Biochemical and Biomolecular Engineering Commons^™

Purifying Circular Rna Therapeutics, Karen Guillen-Cuevas

All Dissertations

Circular RNA (circRNA) has emerged as a promising therapeutic modality due to its enhanced stability and resistance to exonuclease degradation compared with linear mRNA. However, its stability and functionality depend strongly on achieving high purity, and current methods such as size-exclusion chromatography suffer from peak overlap, low yields, and poor scalability. This dissertation addresses these limitations by developing ultrafiltration-based strategies to purify circRNA directly from in vitro transcription (IVT) and self-splicing (circularization) reactions. First, the feasibility of ultrafiltration was demonstrated using polyethersulfone membranes, where sieving coefficients and critical flux values of circRNA, precursor RNA, and nicked RNA were measured to …

Development Of Translational Microscale Systems To Interrogate How Biophysical And Biochemical Cues Alter The Phenotype Of Hormone Positive Breast Cancer, Braulio Ortega Quesada

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Cancer is a complicated disease and one in every three diagnoses are women with breast cancer. Recent research shows that the progression of cancer depends strongly on interactions with other non-cancerous cells and non-cellular components. Metastasis, the spread of cancer to other parts of the body, is a key step in cancer progression and makes treatment harder, leading to poor response to treatments culminating in death in ~90% of patients. This dissertation focused on creating a series of microdevices as novel pre-clinical models of breast cancer. Two different devices were fabricated to (1) study how exposing cancer cells to the …

Magnetic Resonance Imaging Trackable Pathology-Triggered Polymersomes Toward Enzyme Replacement Theranostics For Gm1 Gangliosidosis, Dorian Foster

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This dissertation investigates the development and optimization of pathology-responsive polymersomes for simultaneous enhanced magnetic resonance imaging (MRI) and enzyme replacement therapy (ERT) in the treatment of GM1 gangliosidosis (GM1). While therapeutic strategies exist for similar, non-neuropathic lysosomal storage disorders, implementation towards GM1 faces significant challenges due to the blood-brain barrier.

Herein, we developed and characterized a range of low molecular weight hyaluronic acid-b-polylactic acid (HA-PLA) polymersomes (PSs) capable of encapsulating therapeutic enzymes in their hydrophilic core. These polymersomes exhibit pathophysiology-triggered responsiveness, degrading in the presence of acidic conditions, which is characteristic of lysosomes, and enzymes such as hexosaminidase A, which …

Fabrication And Characterization Of Lignin–Pva Hydrogels With Tunable Network Structures, Keturah Bethel

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The ability to directly tune the crosslinked network structure of hydrogels is crucial for their functional applications in various fields, such as water filtration, protein separation, and tissue engineering. By controlling the crosslink density of the hydrogel, one can directly alter the mesh size – i.e., the end-to-end distance between crosslink junctions – and, subsequently, directly alter the hydrogel performance. This work discusses the fabrication and characterization of soft composites containing the biopolymer, lignin, are discussed. Precisely, physically-crosslinked composite lignin–Poly(vinyl alcohol) (PVA) hydrogels were fabricated via the freeze-thaw (F/T) pathway, whereby solutions containing specified amounts of PVA and lignin were …

Fundamental Study Of Nonlinear Electrophoresis, Joseph Armakan Bentor

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Classical electrophoretic theory describes the linear response of particles suspended in Newtonian fluids under weak electric fields. When the electric field exceeds this weak limit, a nonlinear response arises due to non-uniform surface conduction over the curved particles. This nonlinear behavior introduces dependencies on fluid and particle properties not considered in the classical theory, offering potential advantages for microfluidic particle manipulation. While this phenomenon has been extensively studied theoretically and numerically in Newtonian fluids, experimental investigations have been relatively limited.

Fluid rheology can significantly alter particle dynamics under applied electric fields. Rheological properties like shear-thinning and elasticity which are not …

Elucidating The Mechanical And Transport Properties Of Lignin-Based Hydrogel Composites, Nicholas Gregorich

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The use of lignin in the fabrication of soft composites has become an emerging area of research in polymer science and polymer chemistry. These lignin-based materials present numerous benefits, notably, a reduction in the use of petroleum-based precursor, improved structural benefits to otherwise soft host polymers, as well as the inherent antimicrobial and antioxidant properties of lignin, making it suitable for biomaterials. Herein, we present two chemical reaction pathways of incorporating lignin that was fractionated and cleaned using the Aqueous Lignin Purification with Hot Agents (ALPHA) process into poly(vinyl alcohol) (PVA) hydrogel composites for aqueous-based separations. By leveraging the ALPHA …

Fluorescence Multiplexing With Combination Probes For Biological And Diagnostic Applications, Madeline Mccarthy

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Cancer refers to a group of diseases containing more than 200 different subtypes. Cancer is a heterogeneous disease by nature, meaning that there are differences among tumors of the same type in different patients, and there are differences among cancer cells within a single tumor of one patient. Since cancer is not a single disease, nor does it have a single cause, it proves to be incredibly hard to diagnose and treat. The ability to study cellular markers, cell and tissue spatial arrangement, and gene function are all integral parts of cancer diagnostic and treatment efforts.

Here, I first present …

Metabolic Engineering Of Yarrowia Lipolytica For Enhanced Peroxisomal Production Of Polyhydroxyalkanoates, Michael Spagnuolo

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Traditional chemical processes are made inefficient by the generation of side products and reactions that fail to reach completion. Bioprocesses, on the other hand, lose product potential due to the necessity of growing the organism required to produce the desired compounds. The field of metabolic engineering often seeks to alter this balance between product formation and cell growth to generate more product from the same quantity of feed (reagent). In addition to balance, such organisms may also be engineered to produce the desired products from nontraditional substrates, such as waste compounds. In resource-poor environments, the ability to produce a wanted …

Development Of Enzyme-Responsive Polymersomes As A Drug Delivery System For Gm1 Gangliosidosis, Bipin Chakravarthy Paruchuri

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GM1 gangliosidosis is a lysosomal storage disorder caused by deficiency of β-galactosidase (βgal) and subsequent accumulation of GM1 ganglioside in lysosomes. The rare nature of this disorder presents challenges with prognosis and treatment. Enzyme replacement therapy (ERT) can effectively treat systemic deficiencies, but the intravenously administered enzyme cannot cross the blood-brain barrier (BBB) to treat the central nervous system. A delivery system to encapsulate and deliver the enzyme can facilitate enzyme transport across the BBB with suitable surface modifications. One of the pathological aspects of GM1-affected cells is the upregulation of the lysosomal enzyme hexosaminidase A (HexA). In this dissertation, …

Profiling And Engineering The Unfolded Protein Response In Therapeutic Protein-Producing Cho Cell Lines, Dyllan Rives

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Industrial Chinese hamster ovary (CHO) production of therapeutics requires cell lines with high secretory capacity to avoid an accumulation of improperly folded proteins, or endoplasmic reticulum (ER) stress. This presents a challenging engineering bottleneck. The unfolded protein response (UPR) is initiated to overcome ER stress and reestablish homeostasis. In this dissertation, the impacts of ER stress and the UPR on protein production in mammalian cells are detailed, and both selection- and rational-based strategies for enhancing the ER stress response in productive CHO cell lines are reviewed. This project aims to expand on recent research efforts for engineering ER stress-related responses …

Effects Of Continuous In Situ Low-Dose Ionizing Radiation On Microorganisms, Molly E. Wintenberg

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Precise detection and monitoring of nuclear fuel cycle, enrichment, and weapon development activities are critical for supporting warfighter preparation in chemical, biological, radiological, nuclear, and explosives (CBRNE) operations, clandestine activities, and nuclear compliance. A biological sensing system could serve as an alternative to traditional detection methods by using organic material naturally present in the environment to discreetly detect residual trace nuclear material. Microorganisms provide an optimal platform for an alternative sensing system; however, their response to low levels of ionizing radiation is poorly characterized. Combining the power of next-generation sequencing and transcriptomic analysis, this dissertation takes an approach to obtain …

Towards Understanding Protein Immobilization Rules Through Site-Specific Covalent Immobilization Of T4 Lysozyme, Maxwell Hilbert

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Soluble enzymes have the unique ability to assist the conversion of complex substrates in fewer steps compared to conventional chemical synthesis; however, when immobilized to a surface, the insoluble enzyme's activity and stability can suffer in its new environment. To improve upon future immobilized enzyme efficacy, a fundamental understanding of the interactions across the enzyme, linker, and surface during covalent, site-specific immobilization that can be detrimental to its activity is required. More specifically, using a well-characterized and model enzyme, T4 lysozyme (T4L), 28 different cysteine attachment points' effects on site-specifically immobilized activity are examined. Different combinations of heterobifunctional amine-to-sulfhydryl crosslinkers …