Characterizing microRNA regulators of lung disease

Lung diseases are one of the leading causes of mortality and morbidity worldwide. Understanding these diseases at a molecular level remains a critical component to developing effective therapeutics. Previous work has shown that gene expression alterations play an important role in disease initiation, maintenance, and progression as well as serve as diagnostic tools in disease. However, much remains to be uncovered regarding the role that microRNAs play in both healthy and diseased lung tissue. This thesis seeks to utilize methods of bioinformatics, cell biology, and molecular biology to examine the effect of miR-4423 on lung epithelial cell differentiation (Aim 1), miR-424 on never smoker derived lung adenocarcinoma (Aim 2), and miR-34c isomiRs in interstitial lung disease (ILD) (Aim 3). First, we examined the role of miR-4423 in lung mucociliary epithelium by employing the use of an air-liquid interface culture system, finding miR-4423 has an effect in ciliated cell differentiation and that a loss of miR-4423 is associated with cancer progression. These findings suggest that miR-4423’s actions in airway epithelium differentiation may potentially provide a therapeutic role in lung cancer. Next, we validated transcriptomic differences between lung tumor tissues resected from never and ever smokers. Specifically, miR-424, a predicted regulator of a large number of gene expression changes in never smoker lung adenocarcinoma, was found to regulate cell migration, potentially identifying a novel target and/or pathway for therapeutic action. Lastly, the function of microRNA isomiRs is relatively unknown. We validated miR-34c as upregulated in ILD and modulated both miR-34c and a miR-34c 5’ isomiR in lung relevant cell lines to explore their differing biological roles. We found that they are capable of targeting differing mRNA, indicating an independent role for isomiRs in disease. The studies contained in this dissertation offer valuable insight into the biology of microRNAs in the lung and how they might be employed as therapeutic targets for a number of common lung diseases. In addition, biological insights into the complexity of microRNAs in the lung highlight the need to better understand diseases influenced by microRNA expression and microRNA variants in regards to actionable therapeutics.2017-12-01T00:00:00

MECHANISM OF ASSEMBLY OF ALIGNED EXTRACELLULAR MATRIX FIBRILS

The extracellular matrix (ECM) is a complex three-dimensional network consisting of secreted extracellular macromolecules consisting of fibrous proteins, proteoglycans, and glycoproteins, that provides essential structural support and stability to cells and tissues, as well as regulates vital cell behaviors, such migration, differentiation, and cell division. The physical structure of the ECM is tissue-specific and fundamental to normal tissue function. Parallel alignment of ECM fibers is crucial for the function of a variety of tissues, such as the cornea, tendon, and vasculature. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for the formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils. Microscopic analysis of matrix assembly sites, where FN fibrillogenesis is initiated, revealed that these complexes were oriented in parallel at the cell poles. Utilizing live-cell imaging, we found that these polarized matrix assembly sites progressed into fibrillar adhesions and ultimately into aligned FN fibrils. Using cells that assemble an unaligned, meshwork matrix, we demonstrate that the distribution and orientation of matrix assembly sites can be controlled through micropatterning or mechanical stretch. While elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked since we discovered that transforming growth factor beta (TGF-B1) signals enhance matrix assembly site polarity and assembly of aligned fibrils independently of cell elongation. Our work has revealed that the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites which form during the initial stages of cell-FN interactions and that mechanisms of aligned fibril assembly can be independent of cell elongation. We subsequently utilize our insights into cell and matrix organization in a tissue regeneration application, developing a novel multi-layered scaffold with distinct fiber organizations to recreate smooth muscle and connective tissue layers

“Ha ha ha, very funny”: An Ethnographic Study of Conversational Humor among College Students

In this paper we take up the topic of conversational humor by analyzing instances of teasing, joking, irony and banter in everyday settings in which these integral forms of talk play a part. This paper comes from an ethnographic project that was part of a class in language and culture, taught at a Catholic liberal arts college in a midsized city in Montana. In this exploration of conversational humor among college students – as they interacted for the most part in small groups of friends and acquaintances – we focus on interpretive and functional aspects of conversational humor. Through examining instances of conversational humor, we establish the careful collaboration among participants that maintaining a play frame entails. We also show that participants’ collaboration in seemingly superficial episodes of conversational humor accomplishes a host of relational tasks. In the following sections we draw upon areas of an immense and complex body of scholarship on conversational humor. Then, after explaining our method of gathering and analyzing data, we elaborate three themes we found upon examining more than 140 recorded instances of conversational humor

A multilayered scaffold for regeneration of smooth muscle and connective tissue layers

Tissue regeneration often requires recruitment of different cell types and rebuilding of two or more tissue layers to restore function. Here, we describe the creation of a novel multilayered scaffold with distinct fiber organizations—aligned to unaligned and dense to porous—to template common architectures found in adjacent tissue layers. Electrospun scaffolds were fabricated using a biodegradable, tyrosine-derived terpolymer, yielding densely-packed, aligned fibers that transition into randomly-oriented fibers of increasing diameter and porosity. We demonstrate that differently-oriented scaffold fibers direct cell and extracellular matrix (ECM) organization, and that scaffold fibers and ECM protein networks are maintained after decellularization. Smooth muscle and connective tissue layers are frequently adjacent in vivo; we show that within a single scaffold, the architecture supports alignment of contractile smooth muscle cells and deposition by fibroblasts of a meshwork of ECM fibrils. We rolled a flat scaffold into a tubular construct and, after culture, showed cell viability, orientation, and tissue-specific protein expression in the tube were similar to the flat-sheet scaffold. This scaffold design not only has translational potential for reparation of flat and tubular tissue layers but can also be customized for alternative applications by introducing two or more cell types in different combinations

Proteomic Analysis, Immune Dysregulation, and Pathway Interconnections with Obesity

Proteomic studies can offer information on hundreds to thousands of proteins and potentially provide researchers with a comprehensive understanding of signaling response during stress and disease. Large data sets, such as those obtained in high-dimensional proteomic studies, can be leveraged for pathway analysis to discover or describe the biological implications of clinical disease states. Obesity is a worldwide epidemic that is considered a risk factor for numerous other diseases. We performed analysis on plasma proteomic data from 3 separate sample sets of postmenopausal women to identify the pathways that are altered in subjects with a high body mass index (BMI) compared to normal BMI. We found many pathways consistently and significantly associated with inflammation dysregulated in plasma from obese/overweight subjects compared to plasma from normal BMI subjects. These pathways indicate alterations of soluble inflammatory regulators, cellular stress, and metabolic dysregulation. Our results highlight the importance of high-dimensional pathway analysis in complex diseases as well as provide information on the interconnections between pathways that are dysregulated with obesity. Specifically, overlap of obesity related pathways with those activated during cancer and infection could help describe why obesity is a risk factor for disease and help devise treatment options that mitigate its effect

Augmenting the antinociceptive effects of nicotinic acetylcholine receptor activity through lynx1 modulation.

Neuronal nicotinic acetylcholine receptors (nAChRs) of the cholinergic system have been linked to antinociception, and therefore could be an alternative target for pain alleviation. nAChR activity has been shown to be regulated by the nicotinic modulator, lynx1, which forms stable complexes with nAChRs and has a negative allosteric action on their function. The objective in this study was to investigate the contribution of lynx1 to nicotine-mediated antinociception. Lynx1 contribution was investigated by mRNA expression analysis and electrophysiological responses to nicotine in the dorsal raphe nucleus (DRN), a part of the pain signaling pathway. In vivo antinociception was investigated in a test of nociception, the hot-plate analgesia assay with behavioral pharmacology. Lynx1/α4β2 nAChR interactions were investigated using molecular dynamics computational modeling. Nicotine evoked responses in serotonergic and GABAergic neurons in the DRN are augmented in slices lacking lynx1 (lynx1KO). The antinociceptive effect of nicotine and epibatidine is enhanced in lynx1KO mice and blocked by mecamylamine and DHβE. Computer simulations predict preferential binding affinity of lynx1 to the α:α interface that exists in the stoichiometry of the low sensitivity (α4)3(β2)2 nAChRs. Taken together, these data point to a role of lynx1 in mediating pain signaling in the DRN through preferential affinity to the low sensitivity α4β2 nAChRs. This study suggests that lynx1 is a possible alternative avenue for nociceptive modulation outside of opioid-based strategies

Overall architecture of lynx1 at α4:α4 nAChR interface.

<p>Molecular dynamic simulations of lynx1 binding to α4 nAChR subunits. The cell membrane is represented as a dashed line.</p

Mediation of <i>lynx1</i> through α4β2 nAChRs.

<p>(A) Antinociceptive responses in wt and <i>lynx1</i>KO mice after I.P. injection of the non-selective α4β2* nAChR agonist, epibatidine (5 μg·kg^-1) (n = 24 wt, 21 KO, p = 0.029, Student’s T-test). Mice were tested on the hot-plate 15 minutes after injection. Epibatidine-mediated antinociception is augmented in <i>lynx1</i>KO mice compared to wt mice. Data presented as mean ± SEM time. *P<0.05 compared to wt controls. wt: wild type, KO: <i>lynx1</i> knockout. (B) Antinociceptive responses in wt and <i>lynx1</i>KO mice after I.P. injection of the α4β2 nAChRs inhibitor dihydro-β-erythroidine hydrobromide (DHβE) (3.0 mg·kg^-1) and nicotine (0.5 mg·kg^-1) (nicotine treated <i>lynx1</i>KO mice (n = 8) vs. nicotine+DHβE treated <i>lynx1</i>KO mice (n = 6) using the hot-plate assay. Mice were injected with DHβE 25 minutes and nicotine 15 minutes prior to hot-plate testing. Injections of DHβE blocks the antinociceptive effect of nicotine in <i>lynx1</i>KO mice. Data indicates that lynx1 operates through the α4β2 nAChR to modulate antinociception. Data presented as mean ± SEM time. wt: wild type, KO: <i>lynx1</i> knockout. (C) Schematic of lynx1 binding to the LS stoichiometry of α4β2 nAChRs preferentially over the HS stoichiometry. α4β2 nAChR pentamers shown in the high sensitivity (HS) and low sensitivity (LS) stoichiometry, made up of (α4)₂(β2)₃ vs. (α4)₂(β2)₃ nAChRs respectively. In our model, lynx1 preferentially binds and stabilizes the LS stoichiometry.</p