Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines

The formulation of plasmid DNA (pDNA) in cationic liposomes is a promising strategy to improve the potency of DNA vaccines. In this respect, physicochemical parameters such as liposome size may be important for their efficacy. The aim of the current study was to investigate the effect of vesicle size on the in vivo performance of liposomal pDNA vaccines after subcutaneous vaccination in mice. The tissue distribution of cationic liposomes of two sizes, 500 nm (PDI 0.6) and 140 nm (PDI 0.15), composed of egg PC, DOPE and DOTAP, with encapsulated OVA-encoding pDNA, was studied by using dual radiolabeled pDNA-liposomes. Their potency to elicit cellular and humoral immune responses was investigated upon application in a homologous and heterologous vaccination schedule with 3 week intervals. It was shown that encapsulation of pDNA into cationic lipsomes resulted in deposition at the site of injection, and strongest retention was observed at large vesicle size. The vaccination studies demonstrated a more robust induction of OVA-specific, functional CD8+ T-cells and higher antibody levels upon vaccination with small monodisperse pDNA-liposomes, as compared to large heterodisperse liposomes or naked pDNA. The introduction of a PEG-coating on the small cationic liposomes resulted in enhanced lymphatic drainage, but immune responses were not improved when compared to non-PEGylated liposomes. In conclusion, it was shown that the physicochemical properties of the liposomes are of crucial importance for their performance as pDNA vaccine carrier, and cationic charge and small size are favorable properties for subcutaneous DNA vaccination.

Modelling of induction motors for system faults and transient stability studies

The research carried out in this thesis was mainly concerned with the effects of large induction motors and their transient performance in power systems. Computer packages using the three phase co-ordinate frame of reference were developed to simulate the induction motor transient performance. A technique using matrix algebra was developed to allow extension of the three phase co-ordinate method to analyse asymmetrical and symmetrical faults on both sides of the three phase delta-star transformer which is usually required when connecting large induction motors to the supply system. System simulation, applying these two techniques, was used to study the transient stability of a power system. The response of a typical system, loaded with a group of large induction motors, two three-phase delta-star transformers, a synchronous generator and an infinite system was analysed. The computer software developed to study this system has the advantage that different types of fault at different locations can be studied by simple changes in input data. The research also involved investigating the possibility of using different integrating routines such as Runge-Kutta-Gill, RungeKutta-Fehlberg and the Predictor-Corrector methods. The investigation enables the reduction of computation time, which is necessary when solving the induction motor equations expressed in terms of the three phase variables. The outcome of this investigation was utilised in analysing an introductory model (containing only minimal control action) of an isolated system having a significant induction motor load compared to the size of the generator energising the system.

Topographic mapping and source localization of the pattern onset visual evoked magnetic response

The topography of the visual evoked magnetic response (VEMR) to a pattern onset stimulus was investigated using 4 check sizes and 3 contrast levels. The pattern onset response consists of three early components within the first 200ms, CIm, CIIm and CIIIm. The CIIm is usually of high amplitude and is very consistent in latency within a subject. Half field (HF) stimuli produce their strongest response over the contralateral hemisphere; the RHF stimulus exhibiting a lower positivity (outgoing field) and an upper negativity (ingoing field), rotated towards the midline. LHF stimulation produced the opposite response, a lower negative and an upper positive. Larger check sizes produce a single area of ingoing and outgoing field while smaller checks produce on area of ingoing and outgoing field over each hemisphere. Latency did not appear to vary with change in contrast but amplitudes increased with increasing contrast. A more detailed topographic study incorporating source localisation procedures suggested a source for CIIm - 4cm below the scalp, close to the midline with current flowing towards the lateral surface. Similar depth and position estimates but with opposite polarity were obtained for the pattern shift P100m previously. Hence, the P100m and the CIIm may originate in similar areas of visual cortex but reveal different aspects of visual processing. © 1992 Human Sciences Press, Inc.

Topographic mapping and source localization of the pattern reversal visual evoked magnetic response

The topography of the visual evoked magnetic response (VEMR) to pattern reversal stimulation was studied in four normal subjects using a single channel BTI magnetometer. VEMRs were recorded from 20 locations over the occipital scalp and the topographic distribution of the most consistent component (P100M) studied. A single dipole in a sphere model was fitted to the data. Topographic maps were similar when recorded two months apart on the same subject to the same stimulus. Half field (HF) stimulation elicited responses from sources on the medial surface of the calcarine fissure mainly in the contralateral hemisphere as predicted by the cruciform model. The full field (FF) responses to large checks were approximately the sum of the HF responses. However, with small checks, FF stimulation appeared to activate a different combination of sources than the two HFs. In addition, HF topography was more consistent between subjects than FF for small check sizes. Topographic studies of the VEMR may help to explain the analogous visual evoked electrical response and will be essential to define optimal recording positions for clinical applications.

Differential localization of GABAA receptor subunits in relation to rat striatopallidal and pallidopallidal synapses

As a central integrator of basal ganglia function, the external segment of the globus pallidus (GP) plays a critical role in the control of voluntary movement. The GP is composed of a network of inhibitory GABA-containing projection neurons which receive GABAergic input from axons of the striatum (Str) and local collaterals of GP neurons. Here, using electrophysiological techniques and immunofluorescent labeling we have investigated the differential cellular distribution of a1, a2 and a3 GABAA receptor subunits in relation to striatopallidal (Str-GP) and pallidopallidal (GP-GP) synapses. Electrophysiological investigations showed that zolpidem (100 nm; selective for the a1 subunit) increased the amplitude and the decay time of both Str-GP and GP-GP IPSCs, indicating the presence of the a1 subunits at both synapses. However, the application of drugs selective for the a2, a3 and a5 subunits (zolpidem at 400 nm, L-838,417 and TP003) revealed differential effects on amplitude and decay time of IPSCs, suggesting the nonuniform distribution of non-a1 subunits. Immunofluorescence revealed widespread distribution of the a1 subunit at both soma and dendrites, while double- and triple-immunofluorescent labeling for parvalbumin, enkephalin, gephyrin and the ?2 subunit indicated strong immunoreactivity for GABAAa3 subunits in perisomatic synapses, a region mainly targeted by local axon collaterals. In contrast, immunoreactivity for synaptic GABAAa2 subunits was observed in dendritic compartments where striatal synapses are preferentially located. Due to the kinetic properties which each GABAAa subunit confers, this distribution is likely to contribute differentially to both physiological and pathological patterns of activity.

Predicting and diagnosing faults in wastewater treatment process by Bayesian Belief Networks

Diagnosing faults in wastewater treatment, like diagnosis of most problems, requires bi-directional plausible reasoning. This means that both predictive (from causes to symptoms) and diagnostic (from symptoms to causes) inferences have to be made, depending on the evidence available, in reasoning for the final diagnosis. The use of computer technology for the purpose of diagnosing faults in the wastewater process has been explored, and a rule-based expert system was initiated. It was found that such an approach has serious limitations in its ability to reason bi-directionally, which makes it unsuitable for diagnosing tasks under the conditions of uncertainty. The probabilistic approach known as Bayesian Belief Networks (BBNS) was then critically reviewed, and was found to be well-suited for diagnosis under uncertainty. The theory and application of BBNs are outlined. A full-scale BBN for the diagnosis of faults in a wastewater treatment plant based on the activated sludge system has been developed in this research. Results from the BBN show good agreement with the predictions of wastewater experts. It can be concluded that the BBNs are far superior to rule-based systems based on certainty factors in their ability to diagnose faults and predict systems in complex operating systems having inherently uncertain behaviour.

Selective localization of organoclay and effects on the morphology and mechanical properties of LDPE/PA11 blends with distributed and co-continuous morphology

A study was made on the effect of small amounts of organically modified clay on the morphology and mechanical properties of blends of low-density polyethylene and polyamide 11 at different compositions. The influence of the filler on the blend morphology was investigated using wide angle X-ray diffractometry, scanning and transmission electron microscopy and selective extraction experiments. The filler was found to locate predominantly in the more hydrophilic polyamide phase. Although such uneven distribution does not have a significant effect on the onset of phase co-continuity of the polymer components, it brings about a drastic refinement of the microstructure for the blends both with droplets/matrix and co-continuous morphologies. In addition to the expected reinforcing action of the filler, the resulting fine microstructure plays an important role in enhancing the mechanical properties of the blends. This is essentially because of a good quality of stress transfer across the interface between the constituents, which also seems to benefit for a good interfacial adhesion promoted by the filler. Our results provide the experimental evidence for the capabilities of nanoparticles added to multiphase polymer systems to act selectively as a reinforcing agent for specific domains of the material and as a medium able to assist the refinement of the polymer phases during mixing.

Hypercapnia induces cleavage and nuclear localization of RelB protein, giving insight into CO2 sensing and signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-?B pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-?B family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3ß or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-?B family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

Seventeen a-subunit isoforms of paramecium V-ATPase provide high specialization in localization and function

In the Paramecium tetraurelia genome, 17 genes encoding the 100-kDa-subunit (a-subunit) of the vacuolar-proton-ATPase were identified, representing by far the largest number of a-subunit genes encountered in any organism investigated so far. They group into nine clusters, eight pairs with >82% amino acid identity and one single gene. Green fluorescent protein-tagging of representatives of the nine clusters revealed highly specific targeting to at least seven different compartments, among them dense core secretory vesicles (trichocysts), the contractile vacuole complex, and phagosomes. RNA interference for two pairs confirmed their functional specialization in their target compartments: silencing of the trichocyst-specific form affected this secretory pathway, whereas silencing of the contractile vacuole complex-specific form altered organelle structure and functioning. The construction of chimeras between selected a-subunits surprisingly revealed the targeting signal to be located in the C terminus of the protein, in contrast with the N-terminal targeting signal of the a-subunit in yeast. Interestingly, some chimeras provoked deleterious effects, locally in their target compartment, or remotely, in the compartment whose specific a-subunit N terminus was used in the chimera.

In situ diagnostics and prognostics of wire bonding faults in IGBT modules for electric vehicle drives

This paper presents a diagnostic and prognostic condition monitoring method for insulated-gate bipolar transistor (IGBT) power modules for use primarily in electric vehicle applications. The wire-bond-related failure, one of the most commonly observed packaging failures, is investigated by analytical and experimental methods using the on-state voltage drop as a failure indicator. A sophisticated test bench is developed to generate and apply the required current/power pulses to the device under test. The proposed method is capable of detecting small changes in the failure indicators of the IGBTs and freewheeling diodes and its effectiveness is validated experimentally. The novelty of the work lies in the accurate online testing capacity for diagnostics and prognostics of the power module with a focus on the wire bonding faults, by injecting external currents into the power unit during the idle time. Test results show that the IGBT may sustain a loss of half the bond wires before the impending fault becomes catastrophic. The measurement circuitry can be embedded in the IGBT drive circuits and the measurements can be performed in situ when the electric vehicle stops in stop-and-go, red light traffic conditions, or during routine servicing.

An integrated functional mapping and source localization platform for brain research

The premise of this dissertation is to create a highly integrated platform that combines the most current recording technologies for brain research through the development of new algorithms for three-dimensional (3D) functional mapping and 3D source localization. The recording modalities that were integrated include: Electroencephalography (EEG), Optical Topographic Maps (OTM), Magnetic Resonance Imaging (MRI), and Diffusion Tensor Imaging (DTI). This work can be divided into two parts: The first part involves the integration of OTM with MRI, where the topographic maps are mapped to both the skull and cortical surface of the brain. This integration process is made possible through the development of new algorithms that determine the probes location on the MRI head model and warping the 2D topographic maps onto the 3D MRI head/brain model. Dynamic changes of the brain activation can be visualized on the MRI head model through a graphical user interface. The second part of this research involves augmenting a fiber tracking system, by adding the ability to integrate the source localization results generated by commercial software named Curry. This task involved registering the EEG electrodes and the dipole results to the MRI data. Such Integration will allow the visualization of fiber tracts, along with the source of the EEG, in a 3D transparent brain structure. The research findings of this dissertation were tested and validated through the participation of patients from Miami Children Hospital (MCH). Such an integrated platform presented to the medical professionals in the form of a user-friendly graphical interface is viewed as a major contribution of this dissertation. It should be emphasized that there are two main aspects to this research endeavor: (1) if a dipole could be situated in time at its different positions, its trajectory may reveal additional information on the extent and nature of the brain malfunction; (2) situating such a dipole trajectory with respect to the fiber tracks could ensure the preservation of these fiber tracks (axons) during surgical interventions, preserving as a consequence these parts of the brain that are responsible for information transmission.

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating phosphatidylinositol 3 kinase localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. ^ It was initially found that comparing to wild type cells, gsk3 - cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. ^ I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.^

Design and implementation of a relative localization system for ground and aerial robotic teams

The main focus of this thesis is to address the relative localization problem of a heterogenous team which comprises of both ground and micro aerial vehicle robots. This team configuration allows to combine the advantages of increased accessibility and better perspective provided by aerial robots with the higher computational and sensory resources provided by the ground agents, to realize a cooperative multi robotic system suitable for hostile autonomous missions. However, in such a scenario, the strict constraints in flight time, sensor pay load, and computational capability of micro aerial vehicles limits the practical applicability of popular map-based localization schemes for GPS denied navigation. Therefore, the resource limited aerial platforms of this team demand simpler localization means for autonomous navigation. Relative localization is the process of estimating the formation of a robot team using the acquired inter-robot relative measurements. This allows the team members to know their relative formation even without a global localization reference, such as GPS or a map. Thus a typical robot team would benefit from a relative localization service since it would allow the team to implement formation control, collision avoidance, and supervisory control tasks, independent of a global localization service. More importantly, a heterogenous team such as ground robots and computationally constrained aerial vehicles would benefit from a relative localization service since it provides the crucial localization information required for autonomous operation of the weaker agents. This enables less capable robots to assume supportive roles and contribute to the more powerful robots executing the mission. Hence this study proposes a relative localization-based approach for ground and micro aerial vehicle cooperation, and develops inter-robot measurement, filtering, and distributed computing modules, necessary to realize the system. The research study results in three significant contributions. First, the work designs and validates a novel inter-robot relative measurement hardware solution which has accuracy, range, and scalability characteristics, necessary for relative localization. Second, the research work performs an analysis and design of a novel nonlinear filtering method, which allows the implementation of relative localization modules and attitude reference filters on low cost devices with optimal tuning parameters. Third, this work designs and validates a novel distributed relative localization approach, which harnesses the distributed computing capability of the team to minimize communication requirements, achieve consistent estimation, and enable efficient data correspondence within the network. The work validates the complete relative localization-based system through multiple indoor experiments and numerical simulations. The relative localization based navigation concept with its sensing, filtering, and distributed computing methods introduced in this thesis complements system limitations of a ground and micro aerial vehicle team, and also targets hostile environmental conditions. Thus the work constitutes an essential step towards realizing autonomous navigation of heterogenous teams in real world applications.