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The activity of PEA3, a member of the Ets proto-oncogene family of transcription factors, is associated with various normal and aberrant developmental processes. PEA3 is believed to function in vivo as a transcription factor which regulates the gene expression of its cellular targets. In particular, PEA3 motifs are found in the regulatory regions of several genes implicated in tumor progression and metastasis. Aberrant PEA3 activity has been linked to the development and progression of several types of cancer, particularly, breast cancer and Ewing's sarcoma. To assist in the understanding of the role that PEA3 plays in these processes, constitutively-activated alleles of PEA3 were created by fusing a segment of the EWS gene to murine PEA3; to essentially mimic the EWS-Ets fusion proteins isolated from the human cancer syndrome, Ewing's sarcoma. Five EWS-PEA3 chimeric genes were constructed and one (EWSΔN268PEA3), is an exact murine version of an EWS-human PEA3 fusion gene isolated from Ewing's sarcoma. Transcriptional analysis of the EWS-PEA3 chimeras revealed that with the exception of one mutant, all the chimeras possessed increased transcriptional activity in comparison to normal PEA3 . Furthermore, it was suggested that the transcriptional activity of the chimeras may be regulated by Ras, although this observation requires additional validation. The chimeras were not found to possess any transforming activity, however, this finding does not suggest that the chimeras are not oncogenic in nature. In conclusion, these findings suggest that the EWS-PEA3 chimeras may indeed function as activated versions of the PEA3 transcription factor. / Thesis / Master of Science (MS)

The repair of damaged or diseased bone tissue often requires the use of metallic implants which form an interface with the surrounding bone tissue. Understanding this interface is important for improving the outcomes of implant placement and overall health of patients. Bone is a composite material of organic collagen fibrils and inorganic mineral phases that have structural variations across multiple length scales. This heterogeneous and hierarchical nature poses characterization challenges for (i) understanding bone, (ii) creating biomaterial structures that mimic it, and (iii) approaches for evaluating biomaterials. These challenges formed the basis for the three papers presented in this thesis. In Chapter 3, leporine bone was examined using atom probe tomography (APT) to visualize in vivo mineralized collagen fibrils, their chemical composition, and spatial arrangement in 3D with sub-nanometer accuracy. This provided new insight into the location of biomineral with respect to collagen and demonstrated the power of APT for understanding collagen-mineral arrangement. In Chapter 4, commercially pure titanium was laser ablated to generate periodic surface structures inspired by the periodicity of collagen. Three different periodicities were generated with submicron-scale roughness and a high degree of reproducibility. All the surfaces were non-cytotoxic and encouraged cells to adhere perpendicular to the orientation of the surface structures. In Chapter 5, a simple five-minute room temperature ionic liquid treatment was developed to investigate the same laser-ablated titanium periodic structures with osteoblast-like cells adhered. The development of this technique fulfills an important niche in biological imaging by allowing for simultaneous and repeated visualization of submicron surface features and wet cells. Therefore, the combined impact of this thesis is novel imaging and biomaterials evaluation strategies to (i) improve understanding of bone structure; (ii) leading to bioinspired biomaterials design; and (iii) new methods for simultaneous biological and biomaterials evaluation. / Thesis / Doctor of Philosophy (PhD) / Bone implant devices are required to treat, augment, or replace bone tissue in dental and orthopaedic applications. These, often metallic, implanted devices have success when a structural and functional connection with natural bone tissue is created, a phenomenon known as osseointegration. Good osseointegration is required to ensure stability of the implant without compromising the quality of life of the patient. In order to improve osseointegration of biomaterials, both sides of the interface, i.e. the bone and implant surface, must be better understood. This thesis focuses on exploring methods to improve the evaluation and understanding of both bone structure at the nanoscale and structured metallic implant surfaces for the design of bone-interfacing biomaterials.

Isobutylamine N-hydroxylase (IBAH) and flavin reductase (FRED) from Streptomyces viridifaciens are part of a two-component flavin-dependent monooxygenase enzyme system that catalyze the conversion of isobutylamine (IBA) to isobutylhydroxylamine (IBHA), a key step in the formation of valanimycin, an azoxy antibiotic. In this work, we present the over-expression, purification and biochemical characterization of this two-component enzyme system. IBAH and FRED were expressed and purified to homogeneity as separate proteins. FRED exhibited the oxidoreductase activity by catalyzing the oxidation of NADPH. The hydroxylation activity of IBAH was confirmed using liquid chromatography – mass spectrometry (LC-MS). Steady state kinetic data showed an oxidation activity of the monooxygenase component which proceeded at 1.97 ± 0.06 s⁻¹ as measured from oxygen consumption and in product formation, the rate was 0.012 ± 0.001 s⁻¹ , suggesting a high degree of uncoupling between product formation and oxygen consumption. In pre-steady state kinetic characterization studies, the FRED-catalyzed reduction of FAD by NADPH occurred at a rate of 10.0 ± 0.2 s⁻¹ and the KM was 490 ± 40 µM. The rate of reduction was ~1.5-fold decreased in the presence of substrate IBA whiles the KM was 500 ± 50 µM. NADH showed a markedly reduced rate of reduction with a kred of 0.34 ± 0.03 s⁻¹ with an apparent KM of 3000 ± 500 µM. The rate of flavin re-oxidation in the absence of monooxygenase IBAH was 4.79 × 10⁻⁹ M-1 s⁻¹. Our results suggest a reaction mechanism for the IBAH monooxygenase system controlled by the oxidation half reaction that may be modulated by a complex formation between the reductase and monooxygenase components. / Master of Science in Life Sciences

Software Defined Radios (SDRs) are systems in which components such as filters, modulators, demodulators, etc., typically implemented in hardware are instead implemented in software. Thus, SDR systems shift signal processing from analog to digital domain. The signal processing is performed in reconfigurable devices like General Purpose Processors (GPP) or Field Programmable Gate Arrays (FPGA). The resulting flexibility of SDR systems brings in many advantages to wireless communications such as improved interoperability, adaptation capability and more future-proof hardware. Various prototype/laboratory friendly hardware are available such as Universal Software Radio Peripherals (USRPs), developed by Ettus Research, which in combination with software interfaces such as GNU Radio can be used to design and implement a Software Defined Radio(SDR) system. This has in turn enabled numerous research opportunities and advancements in field of wireless communication. However one major drawback of these simple USRP devices is that they are un-calibrated in terms of power or voltage and hence give the results in relative terms/counts. This poses a disadvantage in real world scenarios where parameters such as power measured in milliwatts or dBm, are used to design, implement, and test devices and systems and to collect and interpret experimental results. Lack of connectivity with these metrics leads to the USRP devices being confined to proof of concept implementation and demonstrations, and limits their utility for experimentation. In this thesis an attempt has been made to calibrate the USRPN210 (with WBX daughter board) devices and derive some calibration factors that would help relate the experimental results obtained from the USRP and GNU Radio combination to real world metrics. This also makes the result-set clearer and easily understandable. Several experiments were conducted to understand and characterize the performance of the USRP under different conditions. Experiments such as determination of 1-dB compression point, Third order intercept point helped understand the linearity range of the device under transmitter and receiver operations. The 3rd order input intercept point for USRPN210 device with WBX board with calibration was found to be around 0.25 dBm, which is closer to the expected value of 0 dBm at 400 MHz. Amplitude and frequency stability tests over the operational frequency range of the daughter board helped to note if change in frequency produced any variation in the received or transmitted power. Also variation with respect to gain helped derive a reference table for transmitted power, which could be used in for future experiments. Conducting these experiments over a sample of 10 USRPs helped in obtaining standardized values and benchmarking them. The factors obtained were also used in some experiments like path loss modeling, position location estimation in order to determine their effectiveness and impact on such applications. Although, these factors obtained may not be directly applicable to all USRP devices, as in any analog circuitry, and also since they are conducted relative to some specific applications such as the predefined spectrum analyzer utility of GNU Radio (UHD_FFT.grc), they do help to understand the basis of derivation of calibration metrics resulting from the various experiments and also provide a platform for future work in this area to obtain more uniform and globally acceptable results. All photos by the author unless cited, 2014. / Master of Science

Seismic time-lapse monitoring of production is an important tool used to efficiently drain a hydrocarbon reservoir. Repeat seismic surveys may be used, because the seismic method is sensitive to the reservoir fluid. A prominent seismic attribute is the reflectivity (or amplitude) as a function of offset (AVO) which strongly depends on material properties, and hence, on the pore fluid. Repeat surveys, however, are very costly. To reduce the risks, the repeat survey is simulated on a computer for a number of different scenarios. Hence, the objectives of this study are to predict the seismic responses after five years of production of the reservoirs at the well locations, correlate the seismic attributes to fluid conditions in the reservoirs, assess the detectability of changes in AVO attributes due to changes in fluid conditions, and determine which attribute is more diagnostic of fluid changes. Petrophysical models were generated for different pore fluids using well logs from a field in the Gulf of Mexico. Synthetic seismograms were then calculated using a layerstack scheme to study the effects of the reservoir fluids on AVO. Compared to idealized half-space models, it was found that the AVO responses are contaminated by the overburden and the thinness of the reservoir. In order to remove transmission loss due to overburden effects, the synthetic AVO curves were scaled by normalizing an overburden-over-half-space model to an idealized analytical Zoeppritz model. In a second step, an offset-dependent overburden correction was applied using a low order polynomial, which was fitted to the amplitude ratios between the overburden/half-space model and the idealized model. Finally, a zero-offset tuning correction was applied. The results of the AVO analyses showed that some errors were unresolved using the applied overburden and tuning corrections, and amplitudes at large offsets were possibly contaminated by multiples and converted waves. Since there is no shallower production or steam injection for this particular field, the repeat surveys should have the same overburden, tuning, multiple-related and converted wave contamination. It appears reasonable to assume that any changes in amplitude between the repeat surveys would be due to fluid saturation changes. Therefore, it was concluded that it is not necessary to attempt to remove the overburden and tuning effects. Results from the AVO analyses of the uncorrected models showed that AVO attributes should be a useful tool to detect reservoir conditions during the production of the field. Generally, the water-flood changes the AVO by decreasing the intercept and increasing the gradient from the in-situ oil/gas cases. The relative changes in both intercept and gradient due to the water-flood are detectable assuming a 20% relative-change detection threshold, and gradient is more diagnostic because the relative change in gradient is very large compared to that for intercept. / Master of Science

Thesis in cotutelle with the University of Gothenburg, Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy Gothenburg, Sweden / Seeing is believing. Our understanding of phenomena often involves their direct observation. However, bone architecture is challenging to visualize given its multi-level hierarchical organization. In this thesis, bone and bone interfaces are characterized via multimodal and multiscale platforms, combining different techniques across several length scales. Imaging techniques across the micro-nano continuum are complemented by spectroscopy methods to explore, respectively, the structure and composition of bone and bone interfaces, using both light and electron probes. By applying a characterization methodology more typical of materials science, this thesis aims to unveil structural and compositional abnormalities of bone induced by disease [Papers I-II], and bone response to functionalized biomaterials in compromised conditions [Papers III-IV]. Additionally, it expands three-dimensional (3D) characterization opportunities at the nanoscale in both native and peri-implant bone [Papers V-VI]. This characterization approach uncovered changes in bone quality (structure and/or composition) in the compromised conditions under investigation in this thesis, i.e., leptin receptor (LepR) deficiency and medication-related osteonecrosis of the jaw (MRONJ) [Papers I-II]. In a preclinical model of LepR deficiency for type 2 diabetes/obesity, multimodal characterization of bone at the microscale showed structural abnormalities indicative of delayed skeletal development, despite unaffected bone matrix composition [Paper I]. A combination of multiscale imaging and spectroscopy techniques spanning the micro-to-nanoscale enabled a detailed study of the interface between necrotic bone and bacteria in a case of MRONJ, shedding light on possible mechanisms of bone degradation. When applied to bone-biomaterial interfaces, the application of a multimodal and multiscale characterization workflow informed perspectives on bone response to novel biomaterial solutions aimed to promote osseointegration in osteoporotic conditions via local drug delivery of phytoestrogens [Paper III] or anabolic agents [Paper IV]. This highlighted the importance of studying peri-implant bone at the mesoscale [Paper III] and of confirming biomaterial behaviour in vivo in the presence of surface functionalization [Paper IV]. Lastly, this thesis emphasized the importance of 3D imaging at the nanoscale with electron tomography to resolve bone ultrastructure at biomaterial interfaces [Paper V] and in native conditions [Paper VI]. Specifically, in Paper VI, artifact-free on-axis electron tomography resolved some long-debated aspects regarding the organization of mineralized collagen fibrils, the fundamental building block units of bone. / Thesis / Doctor of Philosophy (PhD) / Bone is a hierarchical material, meaning that smaller components are progressively organized into larger structures. This multi-level architecture is hard to visualize because different techniques are required to obtain information at different length scales. However, as the proverb says, “seeing is believing”, and our understanding of things is often better accomplished by looking at images. This thesis applies a characterization approach more typical of materials science to study the structure, composition, and repair of bone across multiple length scales. This characterization approach is multimodal because it uses different techniques, and multiscale because it targets different length scales in bone. In particular, this thesis focuses on bone in compromised conditions, namely osteoporosis, diabetes, and medication-related osteonecrosis, to understand the impact of disease-induced changes in bone properties. It also examines how bone organizes itself at a fundamental level, which is a critical aspect to understand since bone is built from the bottom-up.

Estimation of reservoir fracture parameters, fracture orientation and density, from seismic data is often difficult because of one important question: Is observed anisotropy caused by the reservoir interval or by the effect of the lithologic unit or multiple units above the reservoir? Often hydrocarbon reservoirs represent a small portion of the seismic section, and reservoir anisotropic parameter inversion can be easily obscured by the presence of an anisotropic overburden. In this study, I show examples where we can clearly observe imprints of overburden anisotropic layers on the seismic response of the target zone. Then I present a simple method to remove the effect of anisotropic overburden to recover reservoir fracture parameters. It involves analyzing amplitude variation with offset and azimuth (AVOA) for the top of reservoir reflector and for a reflector below the reservoir. Seismic CMP gathers are transformed to delay-time vs. slowness (tau-p) domain. We then calculate the ratio of the amplitudes of reflections at the reservoir top and from the reflector beneath the reservoir. The ratios of these amplitudes are then used to isolate the effect of the reservoir interval and remove the transmission effect of the overburden. The methodology is tested on two sets of models - one containing a fractured reservoir with isotropic overburden and the other containing a fractured reservoir with anisotropic overburden. Conventional analysis in the x-t domain indicates that the anisotropic overburden has completely obscured the anisotropic signature of the reservoir zone. When the new methodology is applied, the overburden effect is significantly reduced. The methodology is also applied to an actual PP surface reflection (Rpp) 3D dataset over a reservoir in the Arabian Peninsula. Ellipse-fitting technique was applied to invert for two Fracture parameters: (1) Fracture density and (2) fracture direction. Fracture density inversion results indicate increased fracturing in the anticline structure hinge zone. Fracture orientation inversion results agree with Formation MicroImaging (FMI) borehole logs showing a WNW-ESE trend. This newly developed amplitude ratio method is suitable for quantitative estimation of fracture parameters including normal and tangential “weaknesses” (ΔN and ΔT respectively). Initially, inversion of conventional AVOA for ΔN and ΔT parameters indicates that the ΔN parameter is reliably estimated given an accurate background isotropic parameter estimation derived from borehole logging data. While ΔN parameter inversion is successful, inversion for ΔT parameter from Rpp information is not, presumably due to the dependence of ΔT estimation on many medium parameters for accurate prediction. The ΔN parameter is then successfully recovered when applied to the amplitude ratio values derived from synthetic data. It is important to recognize that ΔN parameter is directly proportional to fracture density and high ΔN values can be attributed to high crack density values. The ΔN parameter inversion is also applied to the amplitude ratios derived from real seismic data. This inversion requires fracture azimuth data input that is obtained from the fracture direction inversion using ellipse-fitting technique. The background Vp/Vs ratio. / text

The primary objective of this thesis is to develop a methodology for fast, yet accurate temperature estimation during design space exploration. Power and temperature of modern day systems have become important metrics in addition to performance. Static and dynamic power dissipation leads to an increase in temperature, which creates cooling and packaging issues. Furthermore, the transient thermal profile determines temperature gradients, hotspots and thermal cycles. Traditional solutions rely on cycle-accurate simulations of detailed micro-architectural structures and are slow. The thesis shows that the periodic power estimation is the key bottleneck in such approaches. It also demonstrates an approach (FastSpot) that integrates accurate thermal estimation into existing host-compiled simulations. The developed methodology can incorporate different sampling-based thermal models. It achieves a 32000x increase in simulation throughput for temperature trace generation, while incurring low measurement errors (0.06 K- transient,0.014 K- steady-state) compared to a cycle-accurate reference method. / text

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