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The plasminogen activator (PA) system plays an important role in cell adhesion, migration and invasion, and may require the coordinated expression of various proteins. The human urokinase-type plasminogen activator (uPA) receptor (uPAR) is a central protein component of the PA system. By binding its ligand uPA, uPAR can direct proteolysis of the extracellular matrix. Also, it is now apparent that uPAR can initiate proteolytic independent signal transduction to influence angiogenesis, inflammation, wound repair and tumour progression. To determine whether any novel proteins interacted with uPAR, a yeast two-hybrid screening analysis was undertaken using alternate uPAR domain constructs as baits. These included full-length three domain uPAR (uPAR-DIDIIDIII), two domain uPAR (uPAR-DIIDIII), and each individual uPAR domain (uPAR-DI, uPAR-DII and uPAR-DIII). A number of proteins were identified as putative candidate interactors for the alternate constructs, with two of special interest for uPAR-DIDIIDIII. These were the heat shock protein Mrj, and the extracellular matrix protein fibulin-2. The protein Mrj was shown to bind uPAR both in vitro and in vivo using GST-pull down and co-immunoprecipitation assays respectively. The GST-pull down assay identified the interaction between Mrj and uPAR dependent on the C-terminal domain of Mrj and DI of uPAR. Using in vivo co-immunoprecipitation analysis, Mrj also bound to uPAR. Preliminary data suggest the association between uPAR and Mrj may play a role in the regulation of apoptosis. In regard to the uPAR interactor of fibulin-2, a calcium dependent binding interaction with uPAR was identified using the GST-pull down assay. However due to the large molecular weight and stringent conditions needed to solubilise fibulin-2, it was not possible to co-immunoprecipitate both uPAR and fibulin-2. Together, the identification of both Mrj and fibulin-2 amongst other candidate interactors of uPAR presented here provides further insight into the intricate relationship between uPAR and other proteins which may influence a range of biological functions.

This thesis is focused attention on one-dimensional models for fast transient flows in a kinematic non-equilibrium. Besides the thermodynamic non-equilibrium, there is another type of non-equilibrium: the kinematic non-equilibrium, or drift between the phases. Such flow models include bubbly gas/liquid flows which are characterized by strong coupling between the phases, due to the rapid interphase transfers of mass, momentum and energy. As a consequence the assumptions that the phase pressures and the phase temperatures are equal at any cross-section appear consistent with experimental observations. The set of equations includes a momentum equation which has the form of a relaxation law of the drift velocity. This equation is based on a simplified version of the so-called Voinov - Berne equation for the momentum of the gas in a bubbly flow. The ability of the model to predict steady state critical flows is tested first. This is done by means of an analysis of the sensitivity to variations of the main parameters, and also by comparing the results with two sets of original experimental data on air-water critical flows. Finally, the model is tested in transient conditions, modelling the water hammer phenomena.

Mammalian odorant receptors (ORs) are Class I G-protein coupled receptors (GPCRs) located within the nasal epithelium. Odorant receptors interact with Galpha olfactory, a Galpha S type G-protein. Activated Galpha olfactory stimulates adenylate cyclase and the resulting increase in cAMP concentration opens cyclic nucleotide gated channels allowing Ca2+ to enter the cell. The increased Ca2+ then activates a Ca2+ activated Cl- channel which further depolarizes the cell. This depolarization initiates an action potential that reaches the axon of the olfactory sensory neuron located in the main olfactory bulb. Information from the main olfactory bulb is then transmitted to higher regions of the brain. Olfactory information is initially coded through the interaction of odorant molecules with hundreds of distinct ORs, but difficulty in exogenous expression of odorant receptors has delayed the identification of ligands for individual ORs. However, expression of mouse odorant receptors in Xenopus laevis oocytes allows for a systematic screening for potential ligands, as well as for efficient study of the structure-function relationship of the receptors and their ligands. My screening of odorant receptors using Xenopus oocytes included the coexpression of a signal transduction system and the use of robotic two-electrode voltage clamp electrophysiology. In this study, I investigated the structural basis for ligand recognition in mouse odorant receptors. First, I expanded the molecular receptor ranges of seven Class I odorant receptors. By use of a high throughput assay, I was able to expand upon current knowledge in the field for the mouse odorant receptors 23-1, 31-4, 32-11, 40-4, 42-1, 42-2 and 42-3. I then examined one receptor (MOR23-1) in more detail. I used the substituted cysteine accessibility method to identify residues within transmembrane domain five of this receptor that are accessible from the extracellular space. These residues may line the ligand binding site or the ligand access pathway. Conventional mutations of A205 caused little alteration in the molecular receptive range of the receptor, suggesting that this residue may not play a significant role in ligand interaction within the binding pocket. Mutagenesis of G111, a residue within transmembrane domain three caused significant shifts in the molecular receptive range of the receptor, but the location of this residue within the binding pocket could not be confirmed by the substituted cysteine method. Previous reports had suggested significant similarity between the molecular receptive ranges of the seven mouse odorant receptors that I used in my research. By expanding upon the known aliphatic ligands for each receptor identified new ligands for each receptor, I was able to show that the molecular receptive ranges of these receptors are in fact distinct. The experimental identification of residues located within the binding pocket on transmembrane five of mouse odorant receptor 23-1 provides an improved understanding of ligand recognition by this receptor class and will aid in better computer modeling of these receptors. This increased accuracy of the computer models of these basic Class I GPCRs may aid in future drug discoveries. Since GPCRs constitute a significant fraction of current drug targets, understanding the mechanism of ligand interactions with mouse odorant receptors may aid in the development of more efficacious compounds in the treatment of many common ailments.

Large conductance voltage and Ca2+ activated K+ (BK) channels control electrical excitability in many cell types. BK channels have the largest conductance (~250 pS) of all K+ selective channels. To explore whether a large entrance to the inner cavity of BK channels is required for their large conductance, I examined if changing the size of the entrance alters the single-channel current amplitude. Previous studies suggest that residues E321/E324 in BK channels are located at the entrance to the inner cavity. To test if positions 321/324 are accessible to intracellular ions, I compared single-channel outward current before and after attaching thiol reagents at E321C/E324C. Attachment of MBB and MTSET altered single-channel currents, indicating that positions 321/324 are accessible to the conduction pathway. Decreasing the size of the entrance to the inner cavity by substituting residues with larger side chains, such as tyrosine and tryptophan, at positions 321/324 decreased the conductance, whereas increasing the size of the entrance had little effect on conductance. Increasing [K+]i from 0.15 to 2.5 M negated differences in single-channel outward current associated with side chain volume. Substitutions had less effect on inward currents. Plots of conductance vs. substituted side chain volume could be approximated with a simple model for the conduction pathway described by two resistors in series, R1 and R2. R2 is a variable resistor, with the resistance proportional to the inverse of the volume of the entrance to the inner cavity not occupied by the side chains. R1 is a fixed resistor arising from the other parts of the conduction pathway including the selectivity filter. Fitting the experimental observations indicated that R1+R2 ~5.4 GΩ for glycine substitution, with an R1/R2 ratio of ~17, and an effective radius and length of the entrance to the inner cavity of ~9.0 and 5.4 Å, respectively. The volume of K+ and water were not taken into account. Taken together, the above observations suggest that a large entrance to the inner cavity is needed for the large conductance of BK channels, as my study shows that the entrance is large and that decreasing the entrance size decreases the currents.

The slotted plate flow meter has been widely tested as an obstruction flow meter during the past several years. It has been tested for both single-phase flows as well as for two-phase flows. Previous studies have revealed that the slotted plate flow meter is always better in performance and accuracy than the standard orifice plate flow meter. This study is primarily based on how a slotted plate responds to horizontal two-phase flow with air and water being used as the working fluids. The plates under consideration are those with beta ratios of 0.43 and 0.467. Experiments have been performed with six different configurations of the slotted plate test sections. The performances of the slotted plate flow meters will be compared to that of a standard orifice plate flow meter and then with a venturi. The effects of varying the upstream quality of the two-phase flow on the differential pressure and the coefficient of discharge of the slotted plates, the standard orifice plate and the venturi will be evaluated. Response characteristics at low differential pressures will be investigated. Tests for repeatability will be performed by studying the effects of the gas Reynolds number and the upstream quality on the differential pressure. The differential pressures across the slotted plates, the standard orifice plate and the venturi will be compared. Reproducibility will be evaluated by comparing the data obtained from all six different configurations. One of the main objectives of this study is to arrive at the best suitable procedure for accurately measuring the flow rate of two-phase flow using the slotted plate flow meter.

In previous studies the slotted orifice plate has demonstrated superior performance characteristics to those of the standard orifice plate. In this study, these comparisons are investigated further. The response characteristics of the slotted orifice plate to the standard orifice plate and V-Cone for two-phase flows of water and air at various qualities, flow rates, and pressures are shown visually. The effect of oil as it flows through a slotted orifice plate and standard orifice plate are visually documented. The effect of manufacturing accuracy on the slotted orifice plates is investigated as to the effect on the coefficient of discharge, percent change in pressure, and Reynolds number. The slotted orifice plate mixes two-phase flow better than the standard orifice plate and V-Cone. There is a manufacturing effect on the slotted orifice plates; the larger the area of the slots, the larger the discharge coefficient.

The work presented in this dissertation deals with the development of new methodologies for P-C bond formation as well as synthesizing biologically relevant organophosphorus compounds. A distinct emphasis is given to the important synthetic targets, the H-phosphinates. A review of relevant literature is provided in Chapter 1. Chapter 2 describes the synthesis and structural analyses, of triphenylmethyl-containing phosphorus compounds. For the first time, both phosphonothioic and boranophosphonic acids have been characterized by single X-ray diffractometry. The third chapter details the preparation and the reactivity of phosphine-borane complexes. Novel dialkoxyphosphine-borane complexes were introduced, both as general synthetic intermediates for the preparation of H-phosphinates or disubstituted phosphinic acids, and as boranophosphonate precursors. Related to this chemistry, silylation of an H-phosphinate intermediate can also be conducted and the resulting phosphonite protected with borane. This allows the temporary protection of the sensitive P-H group, so that manipulations of the alkyl chain might be conducted. In chapter 4, the palladium-catalyzed cross-coupling reaction of dialkylphosphites with aryl and heteroaryl halides is presented. An efficient, versatile and economically attractive alternative to the original Hirao cross-coupling by using only 1 mol% (or less) Pd(OAc)2/dppf is described. Moreover, first example of palladium-catalyzed P-C bond formation between activated aryl chlorides and a phosphite are herein reported. Chapter 5 focuses on the free-radical hydrophosphinylation of alkynes. The triethylborane-initiated radical addition of sodium hypophosphite to terminal alkyne affords the previously unknown 1,1-bis-H-phosphinates, precursors of the biologically relevant 1,1-bisphosphonates (e.g., treatment of bone diseases). Thus, the oxidative conversion of 1,1-bis-H-phosphinates to the corresponding bisphosphonates, as well as the synthesis of a series of bio-conjugates (steroids, carbohydrates, fluoroquinolones) was investigated. In the last chapter, the palladium-catalyzed hydrophosphinylation of hypophosphorous acid derivatives to terminal alkynes is reported. In an effort to improve the regioselectivity of the reaction, various terminal alkynes were tested, as well as the solvent and catalyst system.

This research examines a two-way relay network consisting of two transceivers and multiple parallel relays, which are equipped with single antennas and operate in a halfduplex mode. In this system, the two transceivers prefer to exchange their information via relays. It is assumed that the relays have the full instantaneous channel state information (CSI) and relay the signals using the amplify-and-forward (AF) method. The performance of two AF bi-directional network beamforming schemes, namely multiple access broadcast channel (MABC) strategy and time division broadcast channel (TDBC) protocol, under joint optimal power control and beamforming design are studied and compared. To do so, we first design a TDBC-based bi-directional network beamformers, through minimization of the total power consumed in the whole network subject to quality of service (QoS) constraints, for the case with a direct link between the two transceivers. The corresponding power minimization problem is carried out over the transceiver transmit powers as well as relay beamforming weights, thus resulting in a jointly optimal power allocation and beamforming approach. We devise optimal secondorder cone programming based solutions as well as fast gradient-based solutions to these problems. Then these solutions are exploited to compare the performance of the underlying TDBC-based approach to that of the MABC-based technique developed in [1]. This comparison is important because the TDBC approach appears to have certain advantages which can be exploited towards improving the performance of two-way network beamforming. These advantages include the additional degree of freedom as well as the possibility of benefitting from the availability of a direct link between the two transceivers. Interestingly, in the absence of a direct link between the two transceivers, we show that when the QoS constraints are imposed to meet certain given probabilities of un-coded error (or, equivalently, to meet certain signal-to-noise ratio constraints), these two schemes perform closely in terms of the minimum total transmit power. However, when the QoS iv constraints are used to guarantee certain given rates, the MABC-based scheme outperforms the TDBC counterpart. In the case when a direct link exists between the two transceivers, the TDBC-based approach can outperform the MABC-based method provided that the direct link is strong enough. / UOIT

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.