Browsing by Subject "Wound healing"
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Item Open Access Distinct Functions and Regulation of Nonmuscle Myosin II Isoforms a and B in Cell Motility(2008-04-23) Sandquist, Joshua CThe ability of cells to migrate is of fundamental importance to a diverse array of biological processes, both physiological and pathological, such as development, the immune response and cancer cell metastasis, to name a few. The process of cell movement is a complicated cycle of coordinated steps involving dynamic and precise rearrangement of the actin-myosin cytoskeleton. As a critical component of the migration machinery, the molecular motor protein nonmuscle myosin II (myosin II) has long been a subject of scientific inquiry. It is now generally accepted that the contractile forces generated by myosin II contribute directly or indirectly to every step in migration. Interestingly, three isoforms of myosin II (myosin IIA, IIB and IIC) have been identified, and although each isoform performs the same basic molecular functions, recent findings suggest that the different myosin II isoforms make unique contributions to the motile process. In this dissertation work I used RNA interference technology to specifically deplete cells of myosin IIA and IIB in order to characterize the distinct migration phenotypes associated with loss-of-function of each individual isoform. Surprisingly, I found that the two myosin II isoforms perform not only distinct but opposing functions in cell migration, with myosin IIA and IIB normally inhibiting and facilitating proper cell movement, respectively. Furthermore, using pharmacological and microscopy techniques, I investigated the cellular mechanisms allowing for isoform-specific function. My results provide evidence for at least two isoform-specific regulatory mechanisms, namely selectivity in signaling pathways and subcellular distribution. A particularly significant finding is the identification of the different assembly properties of myosin IIA and IIB as the key element responsible for directing isoform-distinct distribution. Together the data presented herein represent a considerable advance in our understanding of the distinct functions and regulation of myosin II in cell motility.
Item Embargo Engineering the microstructure and spatial bioactivity of granular biomaterials to guide vascular patterning(2023) Anderson, Alexa R.In tissues where the vasculature is either lacking or abnormal, biomaterial interventions can be designed to induce vessel formation and promote tissue repair. The porous architecture of biomaterials plays a key role in influencing cell infiltration and inducing vascularization by enabling the diffusion of nutrients and providing structural avenues for vessel ingrowth. Microporous annealed particle (MAP) scaffolds are a class of biomaterial that inherently possess a tunable, porous architecture. These materials are composed of small hydrogel particles, or microgels, that pack together to produce an interconnected, porous network. We first demonstrated that the particle fraction in MAP scaffolds serves as a bioactive cue for cell growth. To control this bioactive cue, we developed methods to form MAP scaffolds with user-defined particle fractions to reproducibly assess mechanical properties, macromolecular diffusion, as and cell responses. We then modulated the microstructure of the MAP scaffolds by changing microgel size as well as the spatial bioactivity using heterogeneous microgel populations to promote de novo assembly of endothelial progenitor-like cells into vessel-like structures. Through a combination of in silico and in vitro experimentation, we found that the microstructure (dimension of the void), integrin binding, and growth factor sequestration were all shown to guide vascular morphogenesis. We then demonstrated that the findings produced in a reductionist model of vasculogenesis translated to an in vivo effect on vessel formation in both dermal wounds and glioblastoma tumors.
Item Open Access Exploring the Interface Between Macroorganisms and Microorganisms: Biochemical, Ecological, and Evolutionary Contexts(2015) Essock-Burns, TaraThe focus of this dissertation is the extension of the innate immune response in wound healing and non-wound healing contexts. I am interested in interactions at the interface between macroorganisms and microorganisms from marine/aqueous environments. This dissertation explored two aspects of the interactions: 1) the presence and function of macroorganism secretions and 2) the role of secretions in managing microfouling on macroorganism surfaces. Particularly of interest are how barriers are biochemically reinforced to mitigate microfouling and the potential consequences of a breach in those barriers. The innate immune response, an evolutionary conserved system in vertebrates and invertebrates, provides an evolutionary context for developing the hypotheses.
In this dissertation the biochemical composition and uses of crustacean secretions are explored for barnacles, fiddler crabs and blue crabs. Fluids of interest were secretions released during barnacle settlement and metamorphosis and those collected from living adult barnacles, fluids on fiddler crab sensory appendages including dactyl washings and buccal secretions, and fluids from blue crab egg masses. The biochemical composition was determined using a combination of fluorescent probes and confocal microscopy, proteomics, and enzyme-specific substrates with a spectrophotometer.
I demonstrated that self-wounding is inherent to the critical period of settlement and metamorphosis, in barnacles. Wounding occurs during cuticle expansion and organization and generates proteinaceous secretions, which function as a secondary mode of attachment that facilitates the transition to a sessile juvenile. I showed extensive proteomic evidence for components of all categories of the innate immune response, especially coagulation and pathogen defense during attachment and metamorphosis. This work provides insight into wound healing mechanisms that facilitate coagulation of proteinaceous material and expands the knowledge of potential glue curing mechanisms in barnacles.
In order to test macroorganism secretions in a non-wound healing context, I examined fluids sampled from body parts that macroorganisms must keep free of microorganisms. I showed that two types of decapod crustaceans can physically manage microorganisms on most parts of their body, but certain parts are particularly sensitive or difficult to clean mechanically. I examined sensory regions on the fiddler crab, including dactyls that are important for chemoreception and the buccal cavity that is used to remove microorganisms from sand particles, and blue crab egg mass fluids that protect egg masses from fouling through embryo development.
This dissertation explores organismal interactions across scales in size, space, and time. The findings from the barnacle work inform mechanisms of attachment and glue curing, both central to understanding bioadhesion. The work on fiddler crabs and blue crabs contributes to our understanding of chemoreception of feeding and reproductive behaviors.
The work presented here highlights how biological secretions from macroorganisms serve multifaceted roles. In cases of physical breaches of barriers, or wounding, secretions coagulate to obstruct loss of hemolymph and have antimicrobial capabilities to prevent infection by microorganisms. In non-wounding cases, secretions remove microorganisms from surfaces, whether that is on the body of the macroorganism or in the immediate environment.
Item Open Access Modulating Macrophage Response with Microporous Annealed Particle Scaffolds(2022) Liu, YiningWhen designing biomaterials for clinical applications, the performance of these platforms hinges on their interaction with the host immune system. A failure in engaging and incorporating the correct immune response would lead to foreign body response and subsequent rejection of the materials. To improve the biocompatibility of biomaterials and avoid undesired immune reactions, the key immunomodulatory cell type macrophage needs to be engaged and its phenotype modulated properly and timely. Therefore, the design parameters of biomaterials should be carefully considered in the context of macrophage modulation. Microporous annealed particle scaffolds (MAPS) are a new class of immunomodulatory granular materials generated through the interlinking of microgels. The modular nature of MAPS offers enormous tunability in not only the individual microgel design but also the homogenous or heterogenous microgel assembly into the bulk scaffold. We leveraged the plug-and-play feature of MAPS to study the effect of two design parameters, microgel crosslinking peptide (comprised of L- or D-amino acids) and spatial confinement (achieved through varying microgel size), on macrophage modulation and host responses. We uncovered that a fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes in MAPS with D-amino acid-based crosslinker was an indicator for regenerative scaffolds in a subcutaneous implantation model. We also discovered that scaffolds comprised of large microgels with pore size that can accommodate ~40 µm diameter spheres induced a more balanced pro-regenerative macrophage response and better wound healing outcomes with more mature collagen regeneration and reduced inflammation level. The role of spatial confinement on macrophage response was further explored in vitro, where we demonstrated that size-dependent macrophage response to M1/M2 cytokine stimulations was tied to the change in cell morphology and motility. This work offers valuable insights into the dynamic immune response to synthetic porous scaffolds with a specific focus on macrophages, and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes for would healing and biomaterial implants.