Binding-sites which facilitate the transport of substrates across membranes are ubiquitous in membrane proteins. To understand this fundamental process in cells, we build up a synthetic membrane system consisting of microfluidic channels and colloidal particles. Holographic optical tweezers are used to modulate the potential energy landscape in those channels. We show how to extract the underlying energy potential by analysing local transition probabilities. Our method is applicable both to equilibrium systems and non-equilibrium steady states. Our method offers improved robustness when dealing with fragmented trajectories or small ensembles of data compared to other established approaches, such as probability density function and splitting probability. Meanwhile, we utilise the intensity distribution of the optical traps generated by holographic optical tweezers to estimate energy landscapes featuring high energy barriers where transitions rarely occur. We use this newly developed experimental system to mimic the functionality of membrane protein transporters that are known to alternate their substrate-binding sites between the extracellular and cytosolic side of the membrane. We study particle transport through a channel coupled with an energy well that oscillates its position between the two entrances of the channel deterministically and stochastically. Optimised particle transport across the channel is obtained by adjusting the oscillation frequency. At the optimal oscillation frequency, the translocation rate of particles through the channel is a hundred times higher with respect to free diffusion across the channel. Our findings reveal the effect of time dependent potentials on particle transport across a channel. This work adds a new tool for the investigation of highly controlled membrane transport processes at the micron scale. Our results are relevant for improving our understanding of membrane transport especially for microfluidics application.

Due to the rapid development of semiconductor technology, the number of transistors of integrated circuits in unit area increases by double in roughly every two years. We then can add more circuits and functionality into a single chip. The size of electronic products certainly is reduced. Besides, because of the blooming popularity of wireless network standards in recently year, sensors have been wireless connected to provide more functionality and intelligence. They are, namely, wireless sensor network (WSN). Before long, the integrated circuit design will not only be emphasized on front-end circuits and hardware design, but also integration and functionality, which is so-called the system-on-chip (SOC) design. The first topic of this thesis is the implementation of IEEE 802.15.4 network prototype and hardware design. The main purpose of prototyping is to realize the highly portable IEEE 802.15.4 protocol stack library which can be quickly transferred to different hardwares. Thus, it shortens the time to market. In ASIC hardware design, we use WISHBONE bus as the interconnection architecture which can be easily integrated into current SOC design for an embedded system. The second topic is an application of IEEE 802.15.4 in medical monitoring, including system prototyping and ASIC hardware design, which collects the bladder pressure readings by a wireless link and ECG signals from our ASIC sensors. Finally, we realize the medical monitoring in a prototypical system.

The clock is the important synchronizing element in all synchronous digital systems. The difference in the clock arrival time between sink points is called the clock skew. This uncertainty in arrival times will limit operating frequency and might cause functional errors. Various clock routing techniques can be broadly categorized into 'balanced tree' and 'fixed mesh' methods. The skew and delay using the balanced tree method is higher compared to the fixed mesh method. Although fixed mesh inherently uses more wire length, the redundancy created by loops in a mesh structure reduces undesired delay variations. The fixed mesh method uses a single mesh over the entire chip but it is hard to introduce clock gating in a single clock mesh. This thesis deals with the introduction of 'reconfigurability' by using control structures like transmission gates between sub-clock meshes, thus enabling clock gating in clock mesh. By using the optimum value of size for PMOS and NMOS of transmission gate (SZF) and optimum number of transmission gates between sub-clock meshes (NTG) for 4x4 reconfigurable mesh, the average of the maximum skew for all benchmarks is reduced by 18.12 percent compared to clock mesh structure when no transmission gates are used between the sub-clock meshes (reconfigurable mesh with NTG =0). Further, the research deals with a ‘modified zero skew method' to connect synchronous flip-flops or sink points in the circuit to the clock grids of clock mesh. The wire length reduction algorithms can be applied to reduce the wire length used for a local clock distribution network. The modified version of ‘zero skew method’ of local clock routing which is based on Elmore delay balancing aims at minimizing wire length for the given bounded skew of CDN using clock mesh and H-tree. The results of ‘modified zero skew method' (HC_MZSK) show average local wire length reduction of 17.75 percent for all ISPD benchmarks compared to direct connection method. The maximum skew is small for HC_MZSK in most of the test cases compared to other methods of connections like direct connections and modified AHHK. Thus, HC_MZSK for local routing reduces the wire length and maximum skew.

Power gating is a technique commonly used for runtime leakage reduction in digital CMOS circuits. In microprocessors, power gating can be implemented by using sleep transistors to selectively deactivate circuit modules when they are idle during program execution. In this dissertation, a framework for power gating arithmetic functional units in embedded microprocessors with architecture and compiler support is proposed. During compile time, program regions are identified where one or more functional units are idle and sleep instructions are inserted into the code so that those units can be put to sleep during program execution. Subsequently, when their need is detected during the instruction decode stage, they are woken up with the help of hardware control signals. For a set of benchmarks from the MiBench suite, leakage energy savings of 27% and 31% are achieved (based on a 70 nm PTM model) in the functional units of a processor, modeled on the ARM architecture, with and without floating point units, respectively. Further, the impact of traditional performance-enhancing compiler optimizations on the amount of leakage savings obtained with this framework is studied through analysis and simulations. Based on the observations, a leakage-aware compilation flow is derived that improves the effectiveness of this framework. It is observed that, through the use of various compiler optimizations, an additional savings of around 15% and even up to 9X leakage energy savings in individual functional units is possible. Finally,in the context of multi-core processors supporting multithreading, three different microarchitectural techniques, for different multithreading schemes, are investigated for state-retentive power gating of register files. In an in-order core, when a thread gets blocked due to a memory stall, the corresponding register file can be placed in a low leakage state. When the memory stall gets resolved, the register file is activated so that it may be accessed again. The overhead due to wake-up latency is completely hidden in two of the schemes, while it is hidden for the most part in the third. Experimental results on multiprogrammed workloads comprised of SPEC 2000 integer benchmarks show that, in an 8-core processor executing 64 threads, the average leakage savings in the register files, modeled in FreePDK 45 nm MTCMOS technology, are 42% in coarse-grained multithreading, while they are between 7% and 8% in fine-grained and simultaneous multithreading. The contributions of this dissertation represent a significant advancement in the quest for reducing leakage energy consumption in microprocessors with minimal degradation in performance.

No description available.

Les canaux Ca2+ activés par le voltage (CaV) sont des protéines membranaires qui génèrent des courants Ca2+ dans les cellules excitables suite à une dépolarisation membranaire. Ces complexes oligomériques sont classifiés selon les propriétés structurelles de la sous-unité principale qui forme le pore du canal, soit la sous-unité CaVα1. La sous-unité auxiliaire CaVβ module l’expression membranaire et la dépendance au voltage du « gating » de la sous-unité CaVα1 des canaux HVA (« high-voltage-activated ») CaV1 et CaV2. La sous-unité CaVβ est formée par un domaine SH3 (« Src homology-3 ») connecté à un domaine GK (« guanylate kinase-like ») par le biais d’un domaine variable HOOK. Dans le but d’identifier les résidus dans la CaVβ3 qui sont responsables de la densité membranaire du CaV2.3, nous avons produit des mutants de la sous-unité auxiliaire le long de ses domaines fonctionnels. Cela dit, la délétion complète du domaine SH3 ainsi que la délétion du domaine HOOK n’ont pas modifié la densité membranaire de CaV2.3 ni ses propriétés d’activation. Cependant, la délétion de cinq résidus dans le domaine GK interrompt l’expression membranaire et l’expression fonctionnelle de CaV2.3. La mutation de résidus identifiés précédemment comme soutenant une affinité de liaison de l’ordre du nanomolaire dans le domaine GK de CaVβ n’a pas modifié de manière significative l’adressage membranaire de CaV2.3. Toutefois, les mutations de quatre résidus leucine dans les régions α3, α6, β10 et α9 du domaine GK ont grandement réduit l’adressage membranaire du canal CaV2.3. Nos résultats confirment que le domaine GK contient les déterminants moléculaires responsables de la fonction chaperone de CaVβ. Cela dit, l’adressage membranaire induit par CaVβ semble être déterminé par des éléments structuraux qui ne sont pas strictement dépendants d’une liaison à haute affinité de CaVβ sur CaVα1. / Voltage-activated Ca2+ channels (CaV) are membrane proteins that play a key role in promoting Ca2+ influx in response to membrane depolarization in excitable cells. They form oligomeric complexes that are classified according to the structural properties of the pore-forming CaVα1 subunit. Auxiliary CaVβ subunits modulate cell-surface expression and voltage-dependent gating of high-voltage-activated (HVA) CaV1 and CaV2 α1 subunits. CaVβ subunits are formed by a Src homology-3 (SH3) domain and a guanylate kinase-like (GK) domain connected through a variable HOOK-domain. In order to identify the residues responsible for the CaVβ3-induced membrane density of CaV2.3, we produced mutants along CaVβ3’s fonctionnal domains. Complete deletion of the SH3 domain as well as deletion of the HOOK domain did not alter plasma membrane targeting of CaV2.3 nor its typical activation gating. In contrast, 5-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of CaV2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of CaVβ did not significantly alter cell surface density. Mutations of a quartet of leucine residues in the α3, α6, β10, and α9 regions of the GK domain, each expected to curtail protein-protein interaction, were found to significantly impair cell surface targeting of CaV2.3 channels. Altogether, our results confirm that the GK domain includes the molecular determinants carrying the chaperone function of CaVβ. However, CaVβ-induced cell surface targeting appears to be determined by structural elements that are not strictly dominated by high-affinity binding of CaVβ onto CaVα1.

Les canaux potassiques dépendants du voltage sont formés de quatre sous-unités, chacune possédant six segments transmembranaires (S1-S6) et une boucle (p-loop) qui se trouve entre le cinquième et le sixième segment au niveau du pore. Il est connu que le segment senseur du voltage (S1-S4) subit un mouvement lorsque le potentiel membranaire change. Pour ouvrir le canal, il est nécessaire de transférer l'énergie du senseur du voltage (généré par le mouvement des charges positives de S4) au pore. Le mécanisme exact de ce couplage électromécanique est encore sous étude. Un des points de liaison entre le senseur de voltage et le pore est le lien physique fait par le segment S4-S5 (S45L). Le but de cette étude est de déterminer l'influence de la flexibilité du segment S45L sur le processus de couplage. Dans le S45L, trois glycines sont distribuées dans des positions différentes. Elles sont responsables de la flexibilité des hélices-alpha. Ces glycines (mais pas leurs positions exactes) sont conservées pour tous les canaux potassiques dépendants de potentiel. En utilisant la technique de mutagènes dirigé, la glycine a été remplacée dans chacune de ces différentes positions par une alanine et dans une deuxième étape, par une proline (pour introduire un angle dans l'hélice). Pour étudier le comportement des canaux dans cette nouvelle conformation, on a appliqué la technique de « patch clamp » pour déterminer les effets lors de l'ouverture du pore (courant ionique). Avec le « cut-open oocyte voltage-clamp », nous avons étudié les effets sur le mouvement du senseur de voltage (courant “gating”) et la coordination temporelle avec l'ouverture du pore (courant ionique). Les données ont montré qu’en réduisant la flexibilité dans le S45L, il faut avoir plus d'énergie pour faire ouvrir le canal. Le changement pour une proline suggère que le mouvement du senseur est indépendant du pore pendant l'ouverture du canal. / Voltage-gated potassium channels are formed of four subunits, each one with six transmembrane segments (S1-S6) and a loop (p-loop) between S5 and S6 at the level of the pore. It is known that the voltage sensitive segment (S1-S4) undergoes a movement upon membrane potential changes. To open the channel, it is necessary to transfer the energy of the voltage sensor (generated by the displacement of the positive charges of S4) to the pore. The exact mechanism of this “electromechanical coupling” is still under investigation. The voltage sensor and pore are physically linked by the S4-S5 linker (S45L). The aim of this study is to determine the influence of S45L flexibility on the coupling process. In the S45L, three glycines are distributed at different positions and are responsible for the flexibility of the alpha-helix. These glycines (but not their exact position) are conserved within the potassium voltage-gated ion channels. The glycines were each replaced by an alanine using point mutagenesis. In a second step, a proline was introduced at the position in order to introduce a break in the helix. To study the behaviour of channels in this new conformation, we used the patch clamp technique to determine the effects during the pore opening (ionic current). With the cut-open voltage-clamp we determined the effects on voltage sensor movement (gating current) as well as the temporal correlation with the pore opening (ionic current). The data showed that when the flexibility of the S45L is reduced, the channel needs more energy to open. Exchange with proline suggests that the movement of the sensor is independent of pore opening.

The thesis describes development and application of coupled resonant coil sensors, which is of growing interest for remote monitoring applications. An interrogation technique, which improves the accuracy and interrogation range of coupled resonant coil sensors, is introduced. The method uses time-domain gating to produce measurements that are dominated by the response of the sensor coil and are immune to surrounding object interference. For application in structural health monitoring a low cost embeddable coupled coil sensor, which is able to monitor the corrosion potential of reinforcement steel is presented. Results of an accelerated corrosion test using the sensor indicate that corrosion potential can be monitored with a resolution less than 10 mV and a sensitivity of 0.76 kHz/mV. The last part describes a coupled-coil pH sensor based on pH electrode potential measurement. A linear response over a 4 to 10 pH dynamic range and 50 kHz/pH sensitivity are achieved with a 0.1 pH resolution and 30 s response time.

The thesis describes development and application of coupled resonant coil sensors, which is of growing interest for remote monitoring applications. An interrogation technique, which improves the accuracy and interrogation range of coupled resonant coil sensors, is introduced. The method uses time-domain gating to produce measurements that are dominated by the response of the sensor coil and are immune to surrounding object interference. For application in structural health monitoring a low cost embeddable coupled coil sensor, which is able to monitor the corrosion potential of reinforcement steel is presented. Results of an accelerated corrosion test using the sensor indicate that corrosion potential can be monitored with a resolution less than 10 mV and a sensitivity of 0.76 kHz/mV. The last part describes a coupled-coil pH sensor based on pH electrode potential measurement. A linear response over a 4 to 10 pH dynamic range and 50 kHz/pH sensitivity are achieved with a 0.1 pH resolution and 30 s response time.

CYP17 is an ACTH/cAMP inducible gene in the human adrenal cortex encoding a cytochrome P450 enzyme with sterol 17α-hydroxylase activity and 17,20 lyase activity essential for biosynthesis of cortisol and androgens. Studies carried out during the past decade have shown that acclerated transcription of inducible eukaryotic genes involves sequential chromatin modifications by cooperative promoter-specific transcription factors and the class of proteins called transcriptional coregulators. In the present work, we aimed to first identify important chromatin modifications and chromatin modifying complexes at the CYP17 transcription start site and nearby steroidogenic factor-1 (SF-1) binding site. Then, we asked what modifications to SF-1 occur during the interaction of this nuclear receptor with the CYP17 promoter, and what their function may be. Finally, we asked how ACTH/cAMP signaling affects SF-1-containing chromatin-modifying complexes during the early phase of transcriptional induction of CYP17. Results from chromatin immunoprecipitation (ChIP) and mammalian two hybrid experiments identified complexes including one comprised of SF-1, steroid receptor coactivator-1 (SRC-1), and the histone acetyltransferase general control nonderepressed 5 (GCN5) as cAMP-inducible, but sensitive to the SF-1 antagonist sphingosine, and able to act in stimulating CYP17 transcription. Moreover, ATPases on the promoter coincided with manipulation of nucleosome histone H2 dimer content. Next, we found that SF-1 phosphorylation by glycogen synthase kinase 3beta (GSK3beta), reciprocal dephosphorylation by phosphatase(s), and acetylation by GCN5 at nearby sites at the ligand binding pocket opening were required for efficient CYP17 transcription. This leads us to propose that ligand binding to SF-1 is controlled by these post-translational modifications. Finally, we determined that the corepressors E1A C-terminal binding proteins (CtBP) 1 and 2 are protein kinase A (PKA) targets and are sensitive to PKA-dependent NADH accumulation. These effects of PKA activation by ACTH/cAMP in adrenal cortex cells enforce CYP17 transcription concomitant with dimerization of CtBP1 and CtBP2.