18525 research outputs found
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Fabrication of Endothelialized Capillary-like Microchannel Networks using Sacrificial Thermoresponsive Microfiber Templates
No full textIn general, cells within living tissue can only survive a few hundred microns away from a source of vital gases and nutrients: within this range the cells’ metabolic needs are met effectively by diffusion. Beyond this range cells begin to starve, waste products build up, and the tissue becomes necrotic. This has traditionally limited development of engineered tissues to geometries where the thickest dimension does not exceed the diffusion limit, such as with organoids and thin-walled organs. In order to, develop a greater diversity of artificial tissues and expand their thickness beyond a few hundred microns, a microvascular network must be engineered to carry nutrients throughout, replicating the scale, architecture, and function of capillary beds responsible for tissue support. Current methods of microvascular engineering are able to produce vessels with a high degree of precision and programmability, such as with bioprinting and optical techniques, but fall short when attempting to produce scalable, organic vessel networks in a three-dimensional context. Here we demonstrate a top-down method of patterning microvessels into hydrogels using sacrificial templates formed from thermoresponsive microfibers whose size and architecture approach those of natural capillaries. Artificial microvessels produced by this method were imaged and quantitatively characterized in order to compare against both human capillaries and murine capillaries, which were analysed in an identical manner. Within the resulting microchannels, we cultured endothelial monolayers that remain viable for over three weeks, covered channels smaller than 13 µm in diameter, and exhibited functional barrier properties. Additionally, we cultured endothelialized microchannels within hydrogels containing fibroblasts and characterized the viability of the co-cultures to demonstrate this approach’s potential when applied to cell-laden hydrogels. This method represents a step forward in the evolution of artificial tissues and a path towards producing viable capillary-scale microvasculature for engineered organs
Manganese Exposure Disrupts Glutamatergic Function and Alters Electroencephalogram Phenotypes: Relevance to Alzheimer’s Disease
No full textAlzheimer’s disease (AD) onset and progression can be influenced by modifiable risk factors such as the exposure to neurotoxic metals including manganese (Mn). Mn is an essential metal that serves as a cofactor for metalloenzymes important in modulating glutamatergic function and has been shown to directly impact glutamate clearance when present in excess. Glutamate dyshomeostasis is also observed in AD, with decreased expression in astrocytic glutamate reuptake transporters (GLT-1 and GLAST) being reported in brain tissue and in AD model systems. I hypothesized that Mn exposure in the context of a mouse model of AD would result in disruption of glutamatergic function including impaired glutamate clearance and excitatory/inhibitory imbalance resulting in epileptiform activity. Using the APPswe/PSEN1dE9 mouse model of AD and non-transgenic littermate controls, I observed changes in astrocytic glutamate clearance both in vitro in primary astrocytes and in ex vivo hippocampal slices. Additionally, I determined if Mn exposure increased seizure susceptibility and subclinical epileptiform activity using electroencephalogram recordings and an additional pharmacological challenge of kainic acid, an excitatory agonist. I found that Mn exposure increased seizure susceptibility in APPswe/PSEN1dE9 mice and controls, increased seizure severity in APPswe/PSEN1dE9 mice, and increased subclinical epileptiform activity in control mice. Additionally, I observed increased time awake and less time sleeping in control mice following Mn exposure. Mn exposure also resulted in shifts in different brain wave powerbands (e.g. Alpha, 8-12 Hz) with control mice showing more sensitivity to these changes compared to Mn treated APPswe/PSEN1dE9 mice. However, these changes were not explained by changes in GLT-1 and GLAST protein or RNA expression alone. These findings suggest that Mn exposure does impact glutamatergic homeostasis, and that Mn exposure and AD related pathology may be acting on similar mechanisms based on the changes observed herein between control and AD model mice
In Vitro Modeling for Three-Dimensional Imaging of the Bone Marrow
No full textThe bone marrow microenvironment is responsible for the maintenance of hematopoietic stem cell (HSC) activity, and this HSC niche is characterized by complex cellular, chemical, and structural components necessary to maintain hematopoiesis. While the inaccessibility of living bone marrow hampers the study of its pathophysiology, physical and digital models could be powerful tools to study hematopoiesis and test new therapeutics. Current in vitro bone marrow models utilize either simple microfluidic systems or opaque scaffolds with tortuous pores that do not mimic physiologically relevant bone geometry or do not have the ability for longitudinal imaging. Therefore, there is an unmet need for 3D models that are both perfusable and amenable to fluorescence microscopy to visualize the spatiotemporal dynamics of cells in the bone marrow microenvironment in real time. The primary goal of this work is to create a 3D bone marrow model that is able to capture physiologically relevant features of trabecular bone while still being amenable to longitudinal imaging, easily manufactured, and amenable to human cell culture to study disease and potentially test therapeutics. We developed a hybrid injection molding/stereolithography (SLA) fabrication method for rapid prototyping of polystyrene (PS) perfusion cell culture devices, along with a computational fluid dynamics (CFD) model to predict the collection efficiency of perfused human mesenchymal stem cells (hMSCs) on the collectors. hMSC deposition on the collectors and proliferation of cells over 7 days was visualized by fluorescence microscopy. CFD simulations of collection efficiency agreed with experimental measurements within a factor of two. The effect of collector diameter on simulated and experimental cell collection efficiencies followed a trend similar to that predicted by interception theory corrected for intermolecular and hydrodynamic forces. In addition, engraftment of HSCs and acute myeloid leukemia (AML) cells into a mineralized osteoblastic matrix was visualized in a 2D coculture system. This work highlights the utility of CFD simulations and hybrid injection molding for rapid prototyping and optimization of 3D bone marrow models to study cell dynamics in physiologically relevant bone marrow microenvironments and disease states in real time
Vasculo-metabolic Magnetic Resonance Imaging Markers of Abnormal Brain Aging
No full textCerebrovascular and Alzheimer’s disease (AD) pathology often co-occur, complicating clinical diagnosis, treatment strategies, and understanding of the leading causes of dementia. Cerebral hemodynamic and metabolic rate markers, non-invasively quantifiable on magnetic resonance imaging (MRI), may provide early insight into distinct or co-pathological neurodegenerative and cerebrovascular processes occurring during abnormal brain aging.
The first objective of this project was to characterize associations between cognitive diagnosis and cerebral metabolic rate of oxygen (CMRO2) variables, determining if associations were modified by genetic risk for AD (APOE-ε4 carrier status) or stroke risk. 3T multimodal MRI captured cerebral blood flow (CBF) and oxygen extraction fraction (OEF); CMRO2 was quantified by the Fick principle. Findings indicated that CBF and CMRO2 were lower among individuals with dementia. OEF was lower among APOE-ε4 carriers with mild cognitive impairment (MCI) relative to APOE-ε4 non-carriers with MCI. Interaction models among cognitively impaired individuals suggested that OEF may be lower with greater stroke risk in APOE-ε4 carriers and higher with greater stroke risk in APOE-ε4 non-carriers.
The second and third objectives were to examine the association between a marker of arterial transit time, the arterial spin-labeling spatial coefficient of variation (ASL-sCoV), and neuroimaging markers of cerebral small vessel disease (SVD) and to assess whether ASL-sCoV mediated associations between brain artery lumen diameters and SVD burden. ASL-sCoV, SVD markers, and brain large artery lumen diameters were quantified on 3T multimodal MRI. Higher ASL-sCoV was associated with higher white matter hyperintensity (WMH) and basal ganglia enlarged perivascular spaces volumes. Higher baseline ASL-sCoV was associated with a faster longitudinal increase in WMH volume. The association between larger internal carotid artery lumen diameter and higher anterior WMH volume was partially mediated by higher anterior ASL-sCoV.
Taken together, this research indicates that MRI markers of hemodynamic timing and oxygen metabolism provide useful insights into neurodegenerative and cerebrovascular pathologies which often overlap in the aging brain. Distinct changes in OEF with genetic risk for AD and stroke risk highlight its potential to help disentangle co-pathological mechanisms and assist with differential diagnosis. Finally, hemodynamic timing markers show promise in advancing understanding and early detection of SVD pathophysiology
Structural Optimization of Antioxidant Copolymers for Drug Delivery Applications
No full textOxidative stress, the pathological elevation of reactive oxygen species (ROS), participates in the propagation of many diseases, including osteoarthritis and optic neuropathies. Polymers incorporating ROS-scavenging groups can recapitulate the body’s innate antioxidant defenses to restore oxidative homeostasis and prevent biomolecule oxidation, inflammation, and cell death. Alone or in combination with targeted therapeutics, antioxidant polymers or biomaterials represent a promising avenue for disease treatment. In this work, we first demonstrate the advantages of optimizing a hydrophilic copolymer incorporating the potent ROS-scavenging drug TEMPO for anti-inflammatory activity. The ratio of TEMPO and an inert hydrophilic spacer was varied, and an optimal formulation was determined in vitro. The lead candidate demonstrated ROS scavenging and suppression of cytokine TNFa in a mouse air pouch model of inflammation. In a related project, we formulated antioxidant microparticles for protein and nucleic acid delivery. Candidate polymers were screened for crystallinity and formulation stability, and the optimized structure was first tested for delivery of the protein erythropoietin (EPO) in mouse models of traumatic optic neuropathy and glaucoma. The combined chemical and biological antioxidant activity of the EPO microparticles outperformed the clinical standard poly(lactic-co-glycolic acid) (PLGA) and reduced tissue damage, leading to improved visual function. MP formulations were then adjusted to load small interfering RNA (siRNA) for gene knockdown of pathological targets in a mouse model of osteoarthritis. Sustained release of siRNA in the joint from microparticles resulted in target gene knockdown superior to free siRNA, and knockdown of catabolic protease MMP13 resulted in improved morphology of joint tissues. Overall, this work demonstrates that ROS-scavenging polymers can be used for a variety of applications to improve drug pharmacokinetics and activity, resulting in overall disease mitigation
A clock for all seasons: ecology and evolution of circadian clocks
No full textCircadian clocks are intrinsic biological timers with a period of about 24 hours that can be synchronized to the environmental daily cycle and allow for prediction and anticipation of important cyclic events. These clocks are widespread across the tree of life and are found even in bacteria. Photosynthetic bacteria (i.e. cyanobacteria) have become crucial to understanding the adaptive value of circadian clocks, but much is still unclear about how circadian clocks have evolved and continue to evolve. In this dissertation, I discuss early and future circadian clock evolution, and leverage bacteria – both cyanobacteria and the gut microbiome – as a model system for understanding this evolution. First, I report evidence of bona fide photoperiodism in cyanobacteria, showing that cyanobacteria can use daylength as a cue of future environmental conditions and preemptively change their gene expression and lipid membrane desaturation index in a way that allows for increased survival to cold temperatures. Similar to what is often observed in eukaryotic photoperiodism, this phenomenon takes multiple cycles to develop and requires a functional circadian clock. Next, I use the gut microbiome as a model system to understand how host rhythmicity and arrhythmicity impact the composition of a community. We learned that the gut community can be quite stable to host arrhythmicity, but when the community is perturbed by antibiotics, it can only recover its original composition if the host is rhythmic. We also identify certain bacterial species that appear to be selected for/against in rhythmic conditions. Overall, this dissertation advances the chronobiology field by establishing cyanobacteria as a model for photoperiodic time-measurement and demonstrating the effect that host rhythmicity can have upon the structure of microbial communities
Characterization and Investigation into the Accuracy of Touch-Based Registration Using Fiber Optic Shape Sensing
No full textImage guidance is increasingly used by surgeons to locate specific anatomy more accurately during surgery. Unfortunately, many of the sensing systems currently used for image guidance, such as optical
or magnetic tracking, have limitations that make them incompatible with certain surgical techniques or medical imaging technologies. Recently, fiber optic shape sensing has emerged as an alternate technology that could overcome these challenges. This work investigates whether a commercially available Fiber Optic Shape Sensor (FOSS) can feasibly be used for image guided surgery via touchbased registration. It first aims to characterize the FOSS by testing the effect that conditions common
during touch-based registration have on the sensor’s shape and tip position measurements. Lastly, a method of using the FOSS for touch-based registration that aims to mitigate the effects of these
conditions is presented and evaluated
A generalization of the distortion function and the asymptotic geometry of subgroups
No full textThis thesis introduces and studies a natural generalization of the distortion
function that applies to not necessarily finitely generated subgroups of finitely
generated groups. We begin by computing this function in several natural cases,
and provides an example of a group with an uncountable collection of incom-
parably distorted subgroups.We then show that when we restrict this function
to the case of finitely generated subgroups H of finitely generated groups G,
the generalized distortion function characterizes when a natural subspace of the
asymptotic cone of G corresponding to H is connected. We denote this subspace
by Coneω
G(H) and show that the ordinary distortion function is not sufficient to
detect this subspace’s connectedness. We then study the convexity properties
of Coneω
G(H). We show that a subgroup H of a finitely generated group G
is strongly quasi-convex if and only if Coneω
G(H) satisfies a natural convexity
property in Coneω (G). G acts on Coneω (G) in a natural way. We show that the
stabilizer of Coneω
G(G) is the same as the commensurator of H in G whenever
H is strongly quasi-convex in G. We conclude by providing several applications
of this result to groups with Morse elements
Understanding the mechanisms of HMCES DNA-protein crosslink removal and its importance in human cells
No full textApurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions in mammalian cells. HMCES (5-hydroxymethylcitosine, embryonic cell specific) is an evolutionarily conserved protein found at replication forks that covalently crosslink to AP sites in ssDNA, forming a DNA-protein crosslink (DPC). The HMCES-DPC shields ssDNA AP sites from endonucleases and TLS polymerase activity to help maintain genome integrity. However, how the HMCES-DPC is resolved in human cells and produces better outcomes for genome stability and organism fitness remains unknown. In my thesis project, I describe a system utilizing DNA polymerase alpha inhibitor CD437 to induce the accumulation of ssDNA and AP sites during DNA synthesis. Hence, I use CD437 as a tool to track and quantify the formation and resolution of HMCES-DPCs in human cells. Using this CD437 system, I show that the proteasome and SPRTN are not required to remove HMCES-DPC from human cells. However, mutation of conserved residue Glu127 at HMCES’s SRAP domain strongly delays HMCES-DPC removal in cells. Biochemically, I demonstrate that Glu127 catalysis reversal of the crosslink and how DNA structure conditions HMCES re-crosslink capacity. I also report that deficiency of this crosslink reversal affects cell viability in the absence or presence of DNA stress agents. Taken together, my data suggest that HMCES-DPC removal in human cells can be mediated by the auto-release of the crosslink, which is important to maintain cellular fitness. This mechanism provides a new strategy to protect temporally AP sites, possibly until DNA replication provides a better DNA substrate to repair AP sites in an error-free pathway
Collective Action for Prevention and Health Promotion: Exploring the Role of Community Coalitions in Substance Misuse Prevention Ecosystems
No full textAn approach to addressing complex social issues that has become ubiquitous in recent decades is the establishment of community coalitions and collaboratives. Prevailing theories of community coalition action suggest that the relational processes of community coalitions improve communities’ capacity to address issues and opportunities in their communities, yet the literature linking community coalitions to increases in community capacity is small, scattered, and limited in scope. This manuscript-style dissertation uses qualitative and quantitative data collected from county-level substance misuse prevention coalitions in Tennessee to provide an enriched exploration of coalitions’ relational processes and outcomes. Findings indicate that community coalitions’ actions and development are influenced considerably by the organizational ecosystems and networks of interpersonal relationships in which they are embedded. Network analysis, which foregrounds the relationships that link actors in a system, represents a promising approach to clarifying the mechanisms that link coalitions to increases in community capacity