VictorJuiz/thesis huggingface dataset:
Development of a High-throughput Electrokinetically-controlled Heterogeneous Immunoassay Microfluidic Chip
Keywords: microfluidic
immunoassay
electrokinetic
lab-on-a-chip

This thesis was on the development of a high-throughput electrokinetically-controlled heterogeneous immunoassay (EK-IA) microfluidic chip for clinical application. Through a series of experimental studies, a high-throughput EK-IA was developed. This EK-IA was capable of automatically screening multiple analytes from up to 10 samples in parallel, in only 26 min. Flow control in an integrated microfluidic network was realized by numerical simulation of the transport processes. This EK-IA was successfully applied to detect E. coli O157:H7 antibody and H. pylori antibody from human sera with satisfactory accuracy. Simultaneous screening of both antibodies from human sera was also achieved, demonstrating the potential of this EK-IA for efficiently detecting multiple pathogenic infections in clinical settings. Preliminary work on the application of EK-IA to detect biomarkers of embryo development in embryo culture media also yielded good results. In addition to the experimental studies, the reaction kinetics of this microfluidic EK-IA has also been investigated, using both numerical simulation and a modified Damköhler number. Targeted towards a more sensitive assay, the influences of several important parameters on the reaction kinetics were studied. This EK-IA holds great promise for automated and high-throughput immunoassay in clinical environments.
The Influence of Release Modality on Synaptic Transmission at a Developing Central Synapse
Keywords: Calyx of Held
MNTB
Synaptic Transmission
Calcium Channels
Development
Synaptic Plasticity
Synaptic Delay
Cooperativity
Release Modality
Vesicle Release

The auditory brainstem is comprised of a number of synapses specialized for the transmission of high-fidelity synaptic signals. Within the first three postnatal weeks, these pathways acquire the ability to process high-frequency signals without compromising timing information. However, little is known regarding developmental adaptations which confer this ability. Situated in the sound localization pathway, the calyx of Held-medial nucleus of the trapezoid body synapse provides an ideal model for investigating such adaptations as both the pre- and postsynaptic neurons are accessible to electrophysiological experimentation. Using this synapse, we have shown herein that the spatial coupling between voltage-gated calcium channels (VGCCs) and synaptic vesicles (SVs) tightens during development. Immature synapses use a loosely-coupled arrangement of many N- and P/Q-type VGCCs (“microdomain” modality) while mature synapses use a tightly-coupled arrangement of fewer P/Q-type VGCCs, to release SVs (“nanodomain” modality). As a consequence of this tightening, synaptic delay (SD) shortens. By fluorescence- and electron microscopy of SVs near active zones, we further identified the filamentous protein septin 5 as a molecular substrate, differentiating the two release modalities, which may act as a spatial barrier separating VGCCs and SVs in immature synapses. Finally, we have demonstrated that changes in release modality affect the nature of short-term plasticity observed at this synapse. Using trains of action potentials as presynaptic voltage-commands, we showed that, downstream of calcium influx, the microdomain modality promotes short-term facilitation in excitatory postsynaptic currents (IEPSC), and calcium-dependent decreases in SD, with these being absent in synapses employing the nanodomain modality. In contrast, we found that as a result of depletion of SVs, short-term depression of IEPSC dominates in synapses using the nanodomain modality, and correlates with calcium-dependent increases in SD. These findings imply that the type of release modality has a significant impact on the strength and timing of synaptic responses. The microdomain modality imparts greater dynamic range in timing and strength, but does so at the cost of efficiency and fidelity, while the nanodomain modality is a key accomplishment consolidating the high-fidelity abilities of this synapse.
Mechanisms of Presynaptic CaV2.2 (N-type) Modulation
Keywords: calcium channel
g-protein
voltage-clamp
DRG
presynaptic
CaV2.2
chick
patch-clamp
Munc18
modulation
ion channel

Neurotransmitter release at presynaptic terminals is a complex process involving calcium ion influx through voltage-gated calcium channels (CaV). In addition to their role as entry points through which calcium influx may occur, CaV are now understood to be fundamental components of a common release-site complex that is highly adapted for modulation. Consistent with this model, I investigated mechanisms of modulating a presynaptic calcium channel, CaV2.2, via a heterotrimeric G-protein pathway. Using the patch-clamp technique, I demonstrated in chick dorsal root ganglion (DRG) neurons that the slow kinetics of G-protein inhibition of CaV2.2 via GTPgammaS were limited by the rate of GDP dissociation from the G-protein nucleotide binding site. In addition, I investigated the role of G-protein regulation of CaV2.2 currents evoked by action potential-like stimuli. Here, I characterized an inhibited current that was advanced in time with respect to uninhibited controls. These currents exhibited a shorter latency to current activation and faster deactivation. These findings may have important physiological ramifications on signal transduction and timing. In addition to G-protein regulation, presynaptic CaV2.2 have been demonstrated to exhibit a resistance to voltage-dependent inactivation (VDI), a property thought to be important in determining channel availability and synaptic excitability. I demonstrated a role for dynamic palmitoylation in conferring resistance to VDI in presynaptic terminals of the chick ciliary ganglion. Using tunicamycin, an inhibitor of palmitoylation, I induced a hyperpolarizing shift in the steady-state-inactivation (SSI) profile of presynaptic CaV2.2. Finally, I examined the role of a CaV interacting protein, Munc18, as a potential regulator of CaV. I probed for alterations in CaV2.2 function in DRG neurons that had been transfected with Munc18 or Munc18 siRNA. Despite the intimate interaction between Munc18 and CaV2.2, no major effects on the fundamental characteristics of CaV2.2 function were observed. However, a hyperpolarizing shift in the inactivation profile of CaV2.2 was determined in DRG neurons in which Munc18 was knocked down. It is not clear if this was a direct consequence of Munc18 perturbation.
Development and Application of a Flow-through Sampler for Semi-volatile Organic Compounds in Air
Keywords: Flow-Through Sampler
semi-volatile organic compounds
frontal chromatographic theory
air sampling
Tibet
breakthrough behavior

The investigation of the atmospheric fate and transport of semi-volatile organic compounds (SOCs) often requires the sampling of large volumes of air (>100 m3) in a relatively short period of time. Conventionally high-volume pumps are not suitable for remote areas without access to reliable network power. We have developed a flow through sampler for such situations. It consists of a horizontally-oriented flow-tube, that can collect gaseous and particle-bound SOCs from large volumes of air by turning into the wind and having the wind blow through a porous sampling medium such as polyurethane foam. Through both indoor and outdoor experiments, we quantified its air sampling rate (through battery operated anemometers inside and outside of the flow tube), its sampling efficiency (by theoretical plate number analysis of the break-though curves for PCBs, PAHs, OCPs and PBDEs), and its accuracy (by comparison of concentrations, time trends, temperature dependences and isomer ratios with those obtained by conventional high-volume sampling) under conditions of constant and variable meteorological conditions (wind speed, temperature). The flow-through sampler was deployed to monitor SOC concentrations at a remote Chinese research station located close to Nam Co Lake, Tibet. During the campaign, fifteen 1 month-long samples were taken, corresponding to sample volumes between 5,000 and 20,000 m3. Despite those large sample volumes, only HCB and HCHs experienced break-through, but application of frontal chromatograph theory allows the estimation of breakthrough-corrected air concentrations even for those relatively volatile SOCs. The pesticide levels at Nam Co are generally very low. Most pesticides had higher levels during summer, resulting in a strong temperature dependence. This is correlated with air mass origin across the Himalayas in the Gangetic plains of India and Bangladesh. The flow through sampler constitutes a feasible method for reliably and quantitatively collecting SOCs from large air volumes.
Organic Contaminant Release from Melting Snow
Keywords: organic contaminants
snow

Snow efficiently scavenges organic contaminants from the atmosphere. Those chemicals can be released in short, concentrated pulses during spring snowmelt, potentially affecting both aquatic and terrestrial environments. In order to investigate the behavior of organic contaminants in melting snow laboratory experiments were conducted using artificial snow spiked with the organic target substances naphthalene, phenanthrene, pyrene, benzo(ghi)perylene, lindane, and atrazine. The behavior of those substances during melting is dependent on their partitioning between the different phases present within the bulk snow. Very water soluble organic chemicals, such as atrazine, are preferentially released at an early stage of melting similar to inorganic ions. This first chemical flush becomes more pronounced when a deep, aged, and relatively homogeneous snow pack is exposed to intense melting. Hydrophobic substances attached to particles, such as high molecular weight polycyclic aromatic hydrocarbons, are often released at the very end of the melt period. Dirt cones at the surface of a deep snow pack amplify this late chemical enrichment. Whereas chemicals that are clearly hydrophilic or hydrophobic are likely to be released in pulse loads, the snowmelt behavior of chemicals with intermediate partitioning properties, such as lindane, is more dependent on the varying snow pack and melt characteristics. A notable fraction of volatile chemicals may transfer from the melting snow pack to the lower atmosphere due to evaporation. Shallow snow covers in temperate regions with depths below 10cm that are exposed to recurrent melt phases during the winter are less likely to lead to pulse releases. When the melt water runs off as overland flow over frozen ground, pollutants are directly transferred into catchment streams, and their water quality is affected most. Melt water flow along the soil subsurface dilutes and buffers organic contaminant loads. The findings of this laboratory study may assist in the development of contaminant fate models that includes snowmelt processes. The results may further help to explain observations of organic contaminants in natural snow environments.
Wittgenstein and Köhler on Seeing and Seeing Aspects: A Comparative Study
Keywords: Wittgenstein
Gestalt Psychology
seeing as
Kohler
perception
visual experience
aspect

This thesis examines the relation between philosopher Ludwig Wittgenstein’s 1940s writings on seeing and seeing aspects and Gestalt psychologist Wolfgang Köhler’s theory of perception as set out in his Gestalt Psychology (1929). I argue that much of the existing literature on the Wittgenstein-Köhler relation distorts Köhler’s ideas and thus also Wittgenstein’s engagement with Köhler’s ideas. This double distortion underrates Köhler’s insights, misconstrues Wittgenstein’s complaints against Köhler, and masks points of contact between the two concerning the nature and description of human perceptual experience. In my view, Wittgenstein sympathizes with Köhler’s call to reflect on basic psychological categories such as “experience”, his respect for the “naïve” experience of the layperson, his method of “rediscovering” pervasive features of experience that escape our notice, and his efforts to identify intellectual prejudices that stymie inquiry. But a warning emerges from Wittgenstein’s discussions of seeing and seeing aspects: It is especially difficult to command a clear view of 'seeing' and its interrelations with other everyday, psychological concepts. I argue that Wittgenstein’s far-reaching criticism of Köhler is that the latter's account of visual “organization” overextends an analogy between seeing and seeing aspects and pushes aside other justifiable comparisons, for example between seeing and thinking and seeing and imagining. A consequence of Wittgenstein's criticism is that Kohler falls short of his aim to depict faithfully naïve visual experience. Moreover, despite Kohler's commitment to battling prejudices, the latter's emphasis on similarities between seeing and seeing aspects to the exclusion of their differences is a form of intellectual prejudice. For Wittgenstein various comparisons are justifiable by appeal to the interrelations between ‘seeing’ and other psychological concepts. A perspicuous view of the concept 'seeing' involves steady appreciation of the multitude of justifiable, criss-crossing comparisons. So although Wittgenstein does not deny Köhler’s claim that organization is a feature of visual experience rather than thinking, he does not unqualifiedly endorse it either. We have conceptual grounds for various ways of speaking about our experiences of aspects.
A Modified Yeast One-hybrid Sytem to Investigate Protein-protein and Protein: DNA Interactions
Keywords: protein engineering
yeast one-hybrid assay
protein:DNA interaction
protein-protein interaction

A modified yeast one-hybrid (MY1H) system has been developed for in vivo investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site, whereas our MY1H allows coexpression of two different proteins and examination of their activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H system could be used to examine proteins that contribute inhibitory, repressive, coactivational or bridging functions to the protein under investigation, as well as potential extension toward library screening for identification of novel accessory proteins. After development and validation of the MY1H with the p53/LTAg/53BP2 system, we applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins, the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE). The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false negative observations were turned into true positives by increasing the number of DNA target sites upstream of the reporter genes and increasing the number of activator domains fused to the two monomers. This methodology may help trouble-shooting false negatives stemming from unproductive transcription in yeast genetic assays, which can be a common problem. In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems; these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ) elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription. The present results provide valuable information on DNA-mediated protein heterodimerization and specific DNA binding, as well as the relationship between protein structure and DNA-binding function.
Timed State Tree Structures: Supervisory Control and Fault Diagnosis
Keywords: Discrete Event Systems
Hierarchical Systems
Supervisory Control
Fault Diagnosis
State Tree Structures

It is well known that the optimal nonblocking supervisory control problem of timed discrete event systems is NP-hard, subject in particular to state space explosion that is exponential in the number of system components. In this thesis, we propose to manage complexity by organizing the system as a Timed State Tree Structure (TSTS). TSTS are an adaptation of STS to timed Supervisory Control Theory (SCT). Based on TSTS we present an e±cient recursive symbolic algorithm that can perform nonblocking supervisory control design for systems of state size 10^12 and higher. Failure diagnosis is the process of detecting and identifying deviations of a system from its normal behavior using the information available through sensors. A method for fault diagnosis of the TSTS model is proposed. A state based diagnoser is constructed for each timed holon of TSTS. Fault diagnosis is accomplished using the state estimates provided by the timed holon diagnosers. The diagnosers may communicate among each other in order to update their state estimates. At any given time, only a subset of the diagnosers are operational, and as a result, instead of the entire model of the system, only the models of the timed holons associated with the operational diagnosers are used. It is shown that the computational complexity of constructing and storing the transition systems required for diagnosis in the proposed approach is polynomial in the number of system components, whereas in the original monolithic approach the computational complexity is exponential.
Leaf Conjugacies on the Torus
Keywords: Partially Hypberbolic Systems
Leaf Conjugacies

If a partially hyperbolic diffeomorphism on a torus of dimension d greater than 3 has stable and unstable foliations which are quasi-isometric on the universal cover, and its center direction is one-dimensional, then the diffeomorphism is leaf conjugate to a linear toral automorphism. In other words, the hyperbolic structure of the diffeomorphism is exactly that of a linear, and thus simple to understand, example. In particular, every partially hyperbolic diffeomorphism on the 3-torus is leaf conjugate to a linear toral automorphism.
The Double-edged Sword: A Critical Race Africology of Collaborations between Blacks and Whites in Racial Equity Work
Keywords: Anti-racism
Critical Study of Whiteness
Embodied Knowledge
Collaboration
Afrocentricity
Critical Whiteness Studies
Epistemology of Ignorance
Resistance
African Agency
Racism
Race
Salience of Race
Interest Convergence

In recent years, there has been a significant amount of new attention to white dominance and privilege (or whiteness) as the often unmarked inverse of racial oppression. This interest has spawned the academic domain called Critical Whiteness Studies (CWS). While the critical investigation of whiteness is not new, and has been pioneered by Black scholars beginning at least since the early 1900s in the work of W. E. B. Du Bois, what is notable about this new interest in whiteness is its advancement almost exclusively by white scholars. The paucity of literature centering the Black voice in the study of whiteness both suggests the lack of appreciation for the importance of this perspective when researching the phenomenon of racial dominance, and raises questions about the manner in which racial equity work is approached by some Whites who do work that is intended to advance racial equity. This study investigates the context of racial equity collaborations between Blacks and Whites, responding to this knowledge deficit in two ways: a) it centers the Black voice, specifically and intentionally seeking the perspectives of Blacks about racial equity collaborations b) it investigates the nature and effects of the relationships between Blacks and Whites in these collaborative endeavours. This qualitative research study uses in-depth interview data collected from ten Black racial equity workers who collaborate with Whites in doing racial equity work. The data makes evident that the Black participants find these collaborations to be necessary and strategic while at the same time having the potential to undermine their own agency. The study examines this contradiction, discussing several manifestations of it in the lives of these Black racial equity workers. It outlines the importance of Black embodied knowledge to racial equity work and to these collaborations, and outlines an epistemology of unknowing and a politics of humility that these Blacks seek in their white colleagues. The study also outlines the collective and individual strategies used by these Black racial equity workers to navigate and resist the contradictory terrain of their collaborations with Whites in racial equity work.
Mechanical Forces Regulate Cartilage Tissue Formation by Chondrocytes via Integrin-mediated cell Spreading
Keywords: articular chondrocytes
tissue engineering

In vitro grown cartilage is functionally inferior to native tissue, and improvements in its quality should be attempted so it can be used therapeutically. In these studies we investigated the effects of cell shape on tissue quality through alteration of substrate geometry and application of mechanical stimuli. Articular chondrocytes were isolated and cultured on the surface Ti-6Al-4V substrates with various geometries. When cultured on fully porous titanium alloy substrates, chondrocyte spreading was enhanced over those grown on substrates with solid bases. Chondrocytes which remained round did not synthesize significant amounts of matrix and were thus unable to form cartilaginous tissue. In contrast, chondrocytes which were directed to spread to a limited amount, resulting in a polygonal morphology, accumulated significantly more matrix molecules and in time formed cartilage-like tissue. Computational fluid dynamics analyses demonstrated that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on substrates with solid bases where media flow-through is restricted. Integrin-blocking experiments revealed that integrins are important regulators of cell shape, and appeared to influence the accumulation of collagen and proteoglycans by chondrocytes. Furthermore, compressive mechanical stimulation induced a rapid, transient increase in chondrocyte spreading by 10 minutes, followed by a retraction to pre-stimulated size within 6 hours. This has been shown to be associated with increased accumulation of newly synthesized proteoglycans. Blocking the α5β1 integrin, or its β1 subunit, inhibited cell spreading and resulted in a partial inhibition of compression-induced increases in matrix accumulation, thereby substantiating the role of β1 integrins in this process. These results suggest that both fluid induced shear forces and compressive forces regulate chondrocyte matrix accumulation by altering cell morphology, which is mediated by integrins. Identifying the molecular mechanisms that influence chondrocyte shape and thus tissue formation may ultimately lead to the development of a tissue that more closely resembles native articular cartilage.