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Eukaryotic cells are subdivided into organelles with unique physicochemical compositions and functions. Establishment and maintenance of these organelles requires selective exchange of material by vesicle trafficking. Transport vesicles are formed by cytoplasmic coat protein complexes which shape the membrane, and play a further role in the selection of transmembrane cargo proteins. Transmembrane cargo receptors select cargo in the luminal compartment and bind to these coat proteins through signals in their cytoplasmic domains. Once formed, vesicles recognise and fuse with a target organelle to deliver their content. This final process is tightly regulated by small GTPases of the Rab superfamily. Here, I will describe my work investigating the role of specific Rab GTPase pathways and the KDEL cargo receptor in trafficking in the secretory and endocytic pathways in mammalian cells. There are over 60 Rabs in human cells, each of which is activated by a specific GDP-GTP exchange factor (GEF) and inactivated by a GTPase-activating protein (GAP) at a defined membrane location. For many of these Rabs the GEF and GAP were unknown at the outset of this work in which I have identified and characterised two structurally related families of Rab GEFs, the DENN and tri-longin domain proteins. The DENN family, expressed only in metazoans, which has 17 members acting on 10 different Rabs. The tri-longin family, comprises 3 heterodimeric GEF complexes: Mon1-Ccz1 for the late endosomal, lysosomal Rab7, Hps1-Hps4 (BLOC-3) which activates Rab32/Rab38 in melanogenesis and Intu-Fuz, part of CPLANE complex, which activates Rab23 at cilia. Additionally, I have shown that the unrelated Rab3GAP complex is a GEF for the ancestral Rab18 regulating the structure of the endoplasmic reticulum. In the final part of this work, I have explored how the integrity of the early secretory system depends on selective export and retrieval of proteins between the ER and the Golgi. An essential component of this system is the transmembrane cargo receptor for KDEL retrieval signals. Here I describe the structure of the KDEL receptor and mechanism by which KDEL cargo binding triggers a conformational change exposing a signal for the COP I vesicle coat.

Background and Aim: The International Diabetes Federation suggests in 2017, around 451 million adults around the globe were affected by diabetes mellitus. Worldwide, invasive subcutaneous injection devices remain the standard for diabetes treatment. To increase patient adherence, and to help reduce the anxiety of painful daily administration of insulin this study investigated the prospect of using ultradeformable vesicles, known as transfersomes, to act as carriers and permeation enhancers for the delivery of insulin. The overall aim was the development of a double-layered patch, in which the transfersomes were embedded in the mucoadhesive layer, and the presence of an outer impermeable layer enabled the formation of a novel unidirectional immobilized delivery system for buccal delivery of insulin. Methods: A reverse phase HPLC method was developed and validated, according to ICH guidelines, for the detection and quantification of insulin. Vesicles were formed using a thin-film hydration technique with bath sonication, and manual extrusion was used for further downsizing. Throughout the project, vesicles were evaluated for particle size, polydispersity index, zeta potential and insulin encapsulation efficiency (EE, %). Permeability of insulin was studied across TR146 buccal cell line, and sulforhodamine B (SRB) assay was used for in-vitro cytotoxicity screening. Minitab factorial design was employed to optimise mucoadhesiveness of lyophilised patches. Results and Discussion: Analysis and quantification of insulin with HPLC demonstrated insulin to degrade much faster in acidic conditions. Preliminary studies led to the selection of Span 60, which was combined with the phospholipid DPPE (1,2-dipalmitoyl-3-sn-phosphatidylethanolamine) to produce transfersomes. Membrane ii | P a g e fluidity was enhanced by the addition of Tween 80 and reduction of cholesterol content. Based on toxicity studies, the two promising formulations consisted of Span 60 (40%), DPPE (20%), Tween 80 (20%), cholesterol (15%), with either 5% dicetyl phosphate (D5E) or 5% sodium glycodeoxycholate (S5E). Insulin release from patch S [sodium alginate (2% w/v), HPMC (0.5% w/v), Sorbitol (5% w/v) and PEG 400 (0.25% w/v)] was found to occur as burst release with 75% of the total insulin being released in the first 30 minutes. The most promising percentage drug release (66.5%), in 6 hours, was with patch S containing the transfersomal formulation D5E. Conclusion: This thesis demonstrated an excellent approach in delivering insulin via a non-invasive route by combining novel transfersomes as permeation enhancers within an optimised mucoadhesive buccal patch. Furthermore, the project led to the generation of new data and observations concerning the influence of extrusion and vortexing on vesicle size, use of CytoSMART for cell imaging, use of actinomycin D as positive control in SRB cytotoxicity assays with TR146 cells and influence of the cryoprotectant sorbitol on mucoadhesion combined with HPMC, sodium alginate and chitosan in freeze-dried patches.

Patients with sickle cell disease (SCD) receive regular blood transfusions, which can lead to alloimmunisation due to exposure to different red blood cell (RBC) antigens. The spleen is frequently damaged in these patients, resulting in higher numbers of red cell-derived particles (RCDP) and autophagic vesicles (AV). Most RCDP, produced through membrane budding, are right-side out and expose the external domains of RBC proteins, whereas AV are inside-out, exposing cytoplasmic domains. Since SCD patients have higher numbers of RCDP and AV, we aimed to characterise these particles and compare with healthy donor plasma and with storage vesicles (SV) from outdated blood. We also investigated the use of these particles to induce tolerance to RBC antigens, in a murine model. RCDP and AV from SCD and healthy plasma were isolated using immunomagnetic separation. Results using imaging flow cytometry (IS) showed that RBC markers like glycophorins A and C, band 3, glucose transporter1 and phosphatidylserine, were detected in all particles, confirming they originated from RBC. RCDP were more prevalent and generally larger than AV but both particle types were more abundant in SCD than healthy plasma. IS was more sensitive for small particle detection than flow cytometry. The latter detected large ghost membranes. All particle types were detected by transmission electron microscopy (TEM) and immunogold staining against extracellular or cytoplasmic GPA domains further confirmed their RBC origin. TEM, IS and dynamic light scattering showed that RCDP were larger than AV. A model system was developed to investigate tolerance induction in vivo and indicated that both SV and ghosts could be used to prime host animals. In conclusion, small RCDP and AV from SCD and healthy plasma have been characterised for first time, using IS. It may be possible to use particles from blood cells induce tolerance to RBC antigens and thereby reduce alloimmunisation.

Myelinated axons are crucial for rapid information transmission within the central nervous system (CNS). Myelin injury is a common feature of white matter (WM) pathology in a number of disease states, including ischemic stroke. Myelin disruption can lead to a complete failure in saltatory action potential conduction, resulting in devastating neurological deficits. However, the fundamental mechanism of ischemic myelin injury is controversial. Glutamate-mediated excitotoxicity is now recognised as a crucial event in the development of ischemic WM pathology. This thesis investigates the potential mechanisms of glutamate release in central WM and examines the hypothesis that NMDA receptor over-activation mediates ischemic myelin damage. Using glutamate biosensor microelectrodes and FM-dye imaging, I show that axonal depolarisation in the adult corpus callosum evokes rapid vesicular docking in axons, capable of elevating extracellular glutamate concentration. My findings show that vesicular fusion occurs under the myelin sheath in myelinated axons, which supports the existence of a novel synapse between the axon and overlaying myelin. Simulation of ischemia triggered an early and robust rise in optic nerve extracellular glutamate levels. Unexpectedly, a significant component of ischemic glutamate release also originated from axonal vesicular fusion. Together, these findings show that the axon-myelin synapse represents a significant site of excitotoxic injury during ischemia. Resolving prior conflicting results, I show that NMDA receptor antagonists prevent myelin degradation and improve functional recovery when applied for sufficient time to penetrate the sheath. Finally, I identify a fluorescent myelin stain (QNZ-46) which is a negative allosteric modulator of NR2C/D-containing NMDA receptors. QNZ-46 selectively accumulates in myelinated WM regions of the CNS following systemic administration, and is retained following wash-out. As a result, QNZ-46 provides persistent protection during ischemia by preserving myelin structure and improving functional recovery.

Antibiotic resistance has emerged as one of the World's leading public health challenges. The inexorable emergence of drug resistant pathogens, combined with a steep decline in antibacterial drug discovery, has led to a major crisis. One of the most common drug resistance mechanisms involves bacteria adapting to reduce intracellular drug accumulation. To understand these resistance mechanisms, one needs quantitative information about the membrane permeability of drugs. In this Thesis, we develop a novel optofluidic permeability assay that allows us to quantify the permeability coefficient of drugs crossing lipid membranes. Lipid vesicles are used as model systems and drug molecules are tracked directly using their autofluorescence in the ultraviolet. The permeability coefficient of the drug is inferred by studying the increase in drug autofluorescence intensity within vesicles as they traverse a microfluidic network while exposed to the drug for well defined times. This provides a novel platform from which we can develop membrane models for understanding drug permeability. We incorporate the Escherichia coli outer membrane protein OmpF in vesicles and quantify its role in the transport of fluoroquinolone antibiotics. We provide direct visualisation of OmpF mediated fluoroquinolone transport. We study the pH dependence of antibiotic transport both through pure phospholipid membranes and through OmpF, and present a physical mechanism to explain the pH dependence of E. coli fluoroquinolone susceptibility. We also show the importance of lipid composition on drug permeability - changing the lipid composition of the membrane is shown to change antibiotic permeability by over an order of magnitude. Finally, we report on the discovery of a novel signalling mechanism in E. coli that relies on the transport of small drug-like molecules, and discuss the role it plays in stress response in the microbial community.

The specific interaction between saccharide molecules and saccharide-binding proteins, lectins, can be exploited in order to actively target nanostructures to specific cell types for diagnostic or therapeutic purposes. However, the conjugation of saccharides to nanostructures can be complex, leading to a lack of versatility in the functionalities that can be achieved, or poorly characterized and therefore potentially inconsistent. Herein is reported a new procedure by which novel saccharide-containing molecules, for the functionalization of nanostructures, could be synthesized rapidly and with good yields. The synthesis was found to be versatile, providing coating molecules for magnetite nanoparticles and phospholipid vesicles from a number of different hydrazides and reducing sugars. The stability of the synthesized coating molecules was assessed, both in aqueous solution and when bound to the surface of a magnetite nanoparticle, and found to be satisfactory for cell culture purposes. The availability of the saccharide units for lectin binding was confirmed using a Quartz crystal microbalance (QCM-D) in addition to a number of assays. Finally, microscopy techniques were used to study the interactions between saccharide-functionalized magnetite nanoparticles and two cell types, fibroblasts (3T3) and hepatocytes (HepG2).This work demonstrated the ability of saccharide coatings to improve the uptake of nanostructures in a cell culture environment and highlighted the potential cell manipulation application of saccharide-coated magnetite nanoparticles.

Non-invasive and controlled release of bioactive compounds is an important goal in the development of drug delivery systems and novel biomaterials for tissue engineering. This project aims to exert spatio-temporal control over the release of bioactive compounds from phospholipid vesicle carriers by crosslinking them with superparamagnetic iron oxide nanoparticles to form a magnetic release nanostructure. The magnetic properties of the nanoparticles allow release to be triggered by an alternating magnetic field (AMF), which induces localised heating and “melts” the vesicle membranes. The aggregates can also be manipulated in space by a static magnetic field to create patterned materials. Incorporation of these aggregates into hydrogels has created novel responsive biomaterials. Controlled release of ascorbic acid-2-phosphate has been used to induce collagen production by chondrocytes, demonstrating an AMF triggered cellular response in vitro. The existing system has been redesigned after detailed characterisation and assessment of the performance of each component. Magnetic release has been extensively assessed using fluorescence techniques to quantify release, and optimised through the development of new silica-derived nanoparticle coatings and aggregate formulations informed by quantitative characterisation of nanoparticle functionalisation. The replacement of calcium alginate hydrogels as a 3D cell culture matrix with hyaluronic acid- based hydrogels was found to eliminate gel-induced leakage of vesicle contents and also improves the compatibility of the system with a greater range of cell types. Recently the effective encapsulation and AMF-triggered release of proteins including enzymes has been demonstrated and released enzymes have been demonstrated to retain their activity. Released trypsin was shown to retain proteolytic activity while hyaluronidase released into hyaluronan-derived hydrogels has been demonstrated to influence the rheological properties of the gel. A galactose-terminated lipid has been synthesised that enables specific targeting of the asialoglycoprotein (ASGPR) cell surface receptor receptor found in human hepatocytes, demonstrating the potential for customisation of the MNPV system to particular requirements.

The stability of growing uni-lamellar vesicles is investigated using the formalism of nonequilibrium thermodynamics. The vesicles, which are assumed to be in an otherwise aqueous solution, are growing due to the accretion of lipids to the bilayer which forms the vesicle membrane. The thermodynamic description is based on the hydrodynamics of a water-lipid mixture together with a model of the vesicle as a discontinuous system in the sense of linear nonequilibrium thermodynamics. The approach assumes that the energy of the bilayer membrane is given by the spontaneous curvature model attributed to Helfrich. Furthermore, the rate at which lipids incorporate into the membrane is taken to be proportional to the surface area of the membrane. In this way, the relevant forces and fluxes of the system are identified in the context of a stability analysis. The resulting constitutive equation for the flux of water across the membrane is used to analyse the stability of spherical vesicles that are subject to different perturbations. First, a simplified approach is presented which restricts perturbations to axisymmetric ellipsoids. In that case, the analysis is carried out using an explicit Cartesian parametrisation. A perturbation theory which describes more general deformations is then developed and applied to the case of arbitrary axisymmetric perturbations. It is found that there are generically two critical radii at which changes of stability occur. For the case where the perturbation takes the form of a single zonal harmonic, only one of these radii is physical and is given by the ratio $2L_p / L_\gamma$, where $L_p$ is the hydraulic conductivity and $L_\gamma$ is the Onsager coefficient related to changes in membrane area due to lipid accretion. The stability of such perturbations is related to the value of l corresponding to the particular zonal harmonic: those with lower l are more unstable than those with higher l. The conditions under which general axisymmetric perturbations reduce to explicit ellipsoidal calculations are also found. A heuristic explanation for the results is proposed whilst possible extensions of the current work and the need for experimental input are also discussed.

Dielectrophoresis and electrorotation are receiving increasing attention as useful phenomena for the characterisation and physical manipulation of cells. The primarily concern of this investigation was to determine whether these techniques can interpret accurately the dielectric properties of biological cells with an appropriate dielectric shell model. In this study, synthetic vesicles have been used as the testing samples in electrorotation experiments to verify the reliability of these techniques. By using the electrorotation technique with the dielectric shell models, dielectric properties of vesicles could be analysed very accurately and the results were in agreements with the observed morphology and membrane properties. The physical structure of the vesicles varied from a simple one such as unilamellar vesicle, to a more complex structure such as the oligolamellar and multilamellar form. The morphology and membrane structure of the vesicles were also characterised by fluorescence microscopy, flow cytometry and electron spin resonance using spin probes. This allowed this validification of the application of the dielectric shell theory for analysis of simple cellular systems. The second objective of this work was to extend the potential used of the electrorotation technique, and not only for the analysis of cellular systems. Electrorotation was also performed on single stranded DNA oligonucleotides, covalently bound onto the surface of microscopic-sized latex beads. Different types of the DNA oligonucleotides exhibited different electrorotation responses according to their different base sequences. This has shown that the electrorotation technique can be used as an analytic tool to identify different sequences of DNA oligonucleotide.

Oligomeric Phenylene Ethynylenes (OPEs) are a class of synthetically prepared compounds with the basic structure of phenylene rings connected by triple-bonded carbon atoms, also known as an ethyne link. This structure can be modified by incorporating functional groups onto one or more of the phenylene rings. The addition of functional groups, such as sulfoxide or imidazole, may alter the photophysical properties in detectable ways. The photophysical techniques used in this report include fluorescence, spectroscopy in the ultraviolet and visible light range (UVVIS), and photon-based excitation using a photo chamber. One attractive function of OPEs is their antimicrobial property. Previously published tests have shown that OPEs with different functional groups inactivate bacteria such as E. coli and damage viral components such as the spike proteins of SARS CoV-2 virus (COVID-19) (1). The method of inactivation is still being explored, but activation following exposure to broad-spectrum light seems to be an important component.

Abstract: Life relies on membranes, electron transfer reactions and proton-gradients. To better understand if electron transfer within and proton gradients across a lipid membrane could be achieved with simple, prebiotically plausible components, we synthesized prebiotically plausible neutral metal complexes which are redox active. These metal complexes partitioned to the membranes of lipid vesicles and should be capable of participating in an electron transport chain. Here, different thiol ligands and imidazole ligands were first chosen and used to synthesize Co (II) complexes in water-octanol mixtures. According to the UV-Vis spectra of different Co (II) complex in octanol, the logic and method of octanol-soluble metal complex synthesis was obtained and then applied to the synthesis of Fe (II) complexes. The UV-Vis spectra indicated that octanol soluble Fe (II)-N-Acetyl-L-cysteine-1-butylimidazole complex was successfully synthesized. Subsequently, such complexes were synthesized in the membranes of phospholipid vesicles and detected by 31P NMR spectra. Increased linewidths indicated that Fe (II) complexes successfully migrated to the membranes of phospholipid vesicles.

In this dissertation, we study Stokesian particulate flows. In particular, we are interested in the dynamics of vesicles and red blood cells (RBCs) suspended in Stokes flow. We aim to develop mathematical models and numerical techniques for accurate simulation of their dynamics in microcirculation. Vesicles are closed membranes made of a phospholipid bilayer and are filled with fluid. Red blood cells are highly deformable nucleus-free cells and have rich dynamics when subjected to viscous forcing. Understanding single RBC dynamics is a complex fluid-membrane interaction problem of fundamental importance in expanding our understanding of red blood cell suspensions. For example, one of the fundamental problems is the construction of phase diagrams for the red blood cell shapes as a function of the imposed flow and the mechanical properties of the cell. Accurate knowledge of their shape dynamics has also led to interesting approaches for cell sorting based on mechanical properties in lateral displacement devices. We model an RBC using two different models, namely, “vesicle" and “capsule". We use the term particle to refer to both of them. Vesicles are inextensible surfaces with bending resistance and serve as a good model for RBC in 2D. But in 3D, vesicles miss important features of RBC dynamics because they have zero shear resistance. In contrast, an inextensible capsule resists shear in addition to the bending and is a more accurate model of RBC in 3D. For both the particles, we use a boundary integral formulation to simulate their long time horizon dynamics using spherical harmonics based spectral singular quadratures, differentiation and reparameterization techniques. We demonstrate the full relevance of our simulations using quantitative comparisons with existing experimental results with RBCs and vesicles. Once we have verified and validated our code, we use it to study the bistability (two RBC equilibrium states depending on initial state of RBC) observed under same flow conditions in our simulations. We plot the phase diagrams of equilibrium shapes of vesicles and RBCs in confined and unconfined Poiseuille flow. Finally, we also develop a novel scheme for Stokesian particle simulation using regularized Stokes kernels and overset finite differences based on overlapping patchwise discretization of the surface. Our scheme has lower work complexity than the spherical harmonics based scheme and also exhibits a high order convergence (typically fourth order) than the quadratic convergence of the triangulation based schemes. Furthermore, the patchwise discretization approach allows for more local independent control over resolution of the different parts of the surface than the global spherical harmonics based scheme. We verify this new scheme for extensible capsule simulation by quantitative comparison with the previous results in the literature for extensible capsules. We also demonstrate easy acceleration of singular quadrature using all-pairs evaluation algorithm implemented for the GPU architecture. The GPU acceleration allows us to do long time horizon simulation of capsules of low reduced volume resulting in complex shapes. Our scheme is also easily accessible to further acceleration using the fast multipole methods (FMMs).

Prior to the emergence of cellular life, there must have been an abiotic analogue to the cell -- also known as the protocell. To flourish, they should have performed key functions enabling growth, and ensuring their survival and propagation. While researchers study a variety of model protocell systems, the study of model protocells assembled from fatty acid vesicles is lacking in comparison to the abundant literature on phospholipid vesicles. There is evidence that the existence of fatty acids predated life on Earth. Moreover, the highly dynamic nature of fatty acid vesicles is not well documented in the origins of life context. There is a need to explore the behaviour of fatty acid vesicles in systems which mimic real world environments. I explored the flocculation behaviour of vesicles in environments of mechanical stress, monovalent salt and particles including minerals. I also explored the effect of RNA-relevant divalent cations on fatty acid vesicles, as well as changes due to pH fluctuation. The findings of this project suggest that fatty acid vesicles are able to flocculate under quite low concentrations of monovalent salt, and that such flocs can incorporate particles including mineral particles which are relevant for catalysing RNA-related reactions. Furthermore, I found that after vesicles have been formed, they are surprisingly robust in conditions that inhibit vesicle formation. These results provide evidence that protocells assembled from simple building blocks such as fatty acids are quite robust and may persist in challenging environments, and that their flocculation behaviour may have co-localised other processes pertaining to the origins of life.

The bottom-up generation of lipid membranes from minimal precursors is a key objective in synthetic biology research. In living cells, the synthesis of lipid assemblies is ubiquitously coordinated by several membrane-bound enzymes. However, these pathways are tough to replicate, owing to the difficulty of reconstituting the enzymes in vitro. Lipid membrane synthesis from simple metabolic building blocks remains challenging. We demonstrate a chemoenzymatic schematic for lipid membrane generation, utilizing a bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA as precursors. Palmitoyl-CoA spontaneously reacts with a cysteine-modified lysophospholipid via native chemical ligation (NCL), generating noncanonical amidophospholipids that self-assemble into micron-sized membranes. The results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles. Utilizing this route, we further investigate the application of non-amphiphilic, minimal precursor pathway to generate lipid membranes. We explore the use of acetate as the carbon feedstock for the chemoenzymatic reaction strategy, generation palmitoyl-CoA in situ and coupling it with small molecule head group such as cysteine to form phospholipid analogs. Ubiquitous enzymes acetyl-CoA synthetase (ACS) from E. coli was used to generate acetyl-CoA from acetate and human acetyl-CoA carboxylase (ACC) was used to then synthesize malonyl-CoA from acetyl-CoA. Additionally, cgFAS catalyzed the in situ generation of palmitoyl-CoA from acetyl-CoA and malonyl-CoA. Finally, we explore the formation of a unique analog of sphingolipid membranes from water-soluble precursors that have markedly different biophysical properties to their natural counterparts. We show that numerous transition metal ions, particularly Cu(II), catalyze the selective O-acylation of the biologically occurring single-chain amphiphile sphingosylphosphorylcholine using fatty acyl phosphates or thioesters as acyl donors under mild aqueous conditions. This work demonstrates the value of de novo membrane generation strategies starting from minimal, water-soluble precursors. Utilizing such approaches, we can control both the chemical structures of the lipid analogs as well as their biophysical properties in aqueous media. This effort contributes towards understanding the fundamental requirements for bottom-up generation of lipid membranes, providing alternative strategies to those previously shown.

With increasing industrial and biomedical applications of engineered nanomaterials (ENMs), concerns have been raised regarding increased risk of exposure. Exposure to ENMs can potentially lead to adverse health effects including cell toxicity. The plasma membrane, a lipid-protein bilayer surrounding all cells, is the first cellular entity that comes into direct contact with foreign particles. Membrane damage by ENMs is one of the potential mechanisms through which ENMs induce cytotoxicity. In recent years, significant effort has been focused on elucidating the complex interactions at the particle-plasma membrane interface. Such studies have primarily relied on membrane models to tease out what particle physicochemical properties might perturb the structure and function of the cell plasma membrane. However, the role of membrane lipid asymmetry, the fact that the plasma membrane has a different lipid composition in the exofacial leaflet compared to the cytofacial leaflet, in regulating nanoparticle-membrane interactions has remained obscure. In the first aim of this dissertation research, the role of individual membrane leaflets in regulating the interactions of membrane models and erythrocytes with engineered silica nanoparticles (50 and 100 nm) was examined. It was found that silica nanoparticles bind to and disrupt synthetic vesicles mimicking the exofacial leaflet but not those mimicking the cytofacial leaflet of the erythrocyte plasma membrane. Nanoparticles that disrupted vesicles mimicking the exofacial leaflet also induced hemolysis in erythrocytes, suggesting that the exofacial leaflet is the primary regulator of nanoparticle-induced membrane damage. This was confirmed by demonstrating that nanoparticles caused similar disruptive behavior in symmetric and asymmetric vesicles, which had similar outer leaflet, but different inner leaflet lipid compositions. Together, these studies reveal that membrane lipid asymmetry plays a minor role in nanoparticle-induced membrane disruption with the exofacial leaflet being the primary regulator of interactions.The second aim of this study focused on understanding the mechanisms by which engineered nanomaterials disrupt the cell plasma membrane. Here, we investigated the role of the lipid chemical structure in the disruption of lipid vesicles by unmodified silica, carboxyl-modified silica, and unmodified polystyrene nanoparticles (50 nm) was investigated. The role of the lipid headgroup was examined by comparing nanoparticle effects on vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vs. an inverse phosphocholine (PC) with the same acyl chain structure. The role of acyl chain saturation was examined by comparing nanoparticle effects on saturated vs. unsaturated PCs and sphingomyelins. Nanoparticle effects on PCs (glycerol backbone) vs. sphingomyelins (sphingosine backbone) were also examined. Results showed that the lipid headgroup, backbone, and acyl chain saturation all affect nanoparticle binding to and disruption of the membranes. A low headgroup tilt angle and the presence of a trimethylammonium moiety at the vesicle surface are required for unmodified nanoparticles to induce membrane disruption. Lipid backbone structure significantly affects nanoparticle-membrane interactions, with carboxyl-modified particles only disrupting lipids containing cis unsaturation and a sphingosine backbone. Acyl chain saturation makes vesicles more resistant to particles by increasing lipid packing in vesicles, impeding molecular interactions. Finally, nanoparticles were capable of changing lipid packing, resulting in pore formation in the process. These observations are important in interpreting nanoparticle toxicity to biological membranes.The third aim of this work was to examine the role of plasma membrane lipid chemistry in the endocytosis of ENMs by altering the plasma membrane lipid composition directly in live cells. Using a methyl alpha cyclodextrin (MαCD)-catalyzed lipid exchange method, endogenous membrane lipids were replaced with lipids of choice, and this exchange process was confirmed using chromatography and mass spectrometry methods. It was demonstrated that the lipid composition of the plasma membrane outer leaflet affects cellular internalization of silica nanoparticles (nominal diameter: 50 nm) in serum free medium, depending on the lipids delivered to the plasma membrane. Importantly, changes in membrane lipid composition altered the intracellular fate of nanoparticles, resulting in their displacement from the lysosome. The role of the new lipid microenvironment on function of low-density lipoprotein receptor (LDLR) and macrophage receptor with collagenous structure (MARCO), two well-known scavenger receptors, known to be involved in the uptake of a variety of nanomaterials, was investigated in single cells. It was observed that cells showed high LDLR activity following exchange with 18:1 SM, but low activity following exchange with 36:2 PC and iPC exchange. Little change in the activity of MARCO was observed except after exchange with 36:2 iPC. Using giant plasma membrane vesicles (GPMVs) and anisotropy measurements, it was observed that lipid exchange affected the fluidity of the cell plasma membrane but did not alter receptor localization. Taken together, these findings reveal that the lipid composition of the plasma membrane outer leaflet plays a significant role in ENM endocytosis, by affecting non-specific ENM diffusion into the cell, by changing membrane fluidity, and active endocytosis.

Metal nanoparticles are particles made up of many individual metal atoms ranging in size from one nanometer to hundreds of nanometers in diameter. Originally these particles were used in glazes to make lustrous patterns on pottery. It was not until Michael Faraday’s discovery of colloidal gold in 1856 that nanoparticles were characterized. Since then, nanoparticles of different compositions, sizes, and shapes have been implemented in fields such as catalysis and biotechnology. This dissertation will address two applications of nanoparticles: silver nanoparticles (AgNPs) to amplify signal in a paper-based fluidic device and gold nanoparticles (AuNPs) to quench signal in studies of cell penetrating peptides (CPPs). First, the paper-based device used concentrates a “sandwich-type” immunoassay, made of a capture antibody bound to a magnetic microbead and a detection antibody bound to a AgNP label, at a screen-printed carbon electrode by a magnet. The AgNP labels are oxidized to Ag ions, reduced, and stripped off the electrode in a technique known as anodic stripping voltammetry. Originally, this device could only detect AgNP concentrations as low as 2.1 pM. Scanning electron microscopy (SEM) was used to investigate the percentage of AgNPs oxidized in this detection method enabling optimization of the electrochemical parameters for higher signal output. This lowered the detection limit to 2.6 fM, which is now at a sensitivity comparable to a commercial pregnancy test. Second, this dissertation focuses on the use of AuNPs to suppress signal in fluorescence studies of cell penetrating peptides (CPPs), a class of peptides so effective in crossing cellular membranes that they can be used to carry cargo such as proteins or small molecules into a cell. The mechanism by which CPPs can permeate cell membranes is poorly understood. Here we use a model system composed of phospholipid vesicles and small fluorescent peptides to investigate the transport and mechanism of these peptides interacting with the lipid bilayer. While fluorescence signal can reveal details such as the amount of peptide that transports inside the vesicles, it cannot determine the depth that a peptide penetrates through the vesicles. By growing AuNPs inside these vesicles, however, we can use fluorescence quenching to determine the proximity of the peptide to the center of the vesicles, which will be important as we continue to study CPPs and their permeation through cell membranes.

Membrane proteins constitute a significant fraction of cellular proteins due to their vast diversity in function. However, studies of their native structures, folding mechanisms, and dynamics remain a challenge, in part because of their inherent insolubility. One goal of this project is to enhance the tools to study membrane proteins. Small unilamellar vesicles (SUVs) have been used as a bilayer mimetic to investigate membrane protein structures, dynamics, and folding mechanisms. UV resonance Raman spectroscopy (UVRR) has emerged as a powerful technique for probing structures and dynamics of biomolecules. UVRR is particularly valuable for membrane proteins because of the selective enhancement of signal from key aromatic residues and the ability to probe interactions between membrane protein and the lipid. We present practical considerations and guidelines for UVRR data acquisition, including a detailed description of a typical laser setup as well as the process to analyze the tryptophan vibrational modes of a model β-barrel membrane protein, OmpA, unfolded and folded in SUVs. An additional need in studies of membrane protein folding is alternate membrane mimics. Nanodiscs (NDs) are an excellent bilayer alternative to SUVs because of their experimental benefits including homogeneity, optical clarity, low light scattering, and enhanced stability. We combine SDS-PAGE mobility studies with fluorescence, circular dichroism, and UVRR spectroscopy to confirm and characterize the folding of OmpA into NDs. Our studies show similar secondary and tertiary structures in both SUVs and NDs as well as efficient folding yields greater than 88% in both bilayers. The folding of OmpA into NDs was slower compared to in SUVs, and this difference can be attributed to the different bilayer characteristics. Insights into the folding mechanism of OmpA were gained via bimolecular fluorescence quenching with acrylamide quencher. Stern-Volmer analysis utilizing the sphere-of-action model probed changes in local environment and protein solvation during folding into NDs. An initial fast step after initiation of the folding reaction, associated with a large change in polarity to a hydrophobic environment was attributed to a fast adsorption and interaction with the lipid bilayer. Desolvation kinetics were slower than the formation of tertiary structure, indicating that desolvation may occur in the final steps of folding.

Giant liposomes, or giant unilamellar vesicles (GUVs), are thin, semi-permeable,man-made compartments that often serve as models of the cell plasma membrane due to their sizes (1−100 ?m) and molecular composition (composed of lipids). GUVs have proven useful for understanding a variety of different biophysical phenomena such as lipid membrane organization, membrane protein function, and cytoskeletal mechanics. A variety of different formation methods have been developed to try to optimize the populations of GUVs produced. However, information that allows for the direct comparison of the sizes and yields of the GUVs obtained from the different methods is lacking. In my dissertation, I describe my work on the development of a novel confocal microscopy-based technique that allows for the characterization of the populations of GUVs produced from the most commonly employed surface-assisted assembly methods. Through the development and standardization of careful protocols that allow for the quantification of ?(100,000) vesicles per sample, I characterize the surface-assembled populations of GUVs in comprehensive sets of experiments. From this work, I show novel discoveries including i) the use of nanocellulose paper as a surface to obtain GUVs, ii) the effect of substrate properties on the formation of GUVs, iii) the modulations of ionic strength technique to allow high yields of GUVS to be obtained using physiological salts, and iv) the effect of osmolytes on the formation of GUVs. The results from these quantitative experiments has led to the development of the budding and merging thermodynamic model which describes the mechanism of GUV formation. Overall, the discoveries pave the way for the largescale production of GUVs for biophysical studies as well as towards more practical applications of GUVs such as for compartments for targeted drug delivery or synthetic cells.

Spread of tumor cells from their primary sites to vital distant organs and tissues, i.e. metastasis, is the most devastating stage of cancer development. Metastatic tumors grow rapidly, and they rarely respond to therapeutic interventions. Thus, a better understanding of how aggressive cancer cells get into the circulation and colonize the secondary site is essential for blocking metastasis. In Chapter 2, I describe the role of a lysine deacylase in regulating the ability of breast cancer cells to secrete factors that promote their aggressiveness. Specifically, I will discuss the mechanism by which, breast cancer cells can take advantage of SIRT1 downregulation to promote secretion of nanometer sized vesicles, known as exosomes, as well as protein hydrolases capable of degrading the extracellular matrix. The secretion of these factors results in the ability of other cancer cells in microenvironment to become more invasive. I also show that SIRT1 is in fact regulating the activity of an organelle called lysosomes by influencing the expression of a subunit (i.e. ATP6V1A) of the machinery responsible for lysosomal acidification. In Chapter 3, I found that SIRT1 is modulating the RNA stability of the ATP6V1A transcript through deacetylating IGF2BP2, an RNA binding protein that binds to the 3'- untranslated region (UTR) of this transcript. Mechanistically, acetylation of IGF2BP2, under conditions where SIRT1 is downregulated, results in recruitment of the exonuclease XRN2, which promotes the degradation of the ATP6V1A mRNA. I also show that knocking down IGF2BP2 or XRN2 can reverse the effect of SIRT1 downregulation on the cellular secretome. In the final chapter, I will discuss the implication of my findings in cancer and other biological contexts as well as describing outstanding questions that have emerged from my dissertation.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. As a facultative intracellular pathogen, Mtb is well-adapted to survive within the host and establishes a persistent infection in humans despite a strong immune response. As part of its survival strategy, Mtb releases a wide array of proteins that aid in nutrient acquisition and immune evasion. Broadly, the work presented in this thesis focuses on how an Mtb response regulator, RegX3, regulates protein export by the bacteria, and how export of these proteins impacts Mtb viability. In Chapter 2 we describe that a ∆pstA1 mutant, in which RegX3 is constitutively activated, hyper-secretes a variety of proteins. In previous work, we found that RegX3 induces secretion through the specialized ESX-5 secretion system. We also found hyper-secretion of LpqH, a lipoprotein associated with membrane vesicles (MV) produced by Mtb. Further investigation into this phenotype revealed that ∆pstA1 bacteria release significantly more MV than wild-type (WT) bacteria. We showed that this is a RegX3-dependent phenotype, suggesting a gene(s) within the RegX3 regulon controls MV production. Furthermore, MV release does not depend on ESX-5 activity, so RegX3 induces protein export by at least two different mechanisms. Work in Chapter 3 builds upon the observation that ∆pstA1 bacteria produce more MV. Because this strain constitutively activates RegX3, we leverage it as a tool to identify factors that drive MV release. We hypothesized that the deletion of a specific RegX3-induced gene in the ∆pstA1 background would restore MV production to WT levels, and that the product of this gene would provide information about the physical process of MV release through the Mtb cell wall. We demonstrate that only the deletion of lppF or whiB3 reduce MV production. While WhiB3 was previously reported to transcriptionally regulate the production of cell wall lipids, we found no evidence to support this regulatory mechanism in our ∆whiB3 strains. We conclude that no single gene drives the increased MV production by the ∆pstA1 strain; activation of WhiB3 is responsible for a portion of the phenotype and additional RegX3-dependent factors also contribute. Chapter 4 focuses on the biological functions of ESX-5 activity, specifically to determine how ESX-5 affects Mtb growth and what proteins are secreted by the system. Creating strains harboring tetracycline-repressible copies of ESX-5 components allowed us to circumvent ESX-5 essentiality while still controlling its activity. Using these strains, we found that Mtb requires ESX-5 activity for growth when either glycerol or glucose was the sole carbon source. Additionally, ESX-5 activity was required for optimal growth and survival in both resting and interferon-gamma activated murine macrophages in vitro. It remains to be investigated whether this intracellular survival is connected to ESX-5-dependent nutrient acquisition. These strains also allowed us to perform the first full proteomic analysis to identify protein substrates secreted via ESX-5. Our results uncovered that ESX-5 may play a role in the release of a wider range of proteins than was previously appreciated. We report that release of proteins associated with the cell membrane/cell wall and also proteins secreted via other systems rely on ESX-5 activity to reach the culture filtrate. Taken together, the results presented in Chapter 4 uncover new roles of the ESX-5 system and highlight the importance of this system to Mtb viability.

Lipids, specifically phospholipids like dipalmitoylphosphatidylcholine (DPPC), undergo an order/disorder phase transition between fluid and gel states at ambient temperature and pressure. In the case of lipid-wrapped nanoparticles (LNP), where lipids are wrapped around a nanoparticle core, additional factors like curvature and nano-lipid interaction are present, resulting in a relatively poor understanding of the transition. Computer simulation, specifically molecular dynamics, is an ideal technique to study the fluid/gel transition from the molecular viewpoint. However, simulation of phase transitions is difficult; large free energy barriers separate stable phases, leading to broken ergodicity. Enhanced sampling methods are necessary to study rare events, like phase transitions, which occur on the second, or longer, timescale. Two such methods, the generalized replica exchange method (gREM) and statistical temperature molecular dynamics (STMD), and their recent developments, utilize generalized ensembles to provide enhanced sampling near phase transitions. Using the above methods,we studied flat lamellar bilayers, followed by more complex curved vesicles, and ultimately LNP. In bilayers, the gREM finds a strong coupling between phase transitions of the lipid and water subsystems. Subsequent simulations on an implicit water bilayer revealed distinct subphase transitions between coexisting states of fluid and gel lipids. STMD applied to vesicles shows weakening of the transition and overall lowering of the transition temperature with decreasing diameter, from first order at 40 nm to borderline first/second order at 20 nm and 10 nm. No homogeneous gel phase is formed, instead, low energy structures exhibit a faceted gel phase with gel patches separated by fluid lipid seams. For LNP, we find that curvature promotes the fluid phase but the presence of a core induces order, particularly in the inner layer. This nontrivial balance results in a broad, continuous phase transition for small LNP that becomes a sharp, first order transition between distinct fluid and gel states for large LNP. Overall, LNP hold great promise in a wide range of fields like drug delivery, imaging, and photocatalysis. We aim to provide a first-principles understanding of their properties by utilizing innovative simulation methods to obtain molecular details not available to experimental or traditional methods.

Volcanic eruptive behavior varies from explosive tephra or effusive lava and is controlled largely by subsurface degassing processes. Dense cognate lithic pyroclasts are poorly vesicular products of some eruptions. Their textures and dissolved volatile signatures preserve important information about their formation processes and are thus used to infer pre-eruptive and syn-eruptive conduit dynamics. Dense cognate lithics from the 1991 Mt. Pinatubo pre-climactic surge events were analyzed in terms of their vesicular and crystal textures, and dissolved H₂O and CO₂ concentrations. The ubiquitous occurrence of sub-millimeter-sized crystal fragments in matrix glass is an intriguing feature not observed in the pumice counterpart of the Mt. Pinatubo deposits. Existing models of permeable foam behavior cannot explain the fragmentation and reincorporation processes necessary for the formation of these fragments. On the other hand, sub-millimeter scale heterogeneity in matrix-glass volatile content (0.9 wt.% difference in H₂O within 0.5 mm) is observed. Those heterogeneities argue that different parts of these dense pyroclasts must have formed at various depths and then need to be physically sutured together. Greater vesicularities and increased vesicle sizes and number densities are observed around large crystals and a similar texture was produced by sintering experiments using bimodelly-sized ash particles within a timeframe similar to that of the 1991 Mt. Pinatubo eruptions. Furthermore, an increasingly larger proportion of elongated and stretched vesicles towards the later surge events is observed, which parallels the increasingly shorter hiatus time between Mt. Pinatubo surges. Paired with the lack of equilibration in volatile contents, the textures lead us to propose an ash particle sintering model in deciphering the formation mechanism of these dense lithics in rather short period of time, where ash particles sourced from various depths collide, shear, break up, and sinter on or near the conduit walls above the fragmentation level in a repetitive manner. We conclude that sintering is the main mechanism forming the dense lithic materials from the 1991 Mt. Pinatubo pre-climactic surge deposits, and is responsible for vesicle, crystal, and volatile signatures preserved and observed in this study, and potentially in other volcanic systems around the world.

Biological membranes are heterogeneous structures with complex electrostatic profiles arising from lipids, sterols, membrane proteins, and water molecules. We investigated the effect of cholesterol and its derivative 6-ketocholestanol (6-kc) on membrane electrostatics by directly measuring the dipole electric field (F [arrow above F] [subscript d] ) within lipid bilayers containing cholesterol or 6-kc at concentrations of 0−40 mol% through the vibrational Stark effect (VSE). We found that adding low concentrations of cholesterol, up to ∼10 mol %, increases F [arrow above F] [subscript d], while adding more cholesterol up to 40 mol% lowers F [arrow above F] [subscript d]. In contrast, we measured a monotonic increase in F [arrow above F] [subscript d] as 6-kc concentration increased. We proposed that this membrane electric field is affected by multiple factors: the polarity of the sterol molecules, the reorientation of the phospholipid dipole due to sterol, and the impact of the sterol on hydrogen bonding with surface water. We used molecular dynamics simulations to examine the distribution of phospholipids, sterol, and helix in bilayers containing these sterols. At low concentrations, we observed clustering of sterols near the vibrational probe whereas at high concentrations, we observed spatial correlation between the positions of the sterol molecules. This work demonstrated how a one-atom difference in a sterol changes the physicochemical and electric field properties of the bilayer. Additionally, we set out to understand how a small molecule interacts with the lipid bilayer differently based on its charge. Our laboratory had previously reported that tryptophan permeated through a phosphatidylcholine lipid bilayer membrane at a faster rate when it was positively charged (Trp+) than when negatively charged (Trp−), which corresponded to a lower potential of mean force (PMF) barrier determined through simulations. In the work described here, we demonstrated that Trp+ partitions into the lipid bilayer membrane to a greater degree than Trp− by interacting with the ester linkage of a phosphatidylcholine lipid, where it is stabilized by the electron withdrawing glycerol functional group. These results are in agreement with tryptophan’s known role as an anchor for transmembrane proteins, though the tendency for binding of a positively charged tryptophan is surprising. We discussed the implications of our results on the mechanisms of unassisted permeation and penetration of small molecules within and across lipid bilayer membranes based on molecular charge, shape, and molecular interactions within the bilayer structure.

The biosphere offers many promising economically and environmentally sustainable solutions to humanity’s increasing energy demand such as biomass conversion, chemical production, and pharmaceutical fermentation. These solutions could close the carbon loop and improve manufacturing efficiencies, but they all depend on accurate control of protein expression. To avoid limitations to protein control present in vivo such as membranes, homeostasis, and growth, artificial cell-like systems are being researched. These simplified systems are currently useful to study individual aspects of life such as regulating energy flux across membranes, responding to the environment, replication, and growth. These systems could be made more complex in the future to provide a simplified, engineered, cell-like platform for bioprocessing. Even at the single gene level, control of protein expression is hindered by resource sharing and bursting. To make proteins, genes require many reusable resources such as polymerase, ribosomes, and tRNAs which are shared among different genes. Resource sharing causes correlations in protein populations and limits steady state concentrations of competing genes. Bursty gene expression, periods of high expression, and thus high resource use, separated by periods of no expression, and thus no resource use, is a ubiquitous biological phenomenon that intimately links expression bursting and resource sharing. This dissertation investigates how gene expression bursting and variation is affected by expression resources being shared among genes and how the location of expression resources, either encapsulated or outside permeable lipid membranes, controls the level and the dynamics of cell-free protein expression. Cell-free protein synthesis systems, both crude and PURE, areused in combination with both physical PDMS barriers and defined lipid membranes to study the effects of shared and divided resource pools on gene expression bursting and protein production. Experimental results are supplemented with Gillespie simulations to add further insights. This work provides fundamental knowledge of protein expression and applied knowledge of the effects of resource sharing on cell-free gene expression bursting and variation in protein expression confined to cell-relevant volumes which are important steps toward artificial cell-like systems.

Single molecule studies can provide information of biological molecules which otherwise is lost in ensemble studies. A wide variety of fluorescence-based techniques are utilized for single molecule studies. While these tools have been widely applied for imaging soluble proteins, single molecule studies of transmembrane proteins are much more complicated. A primary reason for this is that, unlike membrane proteins, soluble proteins can be easily isolated from the cellular environment. One approach to isolate membrane proteins into single molecule level involves a very low label expression of the protein in cells. However, cells generate background fluorescence leading to a very low signal to noise ratio. An alternative approach involves isolating membrane proteins in artificial membrane derived vesicles. This approach is limited to proteins which can be solubilized or stabilized in detergent solution. This intermediate step endangers the structural integrity of proteins with multiple subunits. Hence, we isolated transmembrane proteins into cell-derived vesicles which maintain the proteins in their physiological membrane without compromising their functional integrity. We studied the stoichiometric assembly of α3β4 nicotinic receptors which are pentameric receptor with possible stoichiometry of (α3)2(β4)3 and (α3)3(β4)2. We found that (α3)2(β4)3 is the predominant stoichiometry, and we have verified our finding with both single and double color experiments. We have also demonstrated that cell-derived vesicles can be utilized to study ligand receptor interactions. Cell-derived vesicles generated from cellular preparations provide a method to study the overall structural and functional properties of membrane proteins. However, organelle specific information is not available in this approach. Alternatively, separating vesicles based on their original organelle could provide information on the assembly and trafficking of membrane proteins. For example, it has been hypothesized that nicotine acts as a pharmacological chaperone of α4β2 nicotinic receptors and nicotine alters the assembly of the nicotinic receptors towards the high sensitivity isoform in the ER. To validate this hypothesis, we isolated α4β2 nicotinic receptors located on vesicles derived from the ER and plasma membrane origins and utilized single molecule studies to determine the stoichiometric assembly of the receptor. The data suggested that the ER has a higher percentage of the low sensitivity isoform ((α4)3(β2)2) than the plasma membrane indicating that the high sensitivity isoform trafficked more efficiently to the cell surface. When nicotine was added, the distribution of nicotinic receptors changes in those compartments. In both the ER and plasma membrane, the percentage of high sensitivity isoform was greater than the sample without the presence of nicotine. The results suggested that nicotine altered the assembly of nicotinic receptors to form the high sensitivity isoform in the ER and the altered assembly trafficked to the plasma membrane efficiently increasing the ratio of this isoform in the plasma membrane. The cell derived vesicles we utilized to isolate single receptors are structurally similar to liposomes, an FDA approved drug delivery system, which is spherical vesicles composed of at least one lipid bilayer. Hence, cell-derived vesicles possess potential to be utilized as drug delivery vehicles. I explored the applicability of cell-derived vesicles as general delivery vehicles to cultured cells. Additionally, we implanted xenografts into immune compromised nude mice and prepared cell derived vesicles labeled with dye molecules. The vesicles were injected in a mouse containing a xenograft to monitor whether these vesicles can reach to the xenograft. Our data suggested that cell-derived vesicles can successfully reach the xenograft and thus have potential to be utilized as a drug delivery vehicle.

Ascoviruses, family Ascoviridae, are large, enveloped ds DNA viruses that mainly attack lepidopteran larvae of the family Noctuidae causing a chronic, but fatal disease. Their cytopathology differs from other viruses by destroying the nucleus, some species using a caspase, followed by cleavage of the cell into numerous viral vesicles, where most virions are produced. Cell cleavage resembles apoptosis, but differs markedly because mitochondria are not destroyed but rescued by ascoviruses, providing energy for replication and membrane synthesis for viral vesicles and virion envelopes. Ascovirus transmission is also unique in that virions are vectored to hosts on the ovipositor of parasitic wasps. In my research, I used molecular techniques to determine genomic expression patterns underlying ascovirus cytopathology in larvae for two ascoviruses, the Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV). Specifically, through in vivo transcriptome studies, including RNA-Sequencing, qRT-PCR, manual genome curating and bioinformatic analysis, I determined three temporal classes for SfAV and TnAV genes; early, late, and very late. In SfAV, three proteins that inhibit apoptosis were synthesized early, whereas the caspase was synthesized very late, which correlates with apoptotic-like events resulting in vesicle formation. I identified 15 SfAV bicistronic and tricistronic messages, and similar transcripts in TnAV, indicating multi-cistronic transcripts are common in ascoviruses, and may regulate transcription or translation. Analyses of viral vesicle transcripts from Trichoplusia ni hemolymph revealed much higher viral genome expression than in the fat body, epidermis and tracheal matrix, showing larval blood is important for virus reproduction. Host responses to SfAV infection in Spodoptera frugiperda larvae showed mitochondrial gene expression was similar to controls or upregulated slightly during vesicle formation. Interestingly, few cytoskeleton genes known to encode motor proteins were upregulated despite mitochondrial involvement in vesicle formation. Analysis of antimicrobial peptides genes showed several where highly upregulated, as much as 32-fold, for example, moricin and gloverin, whereas innate immunity genes of the Toll, melanization, and phagocytosis pathways were activated only moderately by infection. Phenoloxidase and Toll negative regulators increased 15-fold and 3-fold, respectively. Therefore, conserving mitochondria while balancing immunity gene inducers and repressors may explain the chronic nature of ascovirus diseases.

The coat protein complex II (COPII) achieves cargo sorting and vesicle formation at the endoplasmic reticulum (ER) and aids in protein trafficking by the early secretory pathway. The flexibility of the coat machinery to accommodate diverse size and shaped cargo in order to meet the demands of the cell is not well understood. An example of a large cargo protein is collagen, which is synthesized as 300nm precursor fibrils of procollagen in the endoplasmic reticulum (ER). The molecular mechanism by which large procollagen fibrils are accommodated in canonical COPII vesicles of about 70nm remains unclear. Genetic and biochemical evidence for the role of COPII in collagen trafficking has been established in several studies. Recent collaborative work has shown that mono-ubiquitylation of Sec31 by the ubiquitin ligase complex Cul3-Klhl12 leads to the formation of large COPII vesicles (200-500nm) that drive the secretion of collagen. Amongst the follow-up questions from this study were, whether these enlarged vesicles are bona fide transport carriers of procollagen and what might the three-dimensional ultrastructure be. This dissertation work aims to characterize the role of large COPII coated vesicles in intracellular procollagen-1 trafficking. Using a combination of in vivo imaging techniques and in vitro cell-free reconstitution assays, I present evidence that addresses this question.

Ubiquitously existent in nature, polyamines play an important role as signaling compounds in plant responses to both biotic and abiotic stresses. My research has shown that the clade of Arabidopsis thaliana and rice BIDIRECTIONAL AMINO ACID TRANSPORTERS (BAT) function as antiporters of polyamines and amino acids. The method used in this study takes advantage of the genetic resources of the model organism Escherichia coli. A double knockout E. coli strain that is deficient in all polyamine antiporters was created and then used to heterologously express candidate plant transporters AtBAT1.1, AtBAT1.2, and OsBAT1. Inside-out membrane vesicles of these transgenic E. coli cells were generated by ultrasound sonication or French press. A radioisotope assay was then performed using these vesicles to confirm the specificity of the target proteins as polyamine antiporters. To determine the role of BATs in polyamine homeostasis we tested their GFP fusions by transient expression in Nicotiana benthamiana. The GFP-tagged AtBAT1 and OsBAT1 displayed plastid localization in tobacco leaves by using confocal microscopy. Furthermore, the overexpression of OsBAT1 in A. thaliana wild-type results in novel phenotypes that could have potentially economical usage. This indicates that by altering the expression of a single gene of polyamine transporter, we can manipulate the plants phenotypes similar to natural variations.Metabolic pathways can be localized to one organelle or distributed across several cellular compartments. Previous work has identified only a single cytosolic pathway for putrescine synthesis in Arabidopsis. Here we show that both A. thaliana and soybeans have a plastid-localized putrescine pathway consisting of an arginine decarboxylase and an agmatinase that combine to synthesize putrescine from arginine.

Cellular uptake of clustered α2β1-integrin induces the formation of membrane compartments that subsequently mature into a multivesicular body (MVB). Enhanced internalization mediated by clustered integrins was observed upon infection by the picornavirus echovirus 1 (EVI). We elucidated the structural features of virus-induced MVBs (vMVBs) in comparison to antibody-induced control MVBs (mock infection) by means of high-pressure cryo fixation of cells followed by immuno electron tomography during early entry of the virus. Three-dimensional tomograms revealed a marked increase in the size and complexity of these vMVBs and the intraluminal vesicles (ILVs) at 2 and 3.5 hours post infection (p.i.), in contrast to the control MVBs without virus. Breakages in the membranes of vMVBs were detected from tomograms after 2 and especially after 3.5 h suggesting that these breakages could facilitate the genome release to the cytoplasm. The in situ neutral-red labeling of viral genome showed that virus uncoating starts as early as 30 min p.i., while an increase of permeability was detected in the vMVBs between 1 and 3 hours p.i., based on a confocal microscopy assay. Altogether, the data show marked morphological changes in size and permeability of the endosomes in the infectious entry pathway of this non-enveloped enterovirus and suggest that the formed breakages facilitate the transfer of the genome to the cytoplasm for replication.

Sugar-swollen reverse micelles and alternating polymer vesicles were prepared to examine interfacial self-assembly of both small and macromolecular sugar-based amphiphiles. Anhydrous, glassy sugar-sucrose laurate mixtures spontaneously dissolve in hydrocarbon oil at moderate temperatures and form sugar-swollen reverse micelles. The size of the micelles and the microemulsion viscosity depend on the mass ratio of sugar to surfactant. As sugar loading increases, the micelles increase in size and bulk viscosity decreases. Formation of sugar-oil complex fluids is related to the dynamics of the sugar in the supercooled liquid state, investigated by modulated differential scanning calorimetry.Alternating polymers of N-n-alkylmaleimides and vinyl gluconamide spontaneously form ultra small (10 to 20 nm) and medium (50 to 300 nm) sized vesicles when dissolved in water at room temperature. These materials have molecular weights approximately one hundred times higher than alternating oligomers of alkylmaleate and vinyl ether monomers, and demonstrate conclusively that alternating copolymers can form vesicles. The size and shape of the vesicles are characterized thoroughly by cryogenic-transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), and small angle neutron scattering (SANS). The copolymer vesicles exhibit alkyl chain length dependent release characteristics and bilayer thickness (1.7, 2.0, and 2.6 nm for alkyl chains of 10, 12, and 14 carbons, respectively).These nonionic alternating polymers contain no evident acidic or basic groups yet exhibit pH reversible self-assembly. The polymers form vesicles spontaneously in neutral, deionized water, precipitate under acidic conditions, and re-dissolve into optically clear, blue-tinted vesicular solutions upon neutralization of the turbid mixture. Cryo-TEM and DLS confirm that the size and structure of the vesicles remain the same after pH cycling. In the absence of ionic, acidic, and basic functionalities, the pH reversible self-assembly of this alternating polymer is likely driven by coordinated hydrogen bonding of protons within electron rich pockets of alternating vinyl gluconamide groups. This is consistent with measured titration curves and shifts in the infrared O-H stretch (3380 cm-1) of the gluconamide hydroxyl groups following acidification and neutralizing of the polymer in D2O.

In calcifying cartilage, early bone, and dentin, mineral crystal nucleation and growth are reported to occur first in matrix vesicles and then proceed within the extracellular organic matrices of these tissues (Lowenstam and Weiner, On Biomineralization, Oxford University Press, 1989). In normally mineralizing turkey leg tendons, type I collagen independently initiates mineral formation in areas isolated from matrix vesicles. In this tissue, collagen-mediated nucleation succeeds vesicle-mediated nucleation (Landis and Silver, Comp. Biochem. Physiol. A, 133:1135-1157, 2002). Sesamoid bones of vertebrates are unique in their size and close association with tendon, providing an opportunity to examine possible vesicle- and/or collagen-mediated mineralization in relation to bone and tendon. This study established a maturation timeline of murine metapodophalangeal sesamoid bones and investigated potential matrix vesicle presence and vesicle/collagen/mineral interactions in this tissue. Sesamoid bones, located between the metapodials and proximal phalanges, were examined in one- to six-week-old postnatal normal CD-1 mice through use of radiography, histology, and whole mount staining. These combined data provided the means to define the general morphology and development of the sesamoids, their association with tendon, collagen and proteoglycan presence, and surrounding tissue maturational changes. Murine metapodophalangeal sesamoid bones began mineralization as early as one week of age. The calcification of the epiphyseal plates of the metapodia and proximal phalanges began in advance of sesamoid mineral formation. Mineralization occurred first in the one-week metacarpophalangeal joint of the second digit as a mineral nodule within the cartilaginous core of the lateral-most sesamoid. Complete calcification was obtained between three and four weeks, resulting in a large, oblate sesamoid. Ossification of these small bones occurred before the epiphyseal plates were fully fused. In the TEM analysis of two-week metacarpophalangeal lateral sesamoids, extracellular matrix vesicles were found in small numbers and were not believed to be involved with mineral formation in this bone. In addition, the perpendicular orientation of the collagen fibers in the tissues examined was unexpected. Therefore, the roles of matrix vesicles and type I collagen in the mineralizing sesamoid bones were determined to be inconclusive. It is assumed that extensive proteoglycan networks and type II collagen fibers initiate mineralization in murine metapodophalangeal sesamoid bones.

Liquid-core capsules have wide-ranging applications in the high efficiency encapsulation and controlled delivery of drugs, dyes, enzymes, and many other substrates. These important applications have driven the rapid development of innovative techniques. Here, a direct and scalable approach was presented for encapsulating sub-micron liquid (oil or aqueous) drops within uniform

Quinapril is a nonsulfhydryl angiotensin converting enzyme inhibitor used in hypertension and congestive heart failure. Due to the anionic nature of quinapril at physiologic pH and its tripeptide-like structure, the renal disposition of quinapril may involve organic anion and peptide transport systems. To characterize the mechanism of renal drug transport, we examined the effect of quinapril on organic anion transport in renal basolateral membrane vesicles using p-aminohippurate (PAH) as a model substrate and on peptide transport in renal brush border membrane vesicles using glycylsarcosine (GlySar) as a model substrate. Renal membrane vesicles were prepared from rabbit kidneys and transport measurements were performed using a rapid filtration technique. In basolateral membrane vesicles, PAH uptake was markedly stimulated by an inwardly-directed sodium gradient and the presence of glutarate. Under these conditions, quinapril was as potent as probenecid in cis-inhibiting PAH uptake and more effective than enalapril, quinaprilat and enalaprilat. The dose-response curve for quinapril revealed an IC$\sb{50}$ value of 24.7 $\pm$ 2.5 $\mu$M. Lineweaver-Burk analysis showed that quinapril inhibited PAH transport in a competitive manner. Quinapril significantly increased the $K\sb{\rm m}$ of PAH uptake from 161 $\pm$ 35 $\mu$M to 428 $\pm$ 202 $\mu$M $(p

It is the purpose of this thesis to report further studies on the vesicles appearing in phage lysates of Escherichia coli, phage attachment to these vesicles, and the presence of similar vesicles in lysozyme and penicillin lysed cultures.