In this thesis, new approaches to direct trace metals analysis of solid samples by inductively coupled plasma spectroscopy were investigated using laser ablation and thermal vaporization systems for solid sample introduction of biological, environmental, and pharmaceutical samples. / Laser ablation with inductively coupled plasma atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS) was applied to pharmaceutical tablets. Precision of analysis depended on laser parameters and could be improved using signal ratios. The feasibility of using laser ablation-ICP-MS for detecting natural levels of mercury along a single human hair strand was also demonstrated. / As well, the use of an induction-heating electrothermal vaporizer (IH-ETV) coupled to an ICP-MS was successful in determining mercury concentrations in a single human hair strand. Methodologies for multielement analysis of powdered hair were also explored using IH-ETV-ICP-MS. While calibration by reference hair materials showed promise, calibration methods by liquid standards were not suitable for any element. Detection limits achieved for most elements were below natural levels found in human hair. / IH-ETV-ICP-AES was also applied to the analysis of analyze-laden chromatographic powder. This study showed potential problems that may arise due to the methodology taken to analyze such materials. Finally, recommendations for future investigations and methodologies for laser ablation and thermal vaporization are discussed.

Transforming growth factor beta (TGFbeta) and Angiotensin II (AngII) signaling occurs through two distinct receptor superfamilies, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Through diametric actions, TGFbeta and AngII regulate various biological responses, including cell proliferation and migration. Previously, we identified the G protein-coupled receptor kinase 2 (GRK2), which acts through a negative feedback loop mechanism to terminate Smad signaling. To investigate the impact of TGFbeta-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). We show that activation of the TGFbeta signaling cascade results in increased GRK2 expression levels, consequently inhibiting AngII-induced ERK phosphorylation and antagonizing AngII-induced VSMC proliferation and migration. The inhibitory effect of TGFbeta on AngII signaling occurs at the MEK-ERK interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFbeta signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation. GRK2 is a key factor in mediating this crosstalk.

LPA1 lysophosphatidic acid receptors (LPA1Rs) are normally present on the surface of the cell. Our initial findings were that HMG-CoA reductase inhibitors (atorvastatin and mevastatin) induce the sequestration of the G protein-coupled LPA1R in recycling endosomes, most likely by inhibiting the recycling of tonically internalized receptors. Whereas, co-addition of geranylgeranylpyrophosphate (GGPP) or geranylgeraniol (GGOH) prevented atorvastatin-induced sequestration of LPA1Rs, the geranylgeranyltransferase-I inhibitor, GGTI-298, mimicked atorvastatin and induced LPA1R sequestration. This suggested that statin-induced endosomal sequestration was caused by defective protein prenylation. The likely targets of atorvastatin and GGTI-298 are the Rho family GTPases, RhoC and RhoA, since both inhibitors greatly reduced the abundance of these GTPases and since knockdown of endogenous RhoC or RhoA with small interfering RNAs (siRNAs) led to endosomal sequestration of LPA1R. Knockdown of RhoC was much more potent at inducing endosomal sequestration than knockdown of either RhoA or RhoB. In contrast, atorvastatin, GGTI-298, siRNA against RhoA, B, or C did not alter the internalization or recycling of transferrin receptors, indicating that recycling of transferrin receptors is distinct from LPA1Rs. Thus, these results, for the first time, implicate RhoA and RhoC in endocytic recycling of LPA1Rs and identify atorvastatin and GGTI-298 as novel inhibitors of this process. / Per the request of the author and advisor, and with the approval of the Graduate Education office, the following changes were made to this thesis: Replaced original page 1 with Errata Page 2. Replaced original pages 3-28 with Errata Pages 3 – 16. Replaced original pages 69-71 with Errata pages 17 – 19.

The objective of the research presented in this thesis is to validate the parallel-bonded modeling method in the context of coupled thermo-mechanical simulations. The simulation results were compared with analytical and experimental data, in the attempt to assess the usability of this particular modeling method. Previous studies of numerical approaches that related to the thermal fracturing of hard rock had used continuum-based models with constitutive relations. The simulations in the thesis were conducted using Particle Flow Code (PFC) which was chosen for the research because of its several benefits. The code has unique features such as spontaneous damage development without imposed conditions, and emergent properties such as material heterogeneity, and dynamic behavior giving possibility to monitor synthetic seismic events. The basic code has been available since 1995 and research using the code has produced hundreds of publications. The thermal option for the code is a recent addition and lacked verification, validation and applications. The thesis is the answer for that. In the course of the research work new particle clustering and grouping routines were developed and tested. Three modeling studies were conducted varying from laboratory to field scales. The 2D modeling study of the heated cylinder experiment yielded similar results both in fracture-behavioral and acoustic emission (AE) magnitude ranges when compared with the laboratory data. The 3D cubic numerical specimens, created with breakable particle clusters, were heated, and the induced damage was observed by P wave velocity measurements. The results showed trends comparable to the laboratory data: P wave velocity decreases with rising temperatures of up to 250°C and cluster-boundary cracking occurs, comparable to grain-boundary cracking in the heated rock samples. The large 2D tunnel models captured the phenomena observed in-situ displaying the difference in the damage to the roof and floor regions, respectively. This damage was due to the filling material confinement of about 100 kPa on the tunnel floor. In general, the results of the thermo-mechanical simulations were in accordance with the experimental data. The modeled temperature evolutions during the heating and cooling periods were also in accordance with the experimental and analytical data.

Frequency synthesizers are essential components for modern wireless and wireline communication systems as they provide the local oscillator signal required to transmit and receive data at very high rates. They are also vital for computing devices and microcontrollers as they generate the clocks required to run all the digital circuitry responsible for the high speed computations. Data rates and clocking speeds are continuously increasing to accommodate for the ever growing demand on data and computational power. This places stringent requirements on the performance metrics of frequency synthesizers. They are required to run at higher speeds, cover a wide range of frequencies, provide a low jitter/phase noise output and consume minimum power and area. In this work, we present new techniques and architectures for implementing high speed frequency synthesizers which fulfill the aforementioned requirements. We propose a new architecture and design approach for the realization of wideband millimeter-wave frequency synthesizers. This architecture uses two-step multi-order harmonic generation of a low frequency phase-locked signal to generate wideband mm-wave frequencies. A prototype of the proposed system is designed and fabricated in 90nm Complementary Metal Oxide Semiconductor (CMOS) technology. Measurement results demonstrated that a very wide tuning range of 5 to 32 GHz can be achieved, which is costly to implement using conventional techniques. Moreover the power consumption per octave resembles that of state-of-the art reports. Next, we propose the N-Push cyclic coupled ring oscillator (CCRO) architecture to implement two high performance oscillators: (1) a wideband N-Push/M-Push CCRO operating from 3.16-12.8GHz implemented by two harmonic generation operations using the availability of different phases from the CCRO, and (2) a 13-25GHz millimeter-wave N-Push CCRO with a low phase noise performance of -118dBc/Hz at 10MHz. The proposed oscillators achieve low phase noise with higher FOM than state of the art work. Finally, we present some improvement techniques applied to the performance of phase locked loops (PLLs). We present an adaptive low pass filtering technique which can reduce the reference spur of integer-N charge-pump based PLLs by around 20dB while maintaining the settling time of the original PLL. Another PLL is presented, which features very low power consumption targeting the Medical Implantable Communication Standard. It operates at 402-405 MHz while consuming 600microW from a 1V supply.

The evolution of the blood feeding adaptation has required precise coordination of multiple physiological processes in the insect, such as reproduction, behavior, digestion and diuresis. These processes are under careful synchronous hormonal control. For rapid excretion, multiple diuretic hormones are known. Although originally described based on their ability to stimulate hindgut contractions, the Aedes kinins have been shown to stimulate fluid secretion in female mosquitoes of Aedes aegypti. Aedes kinins are leucokinin-like neuropeptides released from neurosecretory cells in the brain and abdominal ganglia. They act by binding to the Aedes kinin receptor, a G proteincoupled receptor (GPCR). The Aedes kinin receptor has been cloned, sequenced, functionally characterized, and immunolocalized to stellate cells in the Malpighian tubules of Ae. aegypti. In addition to their myotropic and diuretic roles, leucokinin-like peptides and/or their receptors have been also been discovered in the nervous, digestive, and reproductive systems of other arthropod species. Therefore, the Aedes kinins have the potential to function in several simultaneous physiological processes that are stimulated by blood feeding. This thesis aims to understand better their role in the whole mosquito by investigating the Aedes kinin receptor's global expression as well as its in vivo contribution to post-prandial diuresis. Presence of the Aedes kinin receptor was investigated in the head, posterior midgut (stomach), hindgut, ovaries, and Malpighian tubules of both non blood-fed and blood-fed females by western blot using anti-receptor antibodies. The receptor was then immunolocalized in the posterior midgut and rectum. Finally, RNAi was employed to knock down kinin receptor expression, followed by measurement of in vivo urine excretion post blood feeding in a precision humidity chamber. Transcript and protein knockdown were confirmed by qPCR and immunohistochemistry, respectively. Results indicate widespread expression of the Aedes kinin receptor protein in organs novel for hematophagous insects and demonstrate the receptor's fundamental role in rapid diuresis. These findings strongly point to the Aedes kinins as integrative signaling molecules that could coordinate multiple physiological systems. The Aedes kinins could therefore have contributed to the success of the blood feeding adapation in mosquitoes.

The last glacial cycle (c. 115-12 kyr BP) was the most recent in a series of recurring glaciations of the subpolar continents. Massive ice sheets evolved in Eurasia and North America, which, at their maximum, were of continental scale and together lowered the global sea-level by approximately 100 m. The paleo-modelling community has focused on the last glacial maximum (LGM, ~ 20 kyr BP), leaving the longer period when the ice sheets evolved to their LGM configurations largely unexplored. In this thesis we study the mutual interaction between the time-mean atmospheric circulation and the evolution of the Northern Hemisphere ice sheets over the build-up phase of the last glacial cycle. Experiments are conducted with coupled atmosphere-ice-sheet models and a circulation model forced by geologically consistent reconstructions of the ice-sheet topography at key stages of the glacial cycle. The main findings from these studies are that the ice evolution in North America may have been controlled by circulation anomalies induced by the background topography in conjunction with the ice sheets themselves. A geologically consistent pre-LGM ice sheet could only be obtained when including the North American Cordillera. However, the ice sheets' influence on the local climate conditions is also found to be paramount for this configuration. We further suggest that the incipient ice sheets may have had a limited influence on the large-scale winter circulation as a result of their location relative the westerly mean flow. The LGM Laurentide Ice Sheet (LIS) was, however, different because of its continent-wide extent, and it may therefore have had a large influence on the planetary-scale circulation, especially in the Atlantic sector. We find that the planetary waves forced by the LIS were considerably larger than at earlier times, and, as a result of a more frequent planetary wave reflection over the Atlantic Ocean basin, an altered stationary wave field and a zonalised winter jet. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.</p>

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.

No description available.