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The Landscape of Epigenetic and Transcriptional Memory in Pancreatic Cancer Initiation and Progression
Get PDFPancreatic cancer is one of the deadliest malignancies in the United States. In the past several years, it has become clear that the epigenome plays a key role in pancreatic cancer development. However, DNA methylation, one of the key epigenetic modifications, has not been deeply characterized during pancreatic cancer initiation, progression, and metastasis. Thus, this thesis aims to investigate the epigenetic and corresponding transcriptional basis of pancreatic cancer development. In Chapter 1, we investigate the role of the epigenome and transcriptome in mediating a key cell fate change associated with pancreatic cancer initiation. We profile the DNA methylome and transcriptome in mouse models of this cell fate change and find a sustained DNA methylation signature associated with upregulation of oncogenic pathways central to pancreatic cancer. In Chapter 2, we take advantage of the epigenome as a record of cellular memory to investigate the cell-of-origin in human pancreatic cancer development. We examine the DNA methylome of normal cells, precursor lesions, and malignant lesions, which implicate acinar cells as the pancreatic cancer cell-of-origin. Finally in Chapter 3, we characterize the epigenome as a mediator of metastasis using mouse organoid models. We find that the DNA methylome distinguishes primary tumor from metastatic status even in the absence of genetic and transcriptomic differences
An investigation into the mitochondrial quality control functions of Thorase
Get PDFMitochondria play a key role in cell survival due to their importance as key site for ATP production. Maintaining healthy mitochondria is accomplished via multiple quality control mechanisms. One quality attribute to maintain is the proper and timely import of mitochondrial protein. The AAA+ ATPase, Thorase has been identified as a key regulator in maintaining proper import by dislodging protein stuck in the mitochondrial protein importers as well as redirecting mislocalized outer mitochondrial tail-anchored proteins. Separate research has identified Thorase as a key neuroprotective protein acting on synaptic transmission and nutrient dependent mTOR regulation. However, the neuronal specific implications of mitochondrial Thorase function warrant greater investigation. This is especially important given that neurons are highly sensitive to mitochondrial stress given their high energy demands and longevity.
In this investigation, the neuroprotective properties of Thorase are investigated with a focus on its mitochondrial function. Mitochondrial processes affected by Thorase are investigated. It is found that Thorase affects cytochrome c release as well as mitochondrial dynamics by dysregulating phosphorylation levels of DRP1 and MFF in HEK cells. A key finding in this investigation is that the effect of Thorase on cellular functions is highly dependent on the cell study being analyzed and on cell stress conditions introduced. The functionality of Thorase appears to be dependent on the needs of the cell.
A targeted investigation specifically into Thorase interplay with mitophagy regulators PINK1 and Parkin during PFF induced stress led to conclusion that Thorase is ubiquitinated by Parkin and that this ubiquitination regulates the ability of Thorase to promote mitochondrial quality. The results indicate that Thorase improves mitochondrial import efficiency likely through its interaction with TOM proteins. Parkin ubiquitination of Thorase is potentially leading to degradation of Thorase during PFF induced stress impeding the ability of Thorase to rescue mildly damaged mitochondria. Further investigations are needed to ascertain this effect
Effects of walls on turbulent flow: shear perturbations, momentum & vorticity cascades
Get PDFThe ubiquitous nature of wall-bounded turbulent flows in nature and engineering coupled with the breadth of dynamical phenomena involved, implies that researchers from diverse fields have made and continue to make important contributions to the collective knowledge on the subject. In this work, we draw heavily from some older works, like those by Ishihara, Lighthill, Huggins, and Onsager, and take advantage of recent developments in engineering, physics, and mathematics to study various aspects of wall-bounded turbulent flows. We propose a model for the second-order structure function tensor incorporating the anisotropic effect of mean shear as a linear perturbation and compare model predictions with data from channel flow and atmospheric boundary layers. While this model ignores the ‘blocking’ effects of the wall, our focus then shifts to the dynamics responsible for vorticity flux in turbulent channel flow and its relationship with total pressure drop, closely associated with said effects. The validity of Lighthill’s mechanism is investigated therein, with some supporting evidence being found. We document the presence and effect of upgradient ‘U-type’ eddies acting to accumulate vorticity at the wall and opposing downgradient `D-type’ eddies working to drive vorticity away from the wall. Our observations indicate that vorticity flux may also be interpreted as a cascade. We then derive an identity relating the flux of vorticity across a background of suitable potential flow to instantaneous total drag in turbulent flows in symmetric rough channels, known as the Josephson-Anderson relations, originating in the field of superconductivity, and first derived for classical fluids by Huggins. We also discuss other related approaches for calculating drag. Following the recent mathematical developments, we discuss the Onsager theory of wall-bounded turbulence analyzing the momentum dissipation anomaly hypothesized by Taylor. Diverging velocity gradients are eliminated by filtering small-scale motions and windowing out near-wall eddies, thereby introducing two regularization length scales. The regularized governing equations now have an inertial drag force in addition to the usual turbulent stress. This approach allows us to relate the power law variation with Re of skin friction to the power law mean streamwise velocity profile. We also suggest new approaches for large eddy simulation and modeling of sub-grid roughness effects
IMPROVING ALUMINUM ALLOY PERFORMANCE FOR STRUCTURAL AND REACTIVES APPLICATIONS THROUGH STRUCTURE-PROPERTY RELATIONSHIPS
No full textAluminum is one of the most globally ubiquitous materials, useful for structural, electronic, and reactive applications. Leveraging aluminum’s natural advantages requires thoroughly understanding structure-processing-property relationships and tailored alloy formulations. Conventional Al alloy formulations and processing approaches may not be optimized for novel application domains (bio- and chem-agent defeat) or manufacturing methods (Additive Manufacturing (AM)).
Though as-built AM AlSi10Mg includes performance-enhancing refined eutectic cells, these are invariably paired with performance-impairing melt pool boundaries, where the cell network coarsens and breaks down. Eliminating melt pool boundaries and their associated anisotropy motivates conventional heat treatments, but at the cost of removing the AM-specific eutectic cell strengthening. In Chapters 2 and 3, we compare conventional (T6- with a high-temperature solution heat treatment) and alternative (direct aging) thermal treatment impacts on microstructure, supersaturation, mechanical properties, fracture, strain-hardening, and thermal stability. Direct aging complements AlSi10Mg’s as-built microstructure, and achieves superior thermal stability and tensile performance by preserving the as-built cell network.
Retaining AlSi10Mg’s eutectic cells, and the associated melt pool (MP) microstructure, necessitates local characterization of MP impacts. MP center and boundary microstructures can vary significantly, but bulk characterization techniques often lack the resolution to quantify local MP boundary deformation. In Chapter 4, we use in-situ scanning electron microscopy (SEM) microtensile experiments on AlSi10Mg samples to compare the relative yield behavior and deformation between MP boundaries and centers. Differences in the local mechanical response – deformation, strain hardening, and yield – are reported. These differences are correlated to specific microstructural features like cell size, cell connectedness, cell eccentricity, and grain size.
Precision performance is critical for Al-based reactive materials for bio- and chem-agent defeat and neutralization. Effectively neutralizing stockpiles of these harmful agents (without disseminating them) depends on tailored ignition and combustion performance through strategic reactive alloy design. The prodigious alloy design space necessitates efficient, high-throughput experimental techniques for systematic screening. In Chapter 5, we demonstrate a novel combinatorial approach via physical vapor deposition of chemical gradients- to explore optimized reactive alloy compositions in Al-Zr and Al:8Mg-Zr. We characterize the impacts of trace Zr content on ignition and combustion using wire ignition and a novel combustion chamber
MODELING PROTEIN–CARBOHYDRATE COMPLEXES IN ROSETTA
Get PDFCarbohydrates are of fundamental importance in biology. These molecules are essential to life, serving to mediate diverse biological functions. Unraveling the biophysical mechanisms by which carbohydrates operate not only helps complete our understanding of life on Earth but enables rational engineering to fine-tune or even modify their roles in biology. However, carbohydrate molecules are complex, with their conformational diversity and chemical heterogeneity making it notoriously difficult to elucidate their structures experimentally. Yet these models are vital to our mechanistic understanding of carbohydrate-mediated biological functions, making scientific advancements challenging to achieve. Computational methods serve to fill this gap by generating native-like models of protein–carbohydrate systems.
In this dissertation, I describe my advancements to the field of computational modeling with the development of GlycanDock, a protein–carbohydrate docking refinement method in Rosetta. I detail the extensive benchmark I developed to evaluate the effectiveness of the GlycanDock protocol to generate native-like protein–carbohydrate models. Further, I provide residue-level analyses of these models to demonstrate the utility of the protocol toward developing a biophysical understanding of protein–carbohydrate complexes. Finally, I describe an approach utilizing GlycanDock and other computational tools to address the more realistic “blind” docking scenarios.
The development of GlycanDock enabled my work computationally modeling the structures of FpGalNAcDeAc and FpGalNase, two enzymes that together convert A-type blood to the universal O-type. I identified FpGalNAcDeAc residues likely to govern the binding of terminal LacNAc motifs present on the surface of red blood cells, offering mutational sites to modify targeting to the cell surface. Additionally, I identified the FpGalNAcDeAc binding site residues most important for A-antigen recognition, providing a guide to understanding and controlling its enzymatic activity. For FpGalNase, I proposed a sequence- and structure-driven hypothesis regarding its active-site and unique specificity to the terminal α-GalN carbohydrate. My work serves as a blueprint for future experimental studies, including rational engineering toward modifying FpGalNase’s specificity to the B-antigen, which, if achieved, would mean complete conversion of all A, B, and AB blood types to the universal O-type.
In sum, my work advanced our ability to model and dissect protein–carbohydrate systems
ELUCIDATING THE SEX-DEPENDENT REGULATION OF CARDIAC O-GLCNACYLATION DURING INJURY
Get PDFThe post-translational modification of intracellular proteins by monosaccharides of O-linked -N-acetylglucosamine (O-GlcNAc) has emerged as a critical regulator of cardiac function. Enhanced O-GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia-reperfusion (I/R) injury; however, the mechanisms underpinning O-GlcNAc-cycling in response to I/R injury have not been comprehensively assessed. While numerous approaches exist for the detection of O-GlcNAc in cells and tissues, immunoblotting using GlcNAc-specific antibodies is common. Therefore, my initial goal was to optimize the detection of O-GlcNAc in heart lysates. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, we demonstrated that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The aforementioned improvements in O-GlcNAc detection were accompanied by optimization of approaches for assessing the activity of the enzymes that cycle O-GlcNAc, the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), and detection of UDP-GlcNAc in the heart. Overall, the aforementioned techniques aimed to provide a comprehensive assessment of O-GlcNAc-cycling during heart injury.
As baseline heart physiology and pathophysiology are impacted by sex, we hypothesized that sex differences in molecular signaling may target protein O-GlcNAcylation basally and in ischemic hearts. To address this unprecedented question, male and female wild type murine hearts were subjected to ischemia or I/R injury and assessed for protein O-GlcNAcylation, abundance, and activity of OGT and OGA, abundance of GFAT2, the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP), and levels of UDP-GlcNAc, the product of the HBP and a substrate of OGT. Our data demonstrated elevated O-GlcNAcylation in female hearts both basally and during ischemia. Providing a mechanism for these observations, OGT activity was enhanced in females in all treatments. Ischemia also targets OGT, with reduced O-GlcNAcylation and OGT specific activity detected in this treatment.
Collectively, these findings enhance our understanding of molecular mechanisms regulating O-GlcNAcylation in the heart. They also propose the involvement of O-GlcNAc-mediated regulation in sex-dependent cardioprotection. Future investigations into the molecular mechanisms regulating the sex-dependent and injury-associated changes to OGT activity may aid in combating I/R injury in both males and females
The Reclassification of Latine English Language Learners
Get PDFEnglish language learners (ELLs) are the fastest-growing group of students in the United States. Most ELLs are also Latine and low-income. Research has suggested that Latine ELLs are less likely to reclassify or achieve English proficiency than students from other ethnic and linguistic backgrounds. This ethnic disparity is critical to understand as reclassification is positively associated with other consequential outcomes such as high school graduation and college enrollment. Furthermore, educators may struggle to enable Latine ELLs to attain adequate academic progress. A mixed-methods needs assessment study found that, for teachers of Latine ELLs in an urban school district, (a) performative cultural competency and racial bias can coexist, (b) ESOL certification increases self-efficacy for supporting ELLs, and (c) challenges persist in meeting Latine ELL students' unique academic needs in a system that was not created for them. These results inspired the creation of an applied project, an education podcast, that sought to enhance teacher self-efficacy by addressing deficit mindsets about Latine students and their families. The concepts of funds of knowledge, critical pedagogy, and transnational identity informed the podcast. Ideally, the podcast can be used as a teacher development tool to foster critical reflection and an understanding of the societal inequity impacting Latine students and families
SYNTHESIS AND REACTIVITY OF AZABORINE AND SILICON MOLECULES FOR POLYMERIC MATERIALS
No full textOrganometallics are promising synthetic targets due to their desirable electronic and synthetic properties relevant for materials applications. However, due to limitations in commercially available main group reagents, synthesizing polymers with non-carbon elements remains a challenge and therefore creative strategies are necessary. The Klausen group creates novel polymer architectures from organometallic building blocks, specifically silicon and boron. Targeted synthetic strategies and investigations on azaborine (BN) and strained silicon ring reactivities are detailed in this dissertation. BN for CC bond substitution affects the stability of benzylic ions and impacts BN 2-vinylnaphthalene (BN2VN) reactivity toward ionic polymerization. BN2VN’s ionic reactivities demonstrate the effect of resonance in main group aromatics. Ring strain is a valuable tool used by chemists in multiple avenues, especially organic, bioorthogonal, and polymer chemistry. Silicon bond incorporation impacts ring strain due to the length and flexibility of the silicon-silicon bond. Synthetic strategy can be used to synthesize novel silacycloheptenes and silacycloheptyne. Silane polymers made from ring-opening metathesis polymerization (ROMP) allow comparisons of force-responsive behavior examined by single-molecule force spectroscopy. Silacycloheptyne introduces electron donors into a strained alkyne cycle that influence the efficiency of strain-promoted azide-alkyne click chemistry (SPAAC). Overall, the diversity of the science presented emphasizes the value of understanding molecular properties in strategic polymer design
Development of Minimally Invasive Devices and Technologies for Open-Angle Glaucoma Treatment
Get PDFPrimary open-angle glaucoma is a progressive disease affecting nearly 60 million people and is the leading cause of blindness worldwide. People with glaucoma may not notice symptoms or feel damage as it occurs because one of the only major risks and causative factors of the disease is elevated intraocular pressure (IOP). While not part of the clinical definition of glaucoma, prior clinical trials have shown that lowering IOP slows its progression. Therefore, current approaches for treating glaucoma are aimed at reducing IOP by pharmacological or surgical means; however, these methods provide insufficient long-term efficacy in IOP management.
The aim of this work is to develop and demonstrate novel and efficacious therapies for lowering IOP that could be applied under minimally invasive settings. First, I present a semi-permanent, implantable transcorneal duct capable of creating a direct interface between the anterior chamber of the eye and the external environment for aqueous humor outflow. I will discuss the fluid mechanics behind designing and implementing a filter material, which can prevent the ingress of pathogens while modifying aqueous humor outflow resistances to pre-glaucomatous levels, as well as results from its surgical implantation into the eyes of New Zealand White rabbits. Second, I introduce a parylene-based microbore glaucoma drainage device capable of modulating IOP via a minimally invasive implantation procedure and the results from a 6-week study in five healthy New Zealand White rabbits to assess device efficacy in lowering and maintaining IOP. Finally, I introduce my work on an electroceutical therapy approach using a flexible thin-film transistor application specific integrated circuit (ASIC). I validate its capabilities at delivering a range of stimulation parameters through both benchtop and in vivo testing, as well as demonstrate and discuss its potential to be integrated onto a wearable contact lens in the future. Through these technologies, my research demonstrates several promising platforms for lowering and controlling IOP without the shortcomings of current treatments
INTERPLAY OF FIBROBLASTS AND ORGANOIDS: INSIGHTS INTO AGING DYNAMICS AND COLORECTAL CANCER PROGRESSION
Get PDFThe organoid model is a useful tool for modeling the cellular microenvironment of the organ from which it is derived. Organoids recapitulate the self-organization of heterogenous cell types and the microenvironment. Little is known about the interactions between fibroblasts and colon organoids. In order to study this interaction, we cultured organoids in conditioned medium collected from colon derived fibroblasts. Also, since aging is an important risk factor for the development of CRC, aged and young fibroblasts as well as organoids were compared. Additionally, growth characteristics of BRAFV600E organoids with p16 KO, CDX2 KO, DKO, and EV were compared. We found that one week of exposure to colon derived fibroblast conditioned medium was not sufficient to change the methylation state of the organoids. We also developed an algorithm to study the growth characteristics of mouse derived intestinal organoids. We also report that p16 KO significantly alters the morphology of aged organoids in the presence of BRAFV600E