Student Number : 0100281N - MSc(Eng) Research Report - School of Civil and Environmental Engineering - Faculty of Engineering and the Built Environment / Many environmental and engineering projects require prediction of the velocity of flow in river channels, in terms of those channel properties and flow characteristics which induce resisting forces or an energy loss to the flow. Relationships such as the Manning, Chézy and Darcy-Weisbach equations have been in use for a century or more. All of them account for resistance with a single coefficient of resistance, and the central problem is evaluation of this coefficient. Experimental results by different researchers have shown that Manning’s n varies strongly with the ratio of flow depth to roughness height. It is constant for values of this ratio above about 4, but increases significantly for lower values. This suggests that the equation is not suitable in its original form for the case of intermediate-scale roughness. The roughness is intermediate-scale if the relative submergence ratio of flow depth to roughness elements height lies between 1 and 4. The influence of the roughness elements on flow resistance in this regime is caused by a combination of both element drag and boundary shear, or friction. The results of an experimental study with hemispherical roughness elements are presented, showing how the roughness element size, spacing and pattern influence flow resistance. For the range of conditions tested, Manning’s n appears to depend on roughness element size, spacing and pattern.

Organisational excellence stems from the dedicated commitment of peoplewho are motivated to work together and share similar values and visions about their results of their efforts. Organisational communication is linked to managerial effectiveness, the integration of work units across organisational levels and job communication satisfaction. The Department of Social Development can review their levels of communication and the interpersonal relationship in a bid to address their quality of productivity, satisfaction of employees, communication flow, and protocol and to bring changes in the life of the organisation. The success of an organisation is based on the communication levels, flow of information and how people interact. Communication systems within organisations, both human and technological, are responsible for creatively solving increasingly complex problems. The researcher has observed that lack of communication hampers service delivery and production in the Department of Social Development. The study focuses on the perceptions and experiences of employees about the nature of communication at the Department of Social Development in the North RandRegion. A qualitative research approach that was exploratory in nature was followed. The main aim of the study was to determine how the existing communication protocol and communication channels are utilised in the Department of Social Development. Asemi-structured interview schedule and questionnaire were used as research instruments. The methods of data collection were face-toface interviews with 10 participants and questionnaires consisting onlyof open-ended questions that were completed by eight individual respondents. The population for the study was employees of the Department of Social Development in the North Rand Region. The sample represented employees from top management, middle management, line management and junior staff who have been in the employment of the Department of Social Development for more than six months. The study revealed that there is a lack of understanding of relationship roles and that employees communicate without understanding and sometimes they do not seek clarity. All parties involved must be proactive, clear messages should be communicated to all employees, employees have first to seek understanding, allow questions and provide clarity and attempt to understand the relationship role between manager andsubordinate. However, misunderstanding and misinterpretation were identified as major contributing factors to effective communication.

Chloride intracellular channel proteins have homology to ion channels and omega class of glutathione-S-transferases but channel activity is not well established, suggesting roles in other signaling pathways. Among the six CLICs, CLIC1 and CLIC4 are expressed in endothelial cells (EC) and act to promote EC proliferation, capillary-like networks, and lumen formation. We and others determined that Sphingosine-1-phosphate (S1P) signaling promotes transient CLIC4 membrane localization. We report that CLIC1 and CLIC4 have distinct roles in endothelial S1P signaling. In knockdown studies, CLIC1 and CLIC4 were independently required for S1PR1-mediated Rac1 activation, enhanced EC barrier integrity, and EC migration. CLIC1 was uniquely required for S1PR2/3-driven RhoA activation and actin stress fiber formation, while CLIC4 was uniquely required for thrombin/PAR-driven RhoA activation and endothelial permeability. CLICs were not required for other GPCR-mediated pathways measured, including S1PR1-mediated cAMP regulation downstream of Gαi, or Ras and ERK activation downstream of Gβγ. Endothelial β-adrenergic signaling, which uses Gαs, was unaltered by loss of CLICs. Further investigation of receptor tyrosine kinase signaling (VEGF, EGF) in endothelial cells reveals their signaling cascades do not depend on CLICs as well. We conclude that CLICs mediate S1PR-driven RhoA and Rac1 regulation, and thrombin/PAR-driven RhoA activation, and a possible mediator for endothelial GPCR by modulating Rac1 and RhoA. CLIC N-termini contain membrane insertion motifs and the putative ion channel domain, while the C-termini contain two predicted SH domains. Chimeric proteins generated by swapping N and C-termini of CLIC1 and CLIC4 were used in rescue experiments. The C-terminal domain was determined to confer S1PR1-CLIC-Rac1 mediated barrier function and migration. We further characterized N-termini of CLIC4 and membrane localization of by generating CLIC4 C-termini truncated protein, along with CLIC4 C-termini fusing with Lck-peptide for myristylation and plasma membrane re-localization. CLIC4 C-termini alone fails to rescue S1PR1-CLIC-Rac1 mediated barrier function, while membrane localization of the CLIC4 C-terminal domain functions in S1P signaling, suggesting the N-terminal domain confers membrane localization but not signaling function. Thus, we conclude S1P promotes cell localization of CLIC4 to the EC plasma membrane through N-termini, which then regulates Rac1 mediated events through C-termini. Through these findings, our work defines a molecular mechanism through which CLICs function in endothelium.

by Ai-Dong Qi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 219-256). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Ca2+-activated potassium channels (KCa) and canonical transient receptor potential (TRPC) channels are essential to endothelial function. In ischemic heart disease, or in cardiac surgery, coronary endothelium is subjected to ischemia-reperfusion (I-R) / hypoxia-reoxygenation (H-R) injury. Hyperkalemic cardioplegic or organ preservation solutions used in cardiac surgery including heart transplantation also impair endothelial function. The present study was designed to mainly investigate whether endothelial dysfunction occurring in H-R or in hyperkalemic exposure is attributable to alterations of intermediate- and small-conductance KCa (IKCa and SKCa) channels, or TRPC channels, in particular, the TRPC3 channel. / Exposure to 60-min hypoxia followed by reoxygenation inhibited the vasorelaxant response of coronary arteries to IKCa / SKCa activator 1-EBIO. H-R reduced endothelial IKCa and SKCa currents and downregulated IKCa expression in PCECs. 1-EBIO enhanced endothelial K+ current that was blunted by H-R. / Exposure to hyperkalemic solutions decreased Ca2+ influx via TRPC3 in PCECs. The reduced Ca2+ influx in PCECs and the attenuated EDHF-mediated vasorelaxation in porcine coronary arteries, which were caused by hyperkalemic or cardioplegic / organ preservation solutions, were restored by OAG. / In PCECs, hypoxia for 60-min with reoxygenation reduced TRPC3 current and Ca2+ influx via TRPC3, which was accompanied by decreased NO release and endothelium-dependent vasorelaxation of porcine coronary arteries. The compromised endothelial function was restored by OAG. The translocation of TRPC3 to endothelial membrane was inhibited by H-R. / In TRPC3-overexpressing HEK293 cells, followed by reoxygenation, short-time hypoxia (10-min) enhanced, whereas prolonged hypoxia (60-min) reduced the current induced by TRPC3/6/7 activator OAG. / Our results indicate that: (1) Endothelial IKCa, SKCa and TRPC3 play an important role in regulating vascular tone; TRPC3 contributes to NO release from endothelial cells and is also involved in the function of EDHF. (2) H-R (60-30 min) reduces endothelial IKCa and SKCa currents with downregulation ofthe protein expression of IKCa. (3) H-R has dual effect on TRPC3 with short-time hypoxia (lO-min) enhancing whereas prolonged hypoxia (60-min) decreasing the electrophysiological activity of this channel. H-R (60-30 min) inhibits the translocation of TRPC3 to endothelial membrane. Furthermore, H-R inhibits Ca2+ influx via TRPC3 and such inhibition is associated with a decrease of NO production. (4) The activator of IKCa / SKCa or TRPC protects coronary endothelium against H-R injury. In coronary endothelium exposed to hyperkalemic or cardioplegic / organ preservation solutions, TRPC activator also exhibits protective effect. / The above findings are likely to have significant implications in ischemic heart disease and in modem cardiopulmonary surgery. / Whole-cell membrane currents of IKCa, SKCa, or TRPC3 were recorded by patch-clamp in primary cultured porcine coronary endothelial cells (PCECs). TRPC3 current was also studied in human embryonic kidney cells (HEK293 cells) transiently overexpressed with TRPC3 gene. Protein or mRNA expression of these channels was detected by Western blot or RT-PCR. Intracellular Ca2+ concentration was measured by Ca2+ imaging technique. Isometric force study was performed in a wire myograph and endothelial nitric oxide (NO) release was measured electrochemically by using a NO-specific microsensor in porcine coronary small arteries. / Huang, Junhao. / "December 2010." / Adviser: Qin Yang. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 138-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

No description available.

The green algae Chlamydomonas reinhardtii senses light through two photosensory proteins, channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2). The initial discovery of these two photoreceptors introduced a new class of light-gated ion channels. ChR2 is an inwardly-rectified ion channel that is selective for cations of multiple valencies. Similar to microbial-rhodopsin ion pumps, ChR2 has a seven transmembrane domain motif that binds the chromophore all-trans retinal through a protonated Schiff base linkage. Physiologically, ChR2 functions to depolarize the membrane which initiates a signaling cascade triggering phototactic response. This fundamental property has been pivotal in pioneering the field of optogenetics, where excitable cells can be manipulated by light. ChR2 reliably causes neuronal spiking with high spatial and temporal control. Moreover, the recent discovery of new chloride-conducting channelrhodopsins (ChloCs) has further expanded the optogenetic toolbox. Although structurally similar to microbial-rhodopsin ion pumps, ChR2 undergoes more complex conformational rearrangements that lead to ion conductance. Currently, the molecular basis for ChR2 gating remains unresolved. Revealing the specific structural interactions that modulate ChR2 function have important implications in understanding the intricacies of ion transport and molecular differences between ion pumps, channels, and transporters. Here we describe a combined computational and experimental approach to elucidate the mechanism of ion conductance, channel gating, and structure-function relationship of ChR2. Our results have contributed to expanding our understanding of the fundamental properties of ion channels.

Writing concurrent programs using shared memory causes many programmers much trouble, due primarily to unsafe semantics. Memory corruption, race conditions, deadlocks, and even livelocks are trivially easy to introduce into a program and painful to hunt down due the nearly infinite possible interleavings of instructions between the threads. Undergraduate curricula traditionally introduce students to the idea of shared memory multithreading in a systems programming or operating systems class, but rarely expose them to any alternate models of concurrent programming. This leaves them with the idea that shared memory is the only way to do concurrent programming. After students were exposed to alternate models, they came to prefer them to the standard shared memory model. This happened despite their distaste of the programming language that was utilized in performing the study. The students also expressed interest in alternate models of concurrency being taught in the computer science curricula at WPI.

Genetic mutations perturbing inactivation of human cardiac voltage-gated sodium channels (VGSCs), specifically Nav1.5, can cause long QT syndrome type 3 (LQT3). LQT3 is a cardiac disorder in which patients experience syncope and ventricular tachyarrhythmia, and are thus predisposed to sudden cardiac death. Deeper understanding of the structural dynamics of VGSC inactivation is needed to inform treatment of and drug design for potentially life-threatening arrhythmias. A well supported hypothesis is that the VGSC inactivated state is stabilized by hydrophobic interactions between the inactivation gate and an unknown binding site potentially involving the underside of the channel pore, C-terminus (C-T), and auxiliary proteins. Despite advances in biophysical and structural characterization of VGSCs, the specific molecular components and timing of their interactions within the inactivation complex remain unclear. Fluorescence imaging approaches that connect conformational change with channel function in mammalian cells could provide much needed mechanistic insight on the structural dynamics of the VGSC inactivation complex. This thesis describes the development of a site-specific fluorescent unnatural amino acid (UAA) labeling and spectral imaging methodology to probe the cardiac VGSC, Nav1.5, inactivation complex in live mammalian cells. First, UAA mutagenesis experiments were performed to validate orthogonal synthetase-tRNA (aaRS-tRNA) technology for fluorescent labeling of intracellular and membrane proteins in mammalian cells. Next, towards investigating conformational dynamics and intramolecular interactions related to inactivation, the Nav1.5 inactivation gate was labeled with a single environmentally sensitive fluorescent UAA L-anap. While the function of L-anap labeled channels was altered, their function remained within pathophysiological range. Then, imaging of L-anap labeled Nav1.5 in mammalian cells afforded characterization of unique L-anap spectra at different sites in the inactivation gate. Finally, using potassium-depolarization (K-depolarization) as rough means of voltage control, L-anap spectral shifts demonstrated conformational changes between the closed and open-inactivated states, which depended on the presence of the distal C-T (DCT). Site-specific L-anap labeling of the inactivation gate combined with spectral imaging and K-depolarization affords a general imaging assay to directly monitor conformational rearrangements of the Nav1.5 inactivation gate in channels expressed in live mammalian cells. While interactions with the DCT are specifically probed, this general assay provides an opportunity to bring necessary unification of ideas about VGSC inactivation, as well as insight on outstanding questions of VGSC regulation.

In this thesis, I focus on studies of the mechanism of inactivation in KcsA. Allosteric coupling between the pH gate and the selectivity filter in the protein is hypothesized to be the cause of inactivation. Allosteric coupling refers to changes at one site of a protein due to perturbations at a remote site. In chapter 3, I measured the potassium affinities at the selectivity filter at neutral and low pH, which corresponds to the closed and open conformation at the pH gate. The results show a three order of magnitude shift in the potassium affinity. This is direct evidence that the pH gate and the selectivity filter are coupled, in support of the activation-coupled inactivation hypothesis. The allosteric coupling factor, defined as the ratio of the affinities, can be used as a benchmark to study other factors in the allosteric process, such as the membrane and specific residues. Because of the potential deleterious effect of the acidic pH on the protein and membrane, we studied a mutant E118A&H25R, in which the pH gate is mutated to be open. Thus we were able to measure the K+ affinity change in the open and closed conformation at the pH gate at neutral pH. The results confirmed that the opening of the pH gate results in an energetic stabilization of the collapsed (K+-unbound) state, and shifts the K+ affinity towards looser binding. In chapter 4, I tested the important role of residue F103 in mediating allosteric coupling, as suggested by electrophysiology and crystallography studies. I mutated this residue and measured the allosteric coupling factor on the mutant. The affinity at low pH is much tighter than wild-type and the coupling factor is significantly reduced. From the spectra, I observe local structural changes on I100 and T74 as a result of F103A mutation, implying the interaction among F103, I100 and T74 to mediate the allosteric coupling. F103 is distant from the pH gate and the selectivity filter; its effect on the coupling and inactivation behaviors confirms that inactivation involves coupling between the pH gate and the selectivity filter. In chapter 5, I developed a method to probe those allosteric participants, such as F103 in KcsA by NMR measurements. I tested this method on KcsA, dissecting KcsA into various functional compartments. Various allosteric participants T75Cg T74Cg I100 were identified. The importance of residue T74 for the coupling was confirmed by electrophysiology and NMR thermodynamics characterization. In chapter 6, we applied SSNMR to probe the structural and magnetic properties of superatom clusters.