Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1980. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Includes bibliographical references. / by David Bazeley Tuckerman. / M.S.

Dans les organismes multicellulaires, les cellules génèrent et subissent des forces mécaniques qui se propagent aux cellules voisines. Ces forces peuvent déterminer la forme des tissus et organes, et aussi être converties en signaux biochimiques. Dans un épithélium, les cellules forment un tissu en adhérant directement les unes aux autres grâce à des complexes d’adhérence, tels que les Jonctions Adhérentes. Ces Jonctions Adhérentes sont composées de protéines transmembranaires les E-cadhérines, dont la partie cytoplasmique est sous tension générée par le cytosquelette d’actomyosine par un lien assurée par la β-caténine. La β-caténine est aussi un cofacteur de transcription majeur qui régule l’activité de gènes impliqués dans la transition épithélio-mésenchymateuse une fois dans le noyau. L’accumulation nucléaire et l’activité transcriptionnelle de la β-caténine peuvent avoir lieu à la suite de stimulations mécaniques dans des situations physiologiques et pathologiques, et ont été proposées comme la conséquence d’une libération de la β-caténine des Jonctions Adhérentes suite à sa phosphorylation. Néanmoins, les preuves directes de ce phénomène et ses mécanismes manquent, et le rôle qu’y tient la tension des E-cadhérines n’est pas connu.Dans cette thèse, nous avons établi la relation entre la tension des E-cadhérines et la localisation nucléaire et l’activité de la β-caténine, prouvé l’existence d’une translocation de la membrane au noyau de la β-caténine, et caractérisé les mécanismes moléculaires sous-jacents dans des cellules en migration induite par un facteur de croissance ou par blessure sur un épithélium, deux conditions qui récapitulent au moins partiellement une transition épithélio-mésenchymateuse.Nous avons montré que l’accumulation nucléaire de la β-caténine est due à un départ substantiel de celle-ci de la membrane, spécifiquement dans les cellules en migration. Cette translocation a lieu en aval d’une voie de signalisation impliquant les kinases Src et FAK, et qui conduit à une relaxation de tension des E-cadhérines. Le mécanisme sous-jacent implique une réorganisation du cytosquelette d’actine, caractérisé par un enrichissement des fibres des stress ventrales, soutenant les protrusions, en phospho-myosine, au détriment du cortex d’actine des Jonctions Adhérentes. En revanche, les phosphorylations dans le complexe cadhérine/caténine ne sont pas requises. Ces résultats démontrent que les E-cadhérines ont un rôle de senseur de la mécanique intracellulaire, et que les adhésions focales sont impliquées dans l’activation de la voie de signalisation β-caténine / In multicellular organisms, cells generate and experience mechanical forces that propagate between and within cells. These forces may shape cells, tissues and organs, and also convert into biochemical signals. In a simple epithelium, cells form tissue sheets by directly adhering to one another through adhesion complexes, such as the Adherens Junctions. Adherens Junctions comprise transmembrane proteins E-cadherins, which are under actomyosin-generated tension via a link that contains β-catenin. β-catenin is also a major transcription cofactor that regulates gene activity associated with Epithelial-to-Mesenchyme Transition when translocated in the nucleus. β-catenin nuclear localization and transcriptional activity are mechanically inducible in a variety of healthy and disease models and were proposed to follow phosphorylation-induced -catenin release from E-cadherin. However, direct evidence for this translocation and these mechanisms are lacking, and whether E-cadherin tension is involved is unknown.In this thesis, we assess the relationship between E-cadherin tension and β-catenin nuclear localization and activity, determine the relevance of β-catenin shuttling between membrane and nucleus, and characterize the underlying molecular mechanisms in cells migrating in an at least partial EMT-like fashion upon hepatocyte growth factor (HGF) or wound stimulation. We showed that β-catenin nuclear activity follows a substantial release from the membrane that is specific to migrating cells. This translocation occurs downstream of the Src-FAK pathway, which targets E-cadherin tension relaxation. The underlying mechanisms sufficiently involve actomyosin remodeling, characterized by an enrichment of ventral stress fibers that capture phosphomyosin at the expense of the cortex at Adherens Junctions. In contrast, phosphorylations of the cadherin/catenin complex are not substantially required. These data demonstrate that E-cadherin acts as a sensor of intracellular mechanics in a crosstalk with cell-substrate adhesions that targets β-catenin signaling

Gap junctions are a class of membrane proteins that facilitate cell-to-cell communication by forming channels that directly couple the cytoplasm of neighboring cells. The channels are composed of monomers called connexins. Humans express 21 connexin isoforms in a cell-type specific fashion, and each isoform has distinct mechanisms of permeation and regulation. Co-assembly of multiple isoforms into a single intercellular channel can change channel properties, such as conductance and selectivity to substrates (e.g., ions, metabolites and signaling molecules). However, the mechanistic basis for this functional diversity has remained poorly understood. This lack of mechanistic insight has been due in large part to the lack of high-resolution (atomic-level) structural knowledge on this class of proteins. Prior to this work, the only high-resolution information available on gap junction structure came from a single connexin isoform, connexin-26 (cx26). CryoEM has recently transformed from a low-resolution technique into one capable of rivaling the atomic-level resolutions achieved by x-ray crystallography -- but without the necessity for crystal formation, which has hindered progress towards understanding many classes of proteins (ie, membrane proteins, intrinsically disordered cell signaling complexes and other structurally dynamic systems). For my thesis research, I applied novel methods in single particle electron cryo-microscopy (CryoEM) to study a class of membrane proteins called gap junctions isolated from native lens tissue, as well as two signaling complexes not amenable to other structural techniques. I determined the structure of the lens gap junction, which contains connexin-46 (cx46) and connexin-50 (cx50), to a resolution of 3.4 Å and generated atomic models for both connexin isoforms. Structural analysis paired with molecular dynamics gave insight into energetic features of these channels that determine their isoform-specific conductance and selectivity to electrically charged ions. The cx46/50 gating domain was found to be stabilized by hydrophobic anchors, and also seems to adopt a more stable open state than found in cx26. Genetic mutations associated with congenital cataract formation were found to map to hot-spots of conserved structural and functional importance, rationalizing their disease-causing effects. As part of collaborative efforts, I used methods in single particle EM to characterize two separate signaling complexes that had proven difficult to study with x-ray crystallography and NMR spectroscopy. One system, Ca2+/Calmodulin Kinase II (CaMKII), is a signaling complex in the brain involved in memory formation. Characterization of the CaMKII complex by single particle EM revealed an extended state, which was also shown to be prevalent in cells -- giving more depth to our understanding of how this signaling molecule functions. The second collaboration characterized the multimeric binding sites of the hub protein LC8, which interacts with the disordered region of a transcription factor (ASCIZ). This provided support for a novel model of transcription regulation, wherein LC8 fine-tunes its own transcription levels through multi-valent binding to the disordered region of its own regulatory transcription factor.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D. in Cancer Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

The electrochemical reduction of para-substituted aryldiazonium cations is a convenient method of introducing chemical functional groups to a surface. The number of conductive surfaces that have been used for this purpose is rapidly expanding. The body of work presented in this thesis will serve to further investigate this method as it applies to polycrystalline gold surfaces. The stability of diazonium-derived nitroazobenzene (NAB) layers on Au was investigated by subjecting them to a variety of treatments including prolonged exposure to UV radiation, refluxing solvents, ultrasonication, chemical displacement by octadecanethiol (ODT), and the application of negative potentials to -1.5 V vs Ag/AgCl. Infrared reflection-absorption spectroscopy (IRRAS) and electrochemical blocking were used to make the assessments. The films are very resistant to ODT displacement reactions, moderately resistant to ultrasonication and refluxing; but not very resistant to the other treatments. In most cases, quantitative IRRAS measurements indicate that > 50 % of the layer resists the treatments. A direct, side-by-side comparison of the stability of nitrobenzene (NB) layers deposited electrochemically from nitrobenzene diazonium cations to self-assembled monolayers (SAMs) of mercaptonitrobenzene was made. Both types of layers are prone to removal by the various treatments. This is likely due to the presence of weakly bound, physisorbed material in addition to more strongly bound material. Immersion in an ODT solution results in complete displacement of the thiol derived nitrobenzene monolayer but does not completely displace the diazonium-derived layer. Two-component, mixed molecular layers comprised of diazonium-derived NAB and dodecanethiolate (DDT) were prepared using a sequential deposition approach. The aryl component is first deposited electrochemically, followed by immersion in a solution of DDT. We will demonstrate that control over the composition of the layers can be achieved by manipulating the concentration of NAB diazonium cations at the electrochemical grafting step. The mixed layers were characterized by reflection-absorption spectroscopy, atomic force microscopy, electrochemical blocking, and x-ray photoelectron spectroscopy. The electron transfer kinetics of hexaammineruthenium(III) chloride were examined at the mixed layer electrodes. The kinetics are highly dependent on the relative proportions of NAB and DDT present and the thickness of the NAB component. The NAB:DDT mixed films were employed as the molecular layer in molecular electronics junctions. We examined the suitability of Al2O3/Au top contacts for these junctions. Junctions for which the molecular layer was mostly comprised of DDT showed an increased failure rate.

Due to the low current ratings of the currently available silicon carbide (SiC) switches they cannot be employed in high-power converters. Thus, it is necessary to parallel-connect several switches in order to reach higher current ratings. This paper presents an investigation of parallel-connected normally-on SiC junction field effect transistors. There are four crucial parameters affecting the effectiveness of the parallel-connected switches. However, the pinch-off voltage and the reverse breakdown voltage of the gates seem to be the most important parameters which affect the switching performance of the devices. In particular, the spread in these two parameters might affect the stable off-state operation of the switches. The switching performance and the switching losses of a pair of parallel-connected devices having different reverse breakdown voltages of the gates is investigated by employing three different gate-driver configurations. It is experimentally shown that using a single gate-driver circuit the switching performance of the parallel-connected devices is almost identical, while the total switching losses are lower compared to the other two configurations. / <p>QC 20121116</p>

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 109-118).

published_or_final_version / Physics / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy