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Microglia are immunocompetent cells in the central nervous system that have many similarities with macrophages of peripheral tissues. Their activation protects local cells from foreign microbial infection in the CNS. However, “over-activated“ microglia become a “Double-edged sword” which show neuronal toxicity and are implicated in a variety of neurodegenerative diseases. Previous studies have suggested that potassium channels play a role in regulating microglial activation, migration and proliferation. However what kinds of potassium channel subunits are expressed in microglia, whether their expression changes after microglial activation and the functional role of most potassium channels expressed in microglia are still not fully characterized. To address these questions, we used the N9 mouse microglial cell line as a cell model for experiments in vitro. We first optimized the cell culture and lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, mediated stimulation of microglial activation that results in subsequent nitric oxide (NO) release. Using qRT-PCR, we analyzed mRNA expression of >80 potassium channel pore-forming subunits and their regulatory subunits in both LPS-treated (1μg/ml, 24hr) and untreated microglia. The subunits which displayed the highest mRNA expression in resting N9 cells included Kcnma1 (KCa1.1), Kcnk6 (K2p6.1), Kcnc3 (Kv3.3) and Abcc8 (SUR1). In addition, N9 cells also expressed the mRNAs for other channel subunits previously reported in microglia such as Kcnn4 (KCa3.1), Kcna3 (Kv1.3) and Kcna5 (Kv1.5) subunits. Of these channel subunits LPS had no significant effect on mRNA expression except for Kcnk6 which was significantly reduced. We then examined whether pharmacological manipulation of these channels controlled LPS-induced NO release. It was found out that the KCa3.1 selective blocker Tram34 and the Kv1.5 inhibitor propafenone (PPF) significantly decreased LPS-induced NO in agreement with data in primary microglia. Ba2+ that inhibits inwardly rectifying potassium channels as well as K2p6.1 also significantly attenuated LPS-induced microglial activation. Inhibition or activation of KCa1.1 channels by paxilline and NS1619 respectively had no significant effect. However, paxilline significantly attenuated the effect of Tram34, PPF and Ba2+ to control LPSinduced NO release while NS1619 significantly facilitated the effect of Tram34 and PPF. To investigate the major ionic currents expressed in N9 microglia with and without LPS application, we examined whole-cell ionic currents using the patch-clamp technique. Resting N9 cells display a small outward current at positive potentials but a large inwardly rectifying component at negative potentials in physiological potassium gradients. The outward current was dramatically increased by LPS application that was dependent upon the intracellular free calcium concentration. Paxilline or Tram34 was then applied to acutely block this apparent outward KCa current. The result indicated that the LPS triggered KCa current was mainly paxilline sensitive supporting a role for an LPS-induced increase in KCa1.1 channel current. In addition, by using current clamp the mean resting membrane potential of N9 cells was -50.6±6.6mV (N=7) determined in the presence of 1μM [Ca2+]i and -59.4±8.5mV (N=10) with 10nM [Ca2+]i. N9 cells did not display any spontaneous action potentials and the resting membrane potential was not significantly affected by LPS. To conclude, the work presented in this thesis extends the current knowledge regarding potassium channel mRNA expression in microglia and their function in microglial NO release. What is more, it was found that KCa1.1 current expression was increased in LPS-activated N9 cells and revealed KCa1.1 channels as a modulator of NO release by activated microglia.

A review was carried out which revealed that no simple mathematical model was available which could be used to predict the performance of a centrifugal pump when it is operating under two phase flow conditions. Experimental analyses were carried out to aid the development of such a model. A rotating channel test rig was designed to study the structure of airwater flow through an impeller passageway. The observations generated a large amount of qualitative data. A full scale centrifugal pump was also tested which provided data that allowed the results of the rotating channel experiments to be considered in more quantitative terms. These two sets of experimental work allowed a conceptual model of the two phase flow through a centrifugal pump to be constructed. The model assumes that forces act on the bubbles as they pass through the impeller passageway because of the rotation and the curvature of the impeller passageway. These forces cause the bubbles to decelerate and coalesce creating a stationary air void which leads to a partial blockage of the channel. This causes the velocity of the fluid passing through the pump to be modified. The model uses a onedimensional velocity vector approach adjusted for hydraulic losses to calculate the head raised by the pump under such conditions. The model presented provides the basis upon which a predictive tool could be developed and used in the development engineering environment. Currently operational problems that arise because of two phase flow are often quantified using scaled hydraulic models, which are expensive, or within the actual application where costly engineering solutions may be required to give acceptable performance. A mathematical model is a much more cost effective tool and its application allows the engineer to decide whether system performance would be compromised by the two phase conditions encountered and propose possible solutions. In recommending the development of such a model it is necessary to consider where and how in the project cycle it should be used. A number of companies were canvassed and it was concluded that in many cases such models are used too late in the project cycle to provide maximum return. In the case of projects which include participants from a number of companies it is suggested that an inter-company team should be constructed if development models are to be widely used. This team should be used to plan and implement the use of development models efficiently and ensure that the data produced is communicated effectively and is of the maximum value to the participants.

No description available.

ABSTRACT The voltage-gated potassium channel Kv1.2 is a key determinant of cellular excitability in the nervous and cardiovascular systems. In the brain, Kv1.2 is strongly expressed in neurons of the hippocampus, a structure essential for learning and memory, and the cerebellum, a structure essential for motor control and cognition. In the vasculature, Kv1.2 is expressed in smooth muscle cells where it contributes to the regulation of blood flow. Dynamic regulation of Kv1.2 is fundamental to its role in these tissues. Disruption of this regulation can manifest in a range of pathological conditions, including seizure, hypertension and neuropathic pain. Thus, elucidating the mechanisms by which Kv1.2 is regulated addresses fundamental aspects of human physiology and disease. Kv1.2 was the first voltage gated ion channel found to be regulated by tyrosine phosphorylation. The ionic current of Kv1.2 is suppressed following tyrosine phosphorylation by a process involving channel endocytosis. Movement of channel away from the plasma membrane involves many proteins associated with the cytoskeleton, including dynamin, cortactin and RhoA. Because trafficking of Kv1.2 away from the cell surface has emerged as the primary mechanism for its negative regulation, we hypothesized that trafficking of the channel to the cell surface could be a mechanism for positive regulation of the Kv1.2 ionic current. Activation of the cAMP/PKA pathway enhances the ionic current of Kv1.2. We hypothesized that a mechanism for this positive regulation is an increase in the amount of channel protein present at the cell surface. Our data show that cAMP can regulate Kv1.2 surface levels by two opposing trafficking pathways, one PKA-dependent and one PKA-independent. Channel homeostasis is preserved by the dynamic balance between these two pathways. Accordingly, any change in the levels of cAMP causes a net increase in the amount of Kv1.2 present at the cell surface. Specific C-terminal phosphorylation sites of Kv1.2 were identified and shown to have a role in maintaining basal surface channel levels. These findings demonstrate channel trafficking as a mechanism for the positive regulation of the Kv1.2 ionic current. In addition to Kv1.2 trafficking at the plasma membrane, movement of the channel from the biosynthetic pathway to the cell surface is another checkpoint for its regulation. Here we show that the protein arginine methyltransferase 8 (PRMT8) is able to promote the ER exit of Kv1.2, resulting in an increase in Kv1.2 surface expression. PRMT8 not only promoted surface expression of the high mannose glycosylated form of Kv1.2, characteristic of immature, ER-localized channels, but also enhanced Kv1.2 total protein levels, most likely by decreasing the amount of channel protein available for ER-associated degradation (ERAD). These findings highlight biosynthetic trafficking of Kv1.2 as a crucial part of its regulation and identify a novel role for PRMT8, as a regulator of biosynthetic protein trafficking.

Voltage-gated potassium channels are primary determinants of cellular excitability in the mammalian nervous system. The localization of these channels to distinct cellular compartments influences components of neuronal function, including resting membrane potential, action potential characteristics and neurotransmitter release. Thus, understanding the mechanistic basis of ion channel localization can provide fundamental insight into human physiology. The overall goal of this dissertation was to elucidate the regulatory mechanisms governing localization and function of the Kv1.3 voltage-gated potassium channel. The sympathetic branch of the autonomic nervous system innervates many organ systems including the kidneys, heart and blood vessels and was used as a model to study endogenous Kv1.3. We found that postganglionic sympathetic neurons express Kv1.3 and that the channel exhibits a striking pattern of localization to the Golgi apparatus in the soma of these cells. Kv1.3 ionic current was also isolated from the soma of these neurons, indicating the channel is a determinant of the electrophysiological properties of sympathetic neurons. In addition, the specific inhibition of Kv1.3 with margatoxin was found to depolarize neuronal resting membrane potential, decrease the latency to action potential firing and increase nicotinic agonist-induced neurotransmitter release. Collectively, these findings demonstrate that Kv1.3 influences the function of postganglionic sympathetic neurons and led to the hypothesis that regulating channel localization may be a mechanism for modulating the activity of these cells. In this dissertation, we propose that the observed Golgi retention of Kv1.3 may be a trafficking-dependent mechanism of channel regulation. To test this hypothesis, we used HEK293 cells as our model system. Our data show that the degree of Kv1.3 Golgi localization is inversely correlated with the amount of channel at the plasma membrane. In addition, the amplitude of Kv1.3 ionic current measured in cells with low Kv1.3 Golgi localization was significantly greater than the current measured in cells with high Kv1.3 Golgi localization. One mechanism for localizing ion channels to the Golgi apparatus involves the Class I PDZ-binding motif (X-S/T-X-Φ). Deletion of the C-terminal PDZbinding motif of Kv1.3 decreased the intracellular Golgi localization of the channel and increased channel localization at the cell surface. Disrupting this canonical binding motif also increased the amplitude of Kv1.3 ionic current. These findings indicate that regulated subcellular distribution of the channel may be a determinant of Kv1.3 surface expression and function.

Orthogonal frequency-division multiplexing (OFDM) has been employed in several current and future 4-th generation (4G) wireless standards. Frequency offsets in OFDM introduce intercarrier interference (ICI). Channel estimations are also required. This thesis focuses on the channel and frequency offset estimation for OFDM-based systems. For cooperative-relay OFDM with frequency offsets, where inter-relay interference (IRI) exists, channel estimation is developed. Optimal pilot designs are proposed by minimizing the IRI in the mean square error (MSE) of the least square (LS) channel estimation. The impact of frequency offset on the channel estimation accuracy is derived. The pairwise error probability (PEP) with both the frequency offset and channel estimation errors is evaluated. The power allocation is discussed. For multiple-input multiple-output (MIMO) OFDM systems, channel and frequency offset estimation errors are investigated. The signal-to-interference-and-noise ratio (SINR) is first analyzed given channel and frequency offset estimation errors. The bit error rate (BER) is then approximated for multiple-antenna reception with maximal ratio combing (MRC) and equal gain combining (EGC). For orthogonal frequency-division multiplexing access (OFDMA) systems, the variance of the frequency offset estimation is derived as a function of SINR and signal-to- noise ratio (SNR). This variance information is exploited to improve the accuracy of frequency offset estimators. A successive interference cancellation (SIC)-based frequency offset estimator is also developed. The accuracy of frequency offset estimation of the OFDMA uplink can also be improved by using the cooperative relaying. Both conventional amplify-and-forward (AF) relays and new decode-and-compensate-and-forward (DcF) relays are studied. The frequency offset estimate is derived from combining different link estimates. In addition, when CSI is available, a scheme is proposed to adaptively switch between the cooperative and conventional (no relaying) transmissions to optimize the frequency offset estimation. / Wireless Communications

The MotA and MotB proteins of E. coli form a MotA4MotB2 complex. Proton flow through a transmembrane channel in the complex powers flagellar rotation. Protonation of Asp-32 of MotB within the channel is proposed to cause a conformational change in the large cytoplasmic loop of MotA, which pushes against FliG in the rotor. MotB is believed to anchor the complex to the cell wall via a conserved sequence that is found in many proteins that bind peptidoglycan. The research presented in this dissertation focused primarily on the formation and activation of the MotAB proton channel. A proposed amphipathic α-helical region, extending from residue 52 through 65 of the periplasmic domain of MotB, was discovered to block proton flow through the channel in its inactive state prior to incorporation into a flagellar motor. The plug is thought to lie parallel to the periplasmic face of the cell membrane and to be removed from the membrane by a conformational change triggered by contact with the motor. Negatively charged residues near the cytoplasmic C-terminus of MotA and positively charged residues at the cytoplasmic N-terminus of MotB were identified as being important for motility. A mutational analysis and subsequent suppressor analysis suggest that these residues may align MotA and MotB to form the MotA4MotB2 complex in the proper position relative to FliG and the rotor. The underlying mechanism for producing MotA and MotB in a 2:1 ratio was also investigated and found to be primarily due to translational coupling of motA and motB. The stop codon of motA and the start codon of motB overlap, allowing the ribosome that has just completed translation of motA to reinitiate and translate motB. The efficiency of reinitiation is about 66%; presumably degradation of excess MotB not in the MotA4MotB2 complex produces the final 2:1 ratio. Research was also conducted to determine whether MotB binds directly to the peptidoglycan layer of the cell wall. Although inconclusive, the preliminary results appear to support this notion. The overall work provides insights into several aspects of the assembly and subsequent activation of the stator component of the bacterial flagellar motor.

In this thesis, we investigate and implement the channel estimation schemes for digital video broadcasting - handheld (DVB-H) and IEEE 802.16d world interoperability for micro-wave access (WiMAX). Both systems are based on the orthogonal frequency division multiplexing (OFDM) technique. The performance of the channel estimation schemes is first verified by using simulation experiments. Then, the channel estimation algorithms are realized by hardware implementation. For the DVB-H systems, since the mobile device may have a relatively high speed, the channel condition is time-varying, leading to serious degradation in channel estimation. In this thesis, the decision feedback mechanism is adopted to improve the performance of the channel estimation. The adopted structure for channel estimation is realized by using Verilog hardware description language (HDL). Then, all the baseband signal processing related algorithms, which include timing synchronization, frequency offset estimation/compensation, and scattered pilot detection, are integrated together using 0.18 ASIC process. For the IEEE 802.16d WiMAX, since it is dedicated for fixed wireless applications, the channel condition is basically stationary and easier to obtain. Therefore, the decision feedback mechanism is not adopted to save hardware complexity. The system performance is verified by conducting simulation experiments. The adopted channel estimation algorithm is implemented by using Verilog HDL. Finally, the whole baseband receiver is realized by using the field programmable gate array (FPGA) and verified by using the Agilent logic analyzer.

TITLE The Use of Celebrity Endorsement with the Help of Electronic Communication Channel (Instagram): Case Study of Magnum Ice Cream Thailand RESEARCH QUESTION How does the use of celebrity endorsement with the help of electronic communication channel (Social media: Instagram) affect the buying behavior of generation Y consumers in Thailand? STRATEGIC QUESTION How can marketers use this marketing technique in an effective way to increase the number of consumers? PURPOSE OF THE STUDY The purpose of this research paper is to describe the buying behavior of generation Y consumers in Thailand with regards to the use of celebrity endorsement through the electronic communication channel (Instagram). METHODOLOGY In this research study, both primary data and existing secondary data are used. A questionnaire of 436 target respondents was taken into account to answer the research question. CONCLUSION The use of celebrity endorsement through Instagram has a low effect on buying behavior of generation Y in Thailand. However, the result shows that this advertising technique easily reach to consumers and help them to know the product.