We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer's disease (AD), Parkinson's disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained similar to 106 EVs/mu L by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.

In the last few decades, an increasing number of physicists specialized in soft matter, including polymers, have turned their attention to biologically relevant materials. The properties of various molecules and fibres, such as DNA, RNA, proteins, and filaments of all sorts, are studied to better understand their behaviours and functions. Self-assembled biological membranes, or lipid bilayers, are also the focus of much attention as many life processes depend on these. Small lipid bilayers vesicles dubbed liposomes are also frequently used in the pharmaceutical and cosmetic industries. In this thesis, work is presented on both the elastic properties of polymers and the response of lipid bilayer vesicles to extrusion in narrow-channels. These two areas of research may seem disconnected but they both concern deformed soft materials. The thesis contains four articles: the first presenting a fundamental study of the entropic elasticity of circular chains; the second, a simple universal description of the effect of sequence on the elasticity of linear polymers such as DNA; the third, a model of the symmetric thermophoretic stretch of a nano-confined polymer; the fourth, a model that predicts the final sizes of vesicles obtained by pressure extrusion. These articles are preceded by an extensive introduction that covers all of the essential concepts and theories necessary to understand the work that has been done.

Vesicles are simple structures, but display complex, non-linear dynamics in fluid flow. I investigate the deformation of nanometer-sized vesicles, both fully-inflated and those with excess area, as they travel in tightly confined capillaries. By varying both channel size and flow strength, I simulate vesicles as they transition from steady-state to unstable shapes, and then rupture in strong flow fields. By employing a molecular dynamics model of the vesicle, fluid, and capillary system one is able to rupture the lipid bilayer of these vesicles. This is unique in that most other numerical methods for modelling vesicles are unable to show rupture. The rupture of fully-inflated vesicles is applicable to drug delivery in which the release of the encapsulated medicine needs to be controlled. The deformation and rupture of vesicles with excess area could be applicable to red blood cells which have similar rheological properties.

Cortical injury, such as that following stroke, is one of the leading causes of long-term disabilities world-wide. While some neuroprotective agents given within hours of stroke can reduce damage, there are currently no neurorestorative therapeutics that can enhance long-term recovery. To address this, we tested Mesenchymal Stem Cell (MSC) derived Extracellular Vesicles (EVs) as a treatment for cortical injury in rhesus monkeys (Macaca mulatta). Monkeys treated with EVs 24 hours after injury and again at 14 days after injury recovered more completely and more rapidly than monkeys given a vehicle control. However, the cellular changes associated with enhanced recovery remained unknown. In this dissertation, it was hypothesized that EVs modulated cells within the brain to enhance recovery after cortical injury. To explore this hypothesis, three specific aims were tested. Aim 1: To determine the effects of EVs on microglial reactivity. Since EVs in this study were derived from MSCs, it was hypothesized that they would have an immunomodulatory effect. Using immunohistochemistry, image analyses, and 3-D reconstruction, we showed that microglia shifted from reactive, damaging phenotypes towards homeostatic, surveilling functions in EV-treated monkeys. These effects correlated with reduced time to recovery, suggesting that reduced microglial reactivity enhanced recovery. Aim 2: To assess the effects of EVs on myelination. Because MSCs have regenerative effects, it was hypothesized that these MSC-derived EVs would improve neurorestoration. Using immunohistochemistry, qRT-PCR, Spectral Confocal Reflectance microscopy, and ELISA, we assessed myelination after cortical injury with and without EV treatment. EVs limited oligodendrocyte damage and increased densities of mature oligodendrocytes to enhance myelin maintenance. These effects correlated with improved recovery, suggesting the importance of myelination in recovery after cortical injury. Aim 3: To assess the neuroprotective role of EVs on infarct volumes. While it was hypothesized that EVs would reduce the densities of inflammatory cells (astrocytes, macrophages/microglia, T-cells), hemosiderin accumulation, and infarct volume, we found that EVs did not alter these endpoints. Collectively, our results suggest that EVs modulated microglia and oligodendrocytes to promote neurorestoration. Overall, these findings demonstrate the therapeutic potential of EVs for neurorestoration after cortical injury.

Clearance of molecularly damaged and misfolded synaptic vesicle (SV) proteins is vital for the maintenance of healthy, functional synapses. However, this process poses significant trafficking challenges for neurons, as the majority of degradative organelles and machinery are localized in the somatodendritic compartment, far from SV pools in presynaptic terminals. Our previous work showed that SV protein degradation is mediated by the endosomal sorting complex required for transport (ESCRT) pathway in an activity-dependent manner. Moreover, we found that neuronal activity increased ESCRT protein recruitment to axons and SV pools, suggesting a novel mechanism for regulating the trafficking of this critical degradative machinery, whose localization and transport in neurons has been unexplored. Here, we characterize the axonal transport of ESCRT-0 proteins Hrs and STAM1, the first components of the ESCRT pathway, which are critical for initiating SV protein degradation. We find that Hrs- and STAM1-positive transport vesicles exhibit increased anterograde and bidirectional motility in response to neuronal activity, as well as frequent contact with SV pools. ESCRT-0 vesicles typically colocalize with early endosome marker Rab5, but their transport dynamics do not mirror those of the total Rab5 vesicle pool. Moreover, other ESCRT pathway components and effectors do not show activity-dependent changes to motility, indicating that neuronal firing specifically regulates the motility of the ESCRT-0+ subset of Rab5+ structures in axons. Finally, we identify kinesin-3 motor protein KIF13A as essential for the activity-dependent transport of ESCRT-0 vesicles as well as the degradation of SV membrane proteins. Altogether, these studies demonstrate a novel activity-dependent mechanism for mobilizing the axonal transport of a newly characterized endosomal subtype carrying ESCRT machinery. This activity-induced transport is necessary for ESCRT-mediated degradation of synaptic vesicle proteins.

No description available.

No description available.

Alzheimer’s disease is characterized by the formation of two major pathological hallmarks: amyloid plaques and neurofibrillary tangles. Although there have been many studies to understand the role of microglia in Alzheimer’s disease, it is not yet known how microglia can promote disease progression while actively phagocytosing amyloid plaques or phosphorylated tau (p-tau). Through stereotaxic injection of adeno-associated virus expressing mutant P301L tau (AAV-P301L-tau) into the medial entorhinal cortex (MEC) of both wild-type (WT) and APPNL-G-F mice, we demonstrate how amyloid plaques exacerbate p-tau propagation to the granule cell layer (GCL) of the hippocampus. However, in mice receiving the colony-stimulating factor 1 receptor inhibitor (PLX5622), ~95% of microglia were depleted, which dramatically reduced p-tau propagation to the GCL. Although microglia depletion curtailed p-tau propagation, it also led to reduced plaque compaction and an increase in overall amyloid-beta (Aβ) plaque presence. Additionally, we found microglia depletion resulted in greater p-tau aggregation in dystrophic neurites surrounding amyloid plaques. We investigated neurodegenerative microglia (MGnD), which are activated in response to amyloid plaques, for their propensity to release extracellular vesicles in comparison to homeostatic microglia. We discovered that MGnD, identified by Clec7a or Mac2 staining, strongly express Tumor susceptibility gene 101 (Tsg101), which is an ESCRT-1 protein and a marker for extracellular vesicles (EVs). To further investigate EV release and MGnD, a novel lentivirus expressing fluorescent mEmerald conjugated to CD9 (mE-CD9) was constructed and injected into the MEC of both WT and APPNL-G-F mice which allowed for visualization of mE-CD9+ puncta around individual microglia. CD9 is a tetraspanin and also a marker for EVs. We observed that the number of mEmerald+ particles surrounding MGnD was three-fold higher compared to non-diseased, homeostatic microglia. Sequential injection of mE-CD9 and AAV-P301L-tau into the MEC revealed that microglia-derived EVs encapsulate pathologic p-tau, which is augmented by the MGnD phenotype. Taken together, these data provide strong evidence that MGnD exhibit increased secretion of tau-containing EVs, providing a possible mechanism for how amyloid deposition indirectly exacerbates tau propagation.

<div>Quantifying the dynamics and rheology of soft biological suspensions such as red blood cells, vesicles, or capsules is paramount to many biomedical and computational applications. These systems are multiphase flows that can contain a diverse set of deformable cells and rigid bodies with complex wall geometries. For this thesis, we are performing several numerical simulations using boundary element methods (BEM) for biological suspensions in biomedically relevant conditions. Each simulation is devised to answer fundamental questions in modeling these systems.</div><div><br></div><div><br></div><div>Part of this thesis centers around the fluid mechanics of giant unilamellar vesicles (GUVs), fluid droplets surrounded by a phospholipid bilayer. GUVs are important to study because they mimic the dynamics of anuclear cells and are commonly used as a basis for artificial cells. The dynamics of vesicles in simple shear or extensional flows have been extensively studied. However the conditions seen in microfluidic devices or industrial processing are not always described by steady shear or extensional flows alone, and require more investigation. In our first study, we investigate the shape stability of osmotically deflated vesicles in a general linear flow (i.e., linear combinations of extensional and rotational flows). We modeled the vesicles as a droplet with an incompressible interface with a bending resistance. We simulated a range of flow types from purely shear to purely extensional at viscosity ratios ranging from 0.01 to 5.0 and reduced volumes (measured asphericity, higher is more spherical) from 0.60 to 0.70. The vesicle's viscosity ratio appears to play a minimal role in describing its shape and stability for many mixed flows, even in cases when significant flows are present in the vesicle interior. We find in these cases that the bending critical capillary number for shape instabilities collapse onto similar values if the capillary number is scaled by an effective extensional rate. These results contrast with droplet studies where both viscosity ratio and flow type have significant effects on breakup. Our simulations suggest that if the flow type is not close to pure shear flow, one can accurately quantify the shape and stability of vesicles using the results from an equiviscous vesicle in pure extension. Only when the flow type is nearly shear flow, do we start to see deviations in the observations discussed above. In this situation, the vesicle's stationary shape develops a shape deviation, which introduces a stabilizing effect and makes the critical capillary number depend on the viscosity ratio.</div><div><br></div><div><br></div><div>Continuing with our research on single vesicle dynamics, we have performed simulations and experiments on vesicles in large amplitude oscillatory extensional (LAOE) flows. By using LAOE we can probe the non-linear extension and compression of vesicles and how these types of deformation affect dilute suspension microstructure in time-dependent flows through contractions, expansions, or other complex geometries. Our numerical and experimental results for vesicles of reduced volumes from 0.80 to 0.95 have shown there to be three general dynamical regimes differentiated by the amount of deformation that occurs in each half cycle. We have termed the regimes: symmetrical, reorienting, and pulsating in reference to the type of deformation that occurs. We find the deformation of the quasispherical vesicles in the microfluidic experiments and boundary element simulations to be in quantitative agreement. The distinct dynamics observed in each regime result from a competition between the flow frequency, flow time scale, and membrane deformation timescale. Using the numerical results, we calculate the particle coefficient of stresslet and quantify the nonlinear relationship between average vesicle stress and strain rate. We additionally present some results on the dynamics of tubular vesicles in LAOE, showing how the experiments suggest the vesicles undergo a shape transformation over several strain rate cycles. Broadly, our work provides new information regarding the transient dynamics of vesicles in time-dependent flows that directly informs bulk suspension rheology.</div><div><br></div><div><br></div><div>Our most recent project deals with the accuracy of discretized double layer integrals for Stokes flow in the boundary element method.</div><div>In the fluid mechanics literature, the chosen parameterization, meshing procedure, and singularity handling are often selected arbitrarily or based on a convergence study where the number of elements is decreased until the relative error is sufficiently low.</div><div>A practical study on the importance of each of these parameters to the accurate calculation of physically relevant results, such as the particle stresslet, could alleviate some of the guesswork required. The analytical formulas for the eigenfunctions/eigenvalues of the double layer operator of an ellipsoidal particle in a quadratic flow were recently published¹, providing an analytical basis for testing boundary element method discretization accuracy.</div><div>We use these solutions to examine the local and global errors produced by changing the interpolation order of the geometry and the double-layer density. The results show that the local errors can be significant even when the global errors are small, prompting additional study on the distribution of local errors. Interestingly, we find that increasing the interpolation orders for the geometry and the double layer density does not always guarantee smaller errors. Depending on the nature of the meshing near high curvature regions, the number of high aspect ratio elements, and the flatness of the particle geometry, a piecewise-constant density can exhibit lower errors than piecewise-linear density, and there can be little benefit from using curved triangular elements. Overall, this study provides practical insights on how to appropriately discretize and parameterize three-dimensional (3D) boundary-element simulations for elongated particles with prolate-like and oblate-like geometries.</div><div><br></div>

In order to evaluate the short term metabolic effects of gossypol on the testes as well as any possible effects on the secondary sex organs, Balb C mice were injected subcutaneously with various doses of gossypol (0.25‐25.0 mg/kg body weight) in corn oil for 10 days. Wet weights of several different secondary sex reproductive organs decreased during gossypol treatment. However, wet weights of the testes during treatment remained equal to or greater than control values. Following 10 days of gossypol treatment, incorporation of [3H]thymidine or [3H]amino acids into trichloroacetic acid precipitable macromolecules was inhibited in the seminal vesicles and ventral prostates normalized to either DNA or wet weight. Treatment with gossypol also had an inhibitory effect on epididymal sperm count at the two highest doses. These results demonstrate that gossypol will decrease sperm count at high dose levels after treatment of male mice for as short as 10 days. However, its overall effects are not limited to the testes and spermatogenesis but, in addition, it has dramatic inhibitory effects on protein and nuclei acid metabolism in the secondary sex organs.