduction, while AAV6-CAG-GFP and AAV-PHP.S-EF1a targeted arrector pili muscle with a high frequency. The P0 injection of AAV overall showed a significant improvement in transduction efficiency, compared to the adult injection. Importantly, the P0 injection of AAV6-EF1a-DTA-mCherry was highly efficient to transduce APM. In addition, the P0 injection of AAV-PHP.S-EF1a-DTA-mCherry showed tissue-specific transduction in dermal papilla and arrector pili muscle. Our study demonstrates that the combination of different conditions can achieve a cell-type specific transduction of AAV in mice skin. With a variety of conditions, AAV can induce cell-type-specific transduction in the skin, including dermal fibroblasts, adipocytes, APM and DP. Such a system will serve as valuable tool for dissecting the crosstalk among different skin cell populations and provide new windows for conducting future studies in hair growth and wound regeneration.

The development of biosensors for the detection of biological molecules has been subject to an intensive research. Nucleic acid detection of both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) has been the most accurate technique used to date for diseases diagnosis and research purposes. Here, we are presenting a silicon-based nanobiosensor for label-free detection of nucleic acids using aptamers-adsorbed nanostructures such as silicon nanowires (SiNWs). The detector consists of nano-structured materials and will be placed within, a microfluidic embodiment. The application of nano-photonics principles allows for detecting changes in absorption when there is a minute change in the surroundings of the nanostructured material. Using previously developed protocols to fabricate the nanostructured materials with various geometries, and functionalize them with nucleic acid aptamers, we investigated and developed novel structures of solid and hollow silicon nanowires with light absorption that is sensitive to changes in the surrounding molecules. The nanostructured material is first coated by immobilized aptamer, and absorption is measured. A change in absorption occurs when the target nucleic acid fragments hybridize with its complementary immobilized aptamer on the nanostructured material. The nano-biosensor presented here is a novel device with applications in disease diagnostic as well as drug discoveries, as it is an accurate, affordable, rapid and easy-to-use in both clinical and research settings.

While nanoparticles provide an innovative solution for targeting various diseases, including cancer and neurodegenerative disorders, rapid clearance of nanoparticles from circulation after intravenous injection is a major challenge in achieving sufficient nanoparticle accumulation at disease sites. Clearance of nanoparticles from serum is initiated by protein adsorption which subsequently triggers macrophage recognition. While surface modification of nanoparticles by polyethylene glycol (PEG) has been shown to reduce surface protein adsorption on nanoparticles and enhance their circulation, its effectiveness is limited by the generation of anti-PEG antibodies and accelerated clearance after multiple injections. We demonstrate here for the first time that biocompatible ionic liquid (IL) coatings on polymeric nanoparticles dramatically reduce serum protein surface-adsorption. Of 25 ILs designed to coat PLGA nanoparticles and subsequently screened for their ability to reduce surface serum protein adsorption, choline and hexenoic acid at 1:2 ionic ratio (CAHA 1:2) -coated nanoparticles exhibited the highest stability and propensity to reduce protein adsorption compared to PEG while remaining under 200 nm. By electrostatic interactions, CAHA 1:2 formed a surface coating consisting of a minimum of 26 and a maximum of 30 layers each (1:1 layer-by-layer charged assembly) of choline and 2-hexenoic acid (52-60 total layers) around the surface of PLGA nanoparticles. Runner up imidazolium and hexenoic acid (ImHex 1:2) at 1:2 ionic ratio formed an overall thinner coating with equivalent resistance to protein adsorption mediated by 2-hexenoic acid. However, as assembly was mediated by the modified imidazolium cation (delocalized pi bonds), ImHex 1:2 coating assembled with weaker interfacial electrostatic interaction and exhibited weaker surface stability. Several other ILs formed varying ranges of thickness around the surface of the PLGA nanoparticle but did not exhibit equivalent stability nor ability to reduce protein adsorption. Physicochemical mechanistic studies revealed that the ability of IL coatings to reduce protein adsorption correlated with the intensity and structural location of molecular interactions between bovine serum albumin 50 mg/mL and anion-cation combination. The better the ionic liquid interacted with itself, the higher the resistance to protein adsorption was found. Structurally, the length of the fatty acid anion chain was found to tune bulk assembly, as well as the presence of double bonds or “kinks” in the anion tail controlled structural rigidity and availability to bind with serum proteins on the surface, along with providing overall stability and charged repulsion from the IL itself. Importantly, the cation was found to control layered assembly, but the anion structure on the outermost surface mediated the tunability of interaction with serum proteins. Addition of CAHA 1:2 coating was found to be biocompatible and induce low RBC hemolysis (n=4). 24-hour in-vivo pharmacokinetics studies (n=6) indicated CAHAcoated PLGA nanoparticles exhibited significantly prolonged circulation, while significantly reducing IL-6 pro-inflammatory immune activation (n=4). 24- hour in-vivo biodistribution studies (n=6) revealed that whereas unmodified or PEG-coated nanoparticles primarily accumulated in the liver, CAHA-coated nanoparticles exhibited trace accumulation in the liver and dramatic accumulation in the lungs, by hitch-hiking red blood cells intravenously. These results cumulatively suggest CAHA-modified PLGA nanoparticles as a novel potential targeted solution for lung cancer.

L-plastin (LPL) is implicated in regulating innate and adaptive immune responses. Previous work showed that the ocular surface proteomes of Specific Pathogen Free mice had high levels of LPL and neutrophil secreted peptides when compared to the proteomes of Germ Free mice, suggesting that LPL contributed to the responses to commensals, and the response was neutrophil dependent. My goal was to establish conditional knockout mouse strains to assess the role of LPL in different myeloid compartments with respect to susceptibility to bacterial keratitis, identify ocular commensal presence and response of the innate immune cells to commensal sensitization. Comparison of P. aeruginosa susceptibility in LPL conditional knock out mice with specific ablation of LPL under the promoters of LysM Cre (LysM LPL KO), CX3CR1 (CX3CR1 LPL KO)and S100A8 (S100A8 LPL KO) was determined. Both LysM LPL KO and S100 A8 LPL KO mice displayed conjunctival lactic acid bacteria commensal differences in timeline experiments and increased susceptibility to P. aeruginosa-induced keratitis (p=0.0049 and 0.03, respectively). Medium from macrophage training experiments with and without the lactic acid producing commensal, S. ovis, was added to P. aeruginosa challenged PMNs to determine commensal sensitization potential and effect of LPL deficiency on bactericidal activity. Macrophage training inhibited neutrophil bactericidal activity and LPL deficient PMNs had marked reduced killing as compared with wild type. Together these results show that L-plastin controls neutrophil functionality and commensals modulate neutrophil response through macrophage commensal cross-talk and pathogen susceptibility.

It is no surprise that the advent of Artificial Intelligence (AI) is pushing waves across the medical field, amongst others. With the ability to process data in a more robust and efficient manner, mistakes, usually due to human error that are known to cripple the medical diagnostic industry can be somewhat alleviated. However, the cost of AI implementation will not mitigate that which is currently accrued by physicians, due to their expertise. As such, it is not unforeseeable that what is posited as the new age of technological advancement i.e. AI, for the betterment of patient care is nothing more than an addition to patient care cost alongside any potential benefits, to which plenty of patients are already overwhelmed by the status quo (as it pertains to cost). Mind you, this is taking into account only diagnostic modalities and not treatment options, which makes said cost to skyrocket evermore. In epilepsy diagnoses, the implementation of AI can be generally beneficial in processing EEG readings and images generated from fMRI, CT and PET Scans just to name a few (serving as a surface-level diagnostic mechanism); as well as accessing patient records and disease databases. Nevertheless, implementation of advanced imaging techniques can only be so useful, since they do not tell us the underlying cause of epilepsy should it be of pathogenic origin, as has been stipulated in multiple studies as potential causes of epileptic seizures. Therefore, designing an ultra sensitive microfluidic diagnostic chip, with the specific aim to address the question of whether a patient’s seizures are of pathogenic origin seems a better way to effectively confirm symptomatic epilepsy as well as paving the path for well-suited therapy aimed specifically at the identified entity that causes the onset of seizures which could eventually become recurrent. Since epilepsy is due to synchronous rapid firing (electrical conductance) via neurons, we effectively perform our diagnoses by capitalizing on the conductive properties of dendrimers, gold and silicon nanoparticles as our modus operandi.

Non-alcoholic fatty liver disease (NAFLD) is an emerging global health issue that has recently reached 20% in global prevalence and is projected to be the main cause of liver morbidity in the next decade. The lack of accurate human-relevant in vitro systems has caused a barrier in drug development and in the study of cytochrome P450 enzyme expression under hepatosteatotic conditions. Enzyme families CYP1, CYP2, and CYP3 are responsible for the metabolism of more than 60% of all clinically relevant drugs and so, with the growing prevalence of NAFLD, it is likely that NAFLD patients exhibit alterations in these vital drug-metabolizing enzymes and may have risk for harmful drug-drug interactions (DDIs). Here, an in vitro model of NAFLD was engineered using primary human hepatocytes to incorporate clear markers of NAFLD in its progression to non-alcoholic steatohepatitis (NASH) such as inflammation and lipid accumulation. Primary human hepatocytes were cultured in a collagen sandwich configuration that present visible and quantifiable lipid accumulation while maintaining viability and hepatic differentiation over 7 days of cultures and expressing biological markers of NAFLD. Pro-inflammatory cytokines MCP-1, IP-10, IL-8, and VEGFa displayed significant upregulation in the Steatotic states as compared to the Healthy control. These changes were accompanied by significant downregulation of CYP3A4 and CYP1A1 expression and upregulation of CYP2B6 expression in Steatotic cultures upon drug exposure. As there are no approved drug treatments available for NAFLD, this study will give insight into how the diseased state of the liver may affect the metabolism of drugs taken by patients diagnosed with NAFLD.

Here is evaluated a strategy for stem cell repair of nervous tissue with a potential therapeutic application of a transdermal microneedle stromal-derived-factor 1 (SDF-1) chemokine delivery system. The central question of this research focuses on whether enhanced SDF-1 signaling to mesenchymal stem cells (MSCs) at injury sites promotes more effective regeneration of damaged nerves. SDF-1/CXCL12 is a potent chemokine protein known to bind to ligands CXCR4 and CXCR7, both of which are proven to drive processes involved with nervous tissue regeneration (Carbajal 2010). While it is shown that SDF-1 delivery systems can facilitate repair of nerves, many of the mechanisms behind this are still unclear (Purcell 2012). In all studies evaluated in this work, significant neuro-regenerative improvements were seen as: improved MSC chemotaxis, greater volumes of repairing cells at injury sites, and an improved quality of injury repair. Many factors and cell types are also involved in nerve repair and each injury involves a coordination of signaling and checkpoints (Jiang 2017, Sullivan 2016). This study looks closely at specific nervous tissue damage and repair systems, finding that, in all cases, SDF-1 supplementation improves neural regeneration. This study concludes that further investigation of timed-release SDF-1 in microspheres via microneedle patch, using the tested approaches described here, is warranted to understand diffusion rates, cell signaling, inflammation response, fibrosis, and bio-compatibility of such a repair system.

Biological signals interact in complex ways within cells, and can exhibit great cell-to-cell heterogeneity as a function of cell history and state. Therefore, there is increasing desire to use multiple fluorescent sensors to simultaneously image multiple biological signals at the same time in individual cells. For decades the limited number of sensors recorded simultaneously has been due to spectral overlap. To circumvent this limitation of spectrally multiplexing sensors, molecular tools for spatially multiplexing have been engineered. Three biological signals were simultaneously measured with spectrally-overlapping sensors using these novel molecular tools. This initial demonstration of the spatial multiplexing strategy opens the door for the simultaneous imaging of dozens of signals within a physiological cascade as more peptide sequences for clustering are designed.

The focus of this study was to create a PLGA-PEG copolymer nanoparticle (NP), encapsulating the anti-hypertensive drugs valsartan and captopril. The surface of the NP was modified with an anti-ICAM antibody to allow for targeted cell adhesion. DLS data determined the size of the NPs to be between 120 and 150 nm, and the zeta potential had a minimum value of -27 mV. SEM images of the NPs showed smooth spherical morphology consist with all NP formation types. The PEG modification and ICAM antibody conjugation were found successful, and the drug encapsulated NP show a sustained release of both drugs for almost four days. The ICAM NPs were able to specifically bind to the ICAM receptors on the surface of TNF alpha activated Human Endothelial Cells (HUVECs) in static conditions. A flow chamber was designed and constructed to test the capacity of the ICAM NP ability to bind under flow. Initial results demonstrated the possible binding of the NPs under 15 dynes/cm2 shear stress.