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Cytokinesis is an essential event for cell proliferation. The underlying mechanisms and most proteins for cytokinesis are conserved from yeast to humans. Cytokinesis requires multiple coordinated events: division-site selection, actomyosin contractile-ring assembly, ring constriction and disassembly, plasma-membrane deposition, septum formation or extracellular matrix remodeling, and daughter-cell separation. The fission yeast Schizosaccharomyces pombe is an excellent model system to study cytokinesis because it has efficient homologous recombination for gene targeting and is convenient for microscopy and genetics. Extensive studies have been completed to help understand the early stages of cytokinesis involving the actomyosin contractile ring. However, the processes at the later stages of cytokinesis are less well understood. Rho GTPases are important regulators of cytokinesis. Their activities are controlled by Rho guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Rho GAPs hydrolyze GTP to GDP to inactivate Rho GTPases. There are more Rho GAPs compared to Rho GTPases, and Rho GTPases are commonly inactivated by several GAPs. Thus, how Rho GAPs are regulated for specific localization is crucial to their functions. Rga7 is a Rho2 GAP, which functions to ensure actomyosin ring stability, proper ring disassembly, to regulate successful septum formation and cell separation. In my first project, I identified a novel coiled-coil protein Rng10 that regulates the localization and function of the Rho GAP Rga7. Rng10 interacts with Rga7 in vivo. Correct Rga7 localization is abolished without Rng10. Together, Rng10 and Rga7 regulate the accumulation and dynamics of glucan synthases for successful septum formation during cytokinesis. F-BAR proteins are another key player of cytokinesis. They are important for linking membranes to the actin cytoskeleton. The F-BAR domain physically interacts with phospholipids on the plasma membrane. However, whether this intrinsic membrane-binding property of the F-BAR domain is solely sufficient for targeting proteins to their cellular localization is unclear. In my second project, I found that the F-BAR domain of Rga7 directly binds Rng10 simultaneously with the plasma membrane, and that Rng10 also independently binds the plasma membrane. These combined interactions significantly enhance Rga7 F-BAR avidity for membranes. This requirement for the F-BAR domain in Rga7 localization and function is bypassed by tethering a construct of Rga7 lacking its F-BAR to Rng10. Our data suggest that some F-BAR domains are necessary but not sufficient for plasma membrane targeting. Instead they are stably localized to specific cortical positions through protein binding partners.The exocyst is another protein complex at the division site that functions in secretory vesicle tethering and cargo delivery during cytokinesis. Studies have shown that the exocyst works with septins and Mid2 to regulate the localization of glucanases, which are essential for cell separation. However a direct connection that links septins to exocytosis is not established. In my third project, I examined the regulation of the localization of the exocyst by septins. Loss of septin rings at the division site changes the localization pattern of the exocyst complex from a ring to a disk. Septins regulate the exocyst in a specific spatiotemporal manner at the division site during late stages of cytokinesis. Loss of the exocyst ring causes the accumulation of secretory vesicles and their cargos to the center, and a significant reduction at the rim of the division plane. Thus the exocyst depends on septin rings for a stable ring localization to carry out its proper function during cytokinesis.

The microclimate inside microspheres prepared from biodegradable polymers (e.g., poly(lactic-co-glycolic acid) PLGA) often becomes acidic owing to the accumulation of water-soluble polymer degradation products, which can induce the destablization of encapsulated therapeutic agents. The objective of this dissertation was to quantitatively evaluate the microclimate pH (μpH) inside biodegradable polymeric microspheres in order to facilitate the development of microsphere formulations that control μpH and stabilize acid-labile drugs. Chapter 1 presents an overview of the background of these studies with a focus on the most common biodegradable polymer, PLGA. In Chapter 2, the μpH distribution inside protein-encapsulated PLGA microspheres was accurately quantified using a ratiometric method based on confocal laser scanning microscopy (CLSM). The fluorescent response of Lysosensor yellow/blue® dextran used to map acidic µpH in PLGA was influenced by the presence of encapsulated protein. A method for correction of the interference of protein was developed and validated. The µpH kinetics in four different PLGA microsphere formulations during incubation under physiological conditions were determined to be roughly pH 4 to neutral pH depending on the formulation. Based on previous literature findings of enhanced stability of encapsulated proteins and peptides in hydrophilic and biodegradable poly(lactic-co-hydroxymethyl glycolic acid) (PLHMGA) microspheres, the μpH distribution and kinetics in the microspheres prepared from PLHMGAs were evaluated in Chapter 3 by CLSM and compared that with their PLGA counterparts. The PLHMGA microspheres developed a far more neutral μpH than PLGA, which was linked to more rapid diffusion of acidic degradation products out of the polymer. In the last chapter, a mathematical model was developed to simulate the μpH kinetics and spatial distribution inside degrading PLGA microspheres by considering the acid production, mass transfer via diffusion and partition of water-soluble acids that contribute to the development of μpH. Fundamental parameters in the PLGA microspheres were determined from experiments to validate the model. The model successfully predicted the kinetics of μpH development, whereas showing a small difference in distribution compared to experimental results. Hence, these mechanistic approaches may provide valuable experimental and theoretical tools to control μpH inside the most commonly used biodegradable polymer for controlled release of acid-labile therapeutics.

An experimental and modeling study was undertaken to investigate proppant settling and retardation in hydraulic fractures. Experiments were conducted in a fracture flow cell for Newtonian fluids as well as shear thinning fluids with varying viscosities. New empirical and analytical models for proppant transport and settling in hydraulic fractures were developed. These models can be used in any hydraulic fracture simulator. The proppant settling models developed account for changes in the settling velocities and rheology caused by fracture walls, proppant concentration, turbulence effects due to high fluid velocities and inertial effects associated with large relative velocities between the proppant and the fluid. Proppant velocity relative to the fluid in the direction of flow is affected by the fracture walls and can result in significant reduction in the proppant transport. A model was developed to estimate the proppant retardation (ratio of particle velocity to the fluid velocity) due to these effects. All these correlations have been incorporated into a fully 3-D hydraulic fracture code, UTFRAC-3D. The impact of these factors on created fracture geometry is shown for the first time.