Molecular, Cellular, and Tissue Engineering Commons^™

Strategic Enhancement Of Biofuel Production Using Bacillus Subtilis As A Model Organism: A Dual Approach Of Gene Essentiality Mapping And Optimization, Angela A. Zebede

Electronic Theses and Dissertations

With rising global demand for renewable energy, enhancing microbial platforms for biofuel production is vital. This study engineered Bacillus subtilis to improve resilience to solvent stress by targeting membrane lipid biosynthesis pathways essential for integrity. Growth curve analysis (OD₆₀₀) showed that phosphatidylserine (PS) and phosphatidylethanolamine (PE) knockout strains grew significantly slower than wild type (168), indicating the importance of these lipids under stress. Laurdan fluorescence spectroscopy was used to assess membrane properties. Generalized polarization (GP) analysis revealed a concentration-dependent reduction in membrane order upon 1%–2% 1-butanol exposure. GP values declined progressively from control to 2% treatment, reflecting increased membrane fluidity …

Acoustofluidic Delivery Of Biomolecules To Cells: A Novel Approach For Preparing Car-T Cells., Madison C. Colebank

Electronic Theses and Dissertations

The continuous pursuit of effective methods for gene transfection in primary human T cells is crucial for advancing cell-based therapies. Viral vectors, currently used in cell-based therapies, involve a lengthy and costly process. This thesis explores the efficacy of coupling acoustofluidic and sonoporation techniques as an alternative non-viral gene transfection approach with primary T cells. Sonoporation, in conjunction with microbubbles, has been demonstrated to induce cavitation, resulting in the formation of pores in the cell membrane through which molecules can enter. Using flow cytometry, we compared the loading efficiency of primary T cells with that of Jurkat T cells to …

Air-Jet Bioprinting Of Stem Cell-Encapsulated Alginate Micro-Droplets For Organoid Biomanufacturing, Mirabella B. Stump

Electronic Theses and Dissertations

Biomanufactured 3D-organoids show promise for modeling and treating various conditions, particularly insulin-dependent diabetes mellitus (type-1 diabetes). Pancreatic islet organoids serve as sustainable sources of insulin-producing β-cells, potentially restoring insulin regulation in patients and providing a long-term solution for diabetes. Hydrogel droplet printing is an emerging technique in regenerative medicine due to its advantageous tunability and biocompatibility; however, challenges of consistency, quality control, and scalability persist. To address these issues, we developed a novel air-jetting based droplet bioprinting system for the generation of monodispersed alginate micro-droplets. The objective of this project was to incorporate human induced pluripotent stem cells (hiPSCs) into …

Development Of Air-Jetting System For Bioprinting Of Alginate Micro-Droplets, Nathan Lingenfelter

Electronic Theses and Dissertations

Transplantation of organoids has been demonstrated to be a promising cure to many diseases including Type-1 diabetes. However, achieving consistent and robust derivations of mature organoids from human pluripotent stem cells (hPSCs) is challenging. Cellular mechano-transduction, where mechanical stimuli is sensed and converted to biochemical signaling, has been proposed to overcome this challenge. This pathway relies on encapsulating hPSCs in a small volume of a biomaterial such as alginate, which has been widely used as cell encapsulation materials due to its biocompatibility and selective permeability. In this project, we focused on developing an air-jetting based technique to generate small and …

Tumor Targeting Gold Nanoparticles For Delivery Of Rna And Dna Oligonucleotide Therapies For Glioblastoma., Nicholas Allen

Electronic Theses and Dissertations

Glioblastoma (GBM) brain tumors are highly aggressive gliomas due to genetic and cellular heterogeneity. Current GBM treatment consists of surgical resection of the tumor combined with radio- or chemo-therapies. While these treatments have increased the life expectancy for GBM patients up to 20 months, they have had little effect on the 5-year survival rate. The complex cellular and genetic composition of the tumor makes current treatments less effective long term. One approach to developing more effective GBM treatments is to customize nanoparticle-based drug delivery systems that can directly target the aberrant gene expression patterns within a particular GBM tumor. Delivery …

Metabolomic Differentiation Of Tumor Core And Edge In Glioma., Mary E. Baxter

Electronic Theses and Dissertations

Glioma is one of the most aggressive forms of brain cancer. It has been shown that the microenvironments differ significantly between the core and edge regions of glioma tumors. This study obtained metabolomic profiles of glioma core and edge regions using paired glioma core and edge tissue samples from 27 human patients. Data was acquired by performing liquid-liquid metabolite extraction and 2DLC-MS/MS on the tissue samples. In addition, a boosted generalized linear machine learning model was employed to predict the metabolomic profiles associated with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation.

A panel of 66 metabolites was found to be statistically significant …

Establishing The Efficacy Of Non-Cellular Components Of Adipose-Derived Stromal Vascular Fraction In Promoting Angiogenesis., Daniel Benson

Electronic Theses and Dissertations

Microvascular disease is hallmarked by pathophysiological conditions such as endothelial senescence, intimal thickening which impairs vasodilation, and regression of the capillary beds causing tissue ischemia in the myocardium or in peripheral vascular networks. Adipose-derived stromal vascular fraction (SVF) has previously demonstrated the ability to revascularize tissue. Increasing evidence shows that regenerative cells elicit their therapeutic benefit by paracrine mechanisms, leaving open extracellular vesicles (EVs) as a potential crux of the cell therapy paradigm. To test this idea, three types of gelatin methacrylate hydrogels were employed: SVF gels, EV gels derived from SVF, and blank control gels, which were used in-vitro …

Labeling Melanoma Cells With Black Microspheres For Improved Sensitivity In Detection Via Photoacoustic Flow Cytometry, Tori Kocsis

Electronic Theses and Dissertations

Melanoma is an aggressive form of skin cancer known for developing into metastatic disease. Current clinical diagnostics, including medical imaging and tissue biopsy, provide a poor prognosis since the cancer is in the late stages of disease progression. In recent years, photoacoustic flow cytometry has allowed for the detection of circulating melanoma cells within patient blood samples in vitro. Although this method exploits the naturally-produced melanin within the cells, it has only successfully detected highly-pigmented melanoma cell lines. Since various forms of melanoma exist, each with varying melanin concentrations, this research aims to provide a novel method for detecting lightly-pigmented …

A Microfluidic Assay For Single Cell Bacterial Adhesion Studies Under Shear Stress, Amanda Trusiak

Electronic Theses and Dissertations

The study of bacterial adhesion to host cells is important in understanding bacterial pathogenesis and developing new therapeutic approaches. Here, we studied bacterial adhesion under shear stress using a novel microfluidic method. Specifically, the adhesion of a uropathogenic E. coli strain (FimHOn, ATCC 700928/CFT073) to mannose-modified substrates was studied under flow conditions. The FimHOn E. coli strain expresses FimH which is a mannose-specific adhesin found on the fimbriae that binds to glycoproteins on the epithelium. We developed a microfluidic method that mimics bacterial adhesion to urothelial cells. First, the microfluidic channels were modified by sequentially adsorbing BSA-mannose and BSA onto …

Investigating The Effect Of Dissolved Oxygen-Assisted Corneal Cross-Linking (Cxl) On Porcine Corneas, Julianni Dar

Electronic Theses and Dissertations

Corneal cross-linking is a clinical procedure that is known to stop the progression of keratoconus, an eye disease that affects the cornea’s structure, ultimately leading to vision loss in its advanced stages. The typical treatment plan includes riboflavin and UV-A exposure in the hope to increase the mechanical properties of the cornea. There are two types of CXL pathways, with Type-II CXL requiring oxygen. Naturally, the dissolved oxygen is limited in the cornea; therefore, limiting the effect of Type-II CXL. This study proposes to improve the Type-II CXL contribution by integrating dissolved oxygen during the standard CXL treatment used in …

Characterization Of Fundamental Parameters That Modulate Acoustofluidic-Mediated Molecular Delivery To Cells., Connor Sterling Centner

Electronic Theses and Dissertations

Cell-based therapies represent the latest biotechnological revolution in medicine, but significant limitations exist in non-viral intracellular delivery techniques during manufacturing of cell therapies. To address these limitations, a novel acoustofluidic device was developed to deliver ultrasound waves into a flow chamber for rapid molecular delivery to human cells. Our acoustofluidic device delivers biomolecules by inducing expansion and collapse of exogenous gas-filled microbubbles (“cavitation”), which enhances molecular delivery to nearby cells. Experimental studies were conducted to characterize key parameters that influence acoustofluidic-mediated molecular delivery to human cells. A clinical ultrasound transducer was generally utilized with varying cationic microbubble concentrations. Intracellular delivery …

Diagnosis Of Melanoma Disease State From Patient Blood Samples Using Photoacoustic Flow Cytometry, Jacob Salvatore

Electronic Theses and Dissertations

Approximately 1 in 50 Americans develop melanoma in their lifetime. Early detection of melanoma is pivotal to the survival of the patient, with a 99% survival rate for 5 or more years after an early diagnosis. Metastasis, or the spread of cancer, increases the chances of cancerous growth in other parts of the body. In this study, we propose the use of photoacoustic flow cytometry as a diagnostic of patient disease state, by closely monitoring the amount of circulating melanoma cells (CMCs) in a patient’s blood before and after chemotherapy treatments. Using this patient data, we were able to make …

Development And Applications Of In-Vitro And In-Silico Models Of The Cardiovascular System To Study The Effects Of Mechanical Circulatory Support., Moustafa Meki

Electronic Theses and Dissertations

Cardiovascular diseases (CVDs) are the leading cause of mortality globally. With ongoing interest in CVDs treatment, preclinical models for drug/therapeutic development that allow for fast iterative research are needed. Owing to the inherent complexity of the cardiovascular system, current in-vitro models of the cardiovascular system fail to replicate many of the physiological aspects of the cardiovascular system. In this dissertation, the main concern is with heart failure (HF). In advanced HF, patients may receive Left Ventricular Assist Devices (LVADs) as a bridge to transplant or destination therapy. However, LVADs have many limitations, including inability to adapt to varying tissue demand …

Biomechanical Investigation Of Plant Vascular Tissue For Bio-Inspired Design And Flexible Composites, Mukesh Roy

Electronic Theses and Dissertations

Since the inception of evolution, nature has continuously optimized all the natural materials. These optimizations are so efficient that scientists and engineers have started taking inspiration from nature. Such inspiration-based solutions provide sustainable development of new materials with enhanced structural properties and functionalities (such as stiffness, stability, toughness, self-healing etc.). This variety of properties and functionalities is mainly due to the hierarchical organization of such components. Abstraction of the hidden cues of the hierarchical organization, principles and underlying structure-function relations are some of the critical approaches of bioinspired materials design. Inspiration from animals and plants has helped develop the design …

Bacteria Analysis By Using A Supervised Machine Learning Algorithm Based On Droplet Microfluidics, Yulder Daniel Angarita

Electronic Theses and Dissertations

Sepsis is a major medical problem and massive resources have been invested in developing and evaluating alternative treatments. Statistics indicate that sepsis causes between one third and one half of all hospital deaths in the United States. Sepsis has a high impact on health care in the US, with direct sepsis costs in 2009 exceeding $15.4 billion. A research study found that a 1-hour delay in appropriate antimicrobial care resulted in a 7% - 10% rise in mortality. Several professional societies seek to reduce sepsis mortality by targeting the timely use of diagnostic tests and antimicrobial therapy. The diagnostic instruments …

Pilot Study Exploring The Effect Of Targeted Cox-2 Inhibition In Macrophages Responding To Neuronal Injury; Promoting Enhanced Axonal Regeneration, Alyssa Brauckmann

Electronic Theses and Dissertations

Celecoxib nanoemulsion (CXB-NE) has been developed as a macrophage targeted analgesics by Dr. Janjic and her team at Duquesne University, (Janjic et al, 2018; Liu et al, 2020; Saleem et al, 2019b; Vasudeva et al, 2014). The CXB-NE nanoemulsion carrying a Nonsteroidal Anti-inflammatory (NSAID) inhibitor of COX-2 activity result in a reduction in PGE2 expression in macrophages. Using CXB-NE in rats that have peripheral nerve injury constricting the sciatic nerve relieves hypersensitivity, a pain-like behavior. The treatment also decreases inflammation associated with this chronic constriction injury (Janjic et al, 2018; Saleem et al, 2019b; Stevens et al, 2019). In this …

Investigation Of Cardiovascular Tissue Following 56fe-Radiation And Potential Countermeasure Effectiveness., Alexandra R Decarlo

Electronic Theses and Dissertations

Exposure to ionizing radiation is an important medical concern because it can lead to health problems including cancer and cardiovascular issues. In space, astronauts are exposed to ionizing radiation that is not experienced by those who remain on Earth. This radiation can cause health problems such as cardiovascular disease. One way this can happen is through the creation of reactive oxygen species, which can activate TGF-β1, contributing to fibrosis or other cardiovascular problems. Antioxidants can be a potential pharmacological mitigator of these excess reactive oxygen species because of their ability to neutralize reactive oxygen species. One such antioxidant with potential …

Plga-Modified Nanoparticles For The Treatment Of Hypo-Vascularized Hpv-Related Cervical Cancers., Lee B. Sims

Electronic Theses and Dissertations

A major challenge associated with delivery of active agents in the female reproductive tract (FRT) is the ability of agents to efficiently diffuse through the cervicovaginal mucosa (CVM) and reach the underlying sub-epithelial immune cell layer and vasculature. A variety of drug delivery vehicles have been employed to improve the delivery of agents across the CVM and offer the capability to increase the longevity and retention of active agents to treat infections of the female reproductive tract. Nanoparticles (NPs) have been shown to improve retention, diffusion, and cell-specific targeting via specific surface modifications, relative to other delivery platforms. In particular, …

Restoring Cholesterol Homeostasis In Familial Hypercholesterolemia Cell Line Model, Lubna Hindi

Electronic Theses and Dissertations

Familial hypercholesterolemia (FH) is an autosomal dominant disorder that results in elevated levels of low-density lipoprotein cholesterol (LDL-C). This increase in serum cholesterol level has been shown to result in premature coronary artery disease (CAD) with devastating symptoms from a young age. Today, the prevalence of heterozygous FH (HeFH) and homozygous FH (HoFH) is estimated to be 1 in 320 and 1 in 160,000 people, respectively. FH is referred to as an underdiagnosed disease due to the large number of mutations that continues to grow. These mutations often exist in one of or a combination of three genes: low-density lipoprotein …

Development And Evaluation Of A Biocompatible Electroactive Sensor For Continuous Blood Pressure Measurement., Scott D. Cambron

Electronic Theses and Dissertations

Piezo-active composites have been implemented for sensing and transduction for decades. The 0-3 ceramic/polymer composite is one of the most common composite types used for sensing applications, owing to their tailorable properties of the two-phase composition, consisting of a three-dimensionally connected polymer/rubber matrix (inactive phase) with a dispersion of isolated piezo-ceramic particles (active phase). This thesis describes a method to develop novel biocompatible perivascular band comprised of a two-phase piezo-active composite to be fabricated using simple manufacturing processes. Biomaterials such as tissue scaffolds comprised of silk fibroin (SF) and chitosan (CS), and biocompatible soft rubbers will be implemented as the …

Generation Of Recombinant Mouse Embryonic Stem Cell Lines And Theirapplication For In Vivo Bioluminiscence Imaging In The Heart, Ramana Kammili

Electronic Theses and Dissertations

Cardiovascular disease is the major cause of death in the United States, with 80 million people suffering from some form of heart disease each year. One major limitation is the inability of the heart to repair the damaged tissue. Stem cell therapy holds enormous promise to repair and regenerate the damaged myocardium, but there are many technical difficulties that must first be overcome. One such difficulty is the present lack of ability to track and assess transplanted stem cells over time in vivo. The central hypothesis of this thesis is that in vivo bioluminescence imaging is a safe and useful …