Thesis (MScIng)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (Ph.D)--Electrical and Computer Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Cressler, John; Committee Member: Papapolymerou, John; Committee Member: Shen, Shyh-Chiang; Committee Member: Steffes, Paul; Committee Member: Zhou, Hao Min. Part of the SMARTech Electronic Thesis and Dissertation Collection.

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Thesis (Ph. D.)--University of California, Riverside, 2010. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed May 18, 2010). Includes bibliographical references. Also issued in print.

The very low photon backgrounds to be achieved by future cryogenic astronomical telescopes present the ultimate challenge to the sensitivity of infrared detectors and associated readout electronics. Cooled silicon JFETs, operated around 70 K in transimpedance amplifiers, have shown excellent performance and stability. However, due to Johnson noise in the feedback resistor, the read noise in one second achieved by such amplifiers is about 500 electrons per second. A drastic improvement in sensitivity was demonstrated using a simple form of integrating JFET amplifiers. Therefore, the excellent performance obtained with cooled silicon JFETs has led to the investigation of their properties in the temperature range 33-77 K to explore their full potential and improve the performance of the integrating amplifier. The freezeout effect in silicon JFETs has been characterized both experimentally and theoretically using a simple analytical simulation program. The effect of variation in device parameters on the freezeout characteristic has been studied, and test results showed that an effective channel mobility must be used instead of a bulk mobility in order to simulate accurately the device current and transconductance freezeout at low temperatures. Many types of commercially available JFETs have been characterized below 77 K and measurements revealed that a balanced source follower or a common-source amplifier with active load can operate well down to 38 Kelvin with extremely low power dissipation. The open gate equivalent input noise voltage was found to be optimum below 77 K, due to a decrease in the gate leakage current, in agreement with theoretical prediction. Based on the superior performance of the balanced source follower with active load, a single channel hybrid integrating JFET amplifier with a JFET reset and a compensation capacitor was developed for operation in the temperature range 40-77 K. Read noise as low as 10 electrons in 128 seconds integration was achieved when the integrator was operated at an optimum temperature of about 55 K. Using a similar design, a 16-channel monolithic integrating amplifier array was designed and built. Preliminary test results at 77 K showed noise performance comparable to the single channel hybrid integrator.

Diseases caused by pathogenic bacteria continue to be major health concerns. For example, it is estimated that in the year 2000 typhoid fever caused over 21,000,000 illnesses and ~200,000 deaths (Crump et al., 2004). The disease is caused by S. typhi, a closely-related serotype of S. typhiumurium, the salmonella strain in which BipA was first identified. The CDC estimated that in 2013, multidrug resistant bacteria caused over 2 million infections in the United States, ending in more than 23,000 deaths (CDC, 2013). This number is set to rise as more bacteria become resilient to the collection of conventional antibiotics. The increasing number of multidrug resistant bacterial strains necessitates the development of new antimicrobial drugs. BipA is an attractive target for drug research. As mentioned in Section 2.5.2, BipA is ubiquitous in eubacteria and lower eukaryotes such as protozoa, but is absent from higher-order eukaryotes such as humans. Because the protein is essential for bacterial survival, BipA presents a major vulnerability of pathogenic bacteria. A drug targeting the protein itself or its interactions to the ribosome will disable only the bacteria, but have no effect on the eukaryotic host. A comprehensive model of BipA bound to the 70S ribosome will provide unparalleled insight into BipA's binding site and its mechanism. Toward this goal, cryo-EM techniques were employed to visualize the binding site of BipA on the 70S ribosome, characterize its interactions with the ribosome, and elucidate its mechanism on the ribosome. An X-ray structure of isolated BipA-GMPPNP was elucidated, by collaborators, and used for further molecular modeling of the protein to reveal possible atomic interactions between BipA and 70S ribosome. Additional biochemical studies were performed to fully characterize the specific ribosomal complex that optimizes binding of the factor. Together, the cryo-EM reconstruction, the BipA X-ray structure, the subsequent molecular modeling, and the additional biochemical studies provide a comprehensive model for BipA binding. Over the last years, the introduction of new automated algorithms for particle selection (AutoPicker) and classification (RELION) for the cryo-EM technique has revolutionized the workflow of the entire imaging and reconstruction process. The BipA dataset was primed to be used as a test bed for these algorithms and classification technique, respectively. Using old and new techniques to process the dataset allows a discussion of how the single particle reconstruction process can be vastly improved, with greater automation and efficiency.

Cryo-electron microscopy (cryo-EM) is a structural biology technique that determines the structure of proteins and macromolecular complexes using the transmission electron microscope under cryogenic conditions. In my Ph.D. studies, I took advantage of this technique, in the study of dynamical features of ribosomes in both eukaryotes and prokaryotes. In Chapter 2, I report my graduate research on the investigation of ribosomes from the human malaria parasite, Plasmodium falciparum, using single-particle cryo-EM. In collaboration with Dr. Jeffrey Dvorin at Harvard Medical School, we obtained five cryo-EM reconstructions of ribosomes purified from P. falciparum blood-stage schizonts, and discovered structural and dynamical features that differentiate the ribosomes of P. falciparum from those of the mammalian system. Moreover, we discovered that RACK1, a necessary ribosomal protein in eukaryotes, does not specifically co-purify with the 80S fraction in the P. falciparum schizonts stage and would mainly function in a ribosome-unbound, free state during the blood-stage. More extensive studies, using cryo-EM methodology, of translation in the parasite, will provide structural knowledge that could help in the design of effective anti-malaria drugs. In Chapter 3, I describe the cryo-EM studies of the Saccharomyces cerevisiae ribosome in response to a carbon source switch. In collaboration with Dr. Andrew Link at Vanderbilt University, we obtained reconstructions of the 80S ribosomes at selected time points after the glucose-to-glycerol carbon source shift, and observed that a fraction of ribosomes lacked densities for r-proteins, mainly eS1 (yeast rpS1) on the 40S subunit and uL16 (yeast rpL10) on the 60S subunit. We found that the binding ratio of eS1 and uL16 to ribosomes changed as a function of time, consistent with the change in translational activities as gauged by polysome profiling. On the basis of these observations, along with previous structural and genetics studies, we propose that rapid control of translation is exerted through the dissociation of r-protein eS1/rpS1 and uL16/rpL10 from the ribosome. Our studies thus open a new venue on the exploration of S. cerevisiae’s rapid adaption to carbon source shifts at the level of translation. In Chapter 4, I have documented a collaborative work on the development and application of a new technique, time-resolved cryo-EM, which can be used to study processes involving two reaction partners on a sub-second time scale. With my colleagues at the Frank and Gonzalez labs at Columbia University, we successfully applied this method to study the process of E. coli ribosomal subunits association. By mixing and reacting the two subunits for 60 ms and 140 ms, we captured the association reaction in a pre-equilibrium state, and detected different conformations of E. coli 70S ribosomes. With the current capability of this mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, we expect to be able to standardize this method and apply it to more challenging biological processes, such as translation recycling and initiation processes.

The ribosome is a macromolecular machine, present in high copy number in the cell, that synthesizes proteins from information encoded in messenger RNA. It is a universal translator, found in all life forms and in all eras recent enough to bear life. The ribosome is structurally complex and its structure is highly evolutionarily conserved; that conservation reinforces the concept that its function in executing translation is essential. As a subject of study, the ribosome lends itself well to direct imaging, as it is large, asymmetric, dynamic, and it interacts with other heterogeneous agents throughout the translation process; if we are to infer function from structure, then the most certain way to observe the ribosome’s structure is to image it as directly as possible. Cryo-electron microscopy and single particle reconstruction are appropriate tools for this endeavor, as they can produce high-resolution three-dimensional structures of ribosomes or other macromolecular samples, and they can even reveal multiple biologically relevant states of a single sample. Although the ribosome is highly conserved in terms of its presence and core structure and functions, there is considerable variation among taxa, and the function of some of this variation is not yet understood. For example, the ribosome of the unicellular trypanosomatid parasite Leishmania major exhibits unusually large expansion segments of ribosomal RNA, as well as unusual cleavage sites in ribosomal RNA that is otherwise conserved. Here, we present a three-dimensional cryo-electron microscopy reconstruction of the 80S ribosome of Leishmania major and compare it to the available ribosome structures of closely related parasites. There is also structural variation related to the mechanism of translation: certain viruses with RNA genomes employ highly structured segments of RNA called internal ribosome entry sites to initiate translation of viral proteins on host cell ribosomes via noncanonical mechanisms. We explore one instance of this with a reconstruction of the encephalomyocarditis virus internal ribosome entry site bound with necessary protein factors to a eukaryotic 40S ribosomal subunit.

The BICMOS technology which integrates the CMOS technology with bipolar technology has drawn considerable attention as an attractive VLSI technology because of the high speed performance and low power consumption of the BICMOS. However, continued down scaling of CMOS devices has caused increased concerns with problems such as latch up, hot carriers and short channel effect. Most of the above mentioned problems can be avoided by operating the CMOS at liquid-nitrogen temperature(LNT). At low-temperatures, the CMOS exhibits lower sub threshold leakage, higher carrier mobility (which yields improved speed performance), and a steeper logarithmic currentvoltage slope. On the other hand, the low-temperature operation of conventional silicon bipolar circuits has been generally dismissed as impractical because of the well known decrease in the current gain at low temperature. The present interest in integrated bipolarCMOS circuits, plus the prospect of increased reliability, lower wiring delay, and lower noise, has revised interest in low-temperature bipolar devices. In this context, it is therefore important to acquire accurate knowledge of the transistor properties at liquid nitrogen temperature. This can be done in two ways. One is through experimental lowtemperature measurements and the other by low-temperature device simulations. Existing room temperature numerical simulators are typically not useful for low temperature conditions. This is because the physical assumptions such as complete ionization, the parameter models and implementation methods for room temperature condition do not hold at low temperature. Therefore, we used BiLow - a steady state onedimensional Bipolar Low Temperature Simulator for the temperature range of 77K- 300K. This simulator, originally written in FORTRAN, was converted to C for the dual purpose of proper memory management and making further modifications easier. The focus of this research has been to model bandgap narrowing, incomplete ionization and Mott Transition at room and at low-temperature, evaluate the performance of the new BiLow and to derive conclusions on the BIT performance at LNT. It was observed that the bandgap narrowing was independent of temperature for the entire range of majority carrier concentration. The effect of Mott transition on the abrupt decrease in the electron concentration in emitter has been taken care of by smoothing out the concentration profile in the emitter thereby providing a continuity in the region of Mott transition. Both the current gain(~) and the frequency(ft) values obtained from simulating the two new profiles were found to be smaller than those obtained using the original BiLow simulator, as the doping in the base is higher and the device sizes were smaller. Most of the degradation in 13 and ft was found to occur below 150K. From the plots of the charge characteristics, we found that the total charge which is a strong function of temperature is more in the case of the profiles studied for this work than the total charge from the original BiLow simulator.

Thesis (Ph. D.)--Mississippi State University. Department of Electrical and Computer Engineering. / Title from title screen. Includes bibliographical references.