A genetic and biochemical analysis of pre-mRNA splicing in Saccharomyces cerevisiae

Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.

Yeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.

To study trans -acting splicing factors we generated and screened a bank of temperature- sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP 18 gene has been cloned and its polyadenylated transcript identified.

Beyond the blur: Construction and characterization of the first autonomous AO system and An AO survey of magnetar proper motions

Adaptive optics (AO) corrects distortions created by atmospheric turbulence and delivers diffraction-limited images on ground-based telescopes. The vastly improved spatial resolution and sensitivity has been utilized for studying everything from the magnetic fields of sunspots upto the internal dynamics of high-redshift galaxies. This thesis about AO science from small and large telescopes is divided into two parts: Robo-AO and magnetar kinematics.

In the first part, I discuss the construction and performance of the world’s first fully autonomous visible light AO system, Robo-AO, at the Palomar 60-inch telescope. Robo-AO operates extremely efficiently with an overhead < 50s, typically observing about 22 targets every hour. We have performed large AO programs observing a total of over 7,500 targets since May 2012. In the visible band, the images have a Strehl ratio of about 10% and achieve a contrast of upto 6 magnitudes at a separation of 1′′. The full-width at half maximum achieved is 110–130 milli-arcsecond. I describe how Robo-AO is used to constrain the evolutionary models of low-mass pre-main-sequence stars by measuring resolved spectral energy distributions of stellar multiples in the visible band, more than doubling the current sample. I conclude this part with a discussion of possible future improvements to the Robo-AO system.

In the second part, I describe a study of magnetar kinematics using high-resolution near-infrared (NIR) AO imaging from the 10-meter Keck II telescope. Measuring the proper motions of five magnetars with a precision of upto 0.7 milli-arcsecond/yr, we have more than tripled the previously known sample of magnetar proper motions and proved that magnetar kinematics are equivalent to those of radio pulsars. We conclusively showed that SGR 1900+14 and SGR 1806-20 were ejected from the stellar clusters with which they were traditionally associated. The inferred kinematic ages of these two magnetars are 6±1.8 kyr and 650±300 yr respectively. These ages are a factor of three to four times greater than their respective characteristic ages. The calculated braking index is close to unity as compared to three for the vacuum dipole model and 2.5-2.8 as measured for young pulsars. I conclude this section by describing a search for NIR counterparts of new magnetars and a future promise of polarimetric investigation of a magnetars’ NIR emission mechanism.

A retrofitting framework for pre-Northridge steel moment-frame buildings

In the 1994 M_w 6.7 Northridge and 1995 M_w 6.9 Kobe earthquakes, steel moment-frame buildings were exposed to an unexpected flaw. The commonly utilized welded unreinforced flange, bolted web connections were observed to experience brittle fractures in a number of buildings, even at low levels of seismic demand. A majority of these buildings have not been retrofitted and may be susceptible to structural collapse in a major earthquake.

This dissertation presents a case study of retrofitting a 20-story pre-Northridge steel moment-frame building. Twelve retrofit schemes are developed that present some range in degree of intervention. Three retrofitting techniques are considered: upgrading the brittle beam-to-column moment resisting connections, and implementing either conventional or buckling-restrained brace elements within the existing moment-frame bays. The retrofit schemes include some that are designed to the basic safety objective of ASCE-41 Seismic Rehabilitation of Existing Buildings.

Detailed finite element models of the base line building and the retrofit schemes are constructed. The models include considerations of brittle beam-to-column moment resisting connection fractures, column splice fractures, column baseplate fractures, accidental contributions from ``simple'' non-moment resisting beam-to-column connections to the lateral force-resisting system, and composite actions of beams with the overlying floor system. In addition, foundation interaction is included through nonlinear translational springs underneath basement columns.

To investigate the effectiveness of the retrofit schemes, the building models are analyzed under ground motions from three large magnitude simulated earthquakes that cause intense shaking in the greater Los Angeles metropolitan area, and under recorded ground motions from actual earthquakes. It is found that retrofit schemes that convert the existing moment-frames into braced-frames by implementing either conventional or buckling-restrained braces are effective in limiting structural damage and mitigating structural collapse. In the three simulated earthquakes, a 20% chance of simulated collapse is realized at PGV of around 0.6 m/s for the base line model, but at PGV of around 1.8 m/s for some of the retrofit schemes. However, conventional braces are observed to deteriorate rapidly. Hence, if a braced-frame that employs conventional braces survives a large earthquake, it is questionable how much service the braces provide in potential aftershocks.

Battery-Powered RF Pre-Ionization System for the Caltech Magnetohydrodynamically-Driven Jet Experiment: RF Discharge Properties and MHD-Driven Jet Dynamics

This thesis describes investigations of two classes of laboratory plasmas with rather different properties: partially ionized low pressure radiofrequency (RF) discharges, and fully ionized high density magnetohydrodynamically (MHD)-driven jets. An RF pre-ionization system was developed to enable neutral gas breakdown at lower pressures and create hotter, faster jets in the Caltech MHD-Driven Jet Experiment. The RF plasma source used a custom pulsed 3 kW 13.56 MHz RF power amplifier that was powered by AA batteries, allowing it to safely float at 4-6 kV with the cathode of the jet experiment. The argon RF discharge equilibrium and transport properties were analyzed, and novel jet dynamics were observed.

Although the RF plasma source was conceived as a wave-heated helicon source, scaling measurements and numerical modeling showed that inductive coupling was the dominant energy input mechanism. A one-dimensional time-dependent fluid model was developed to quantitatively explain the expansion of the pre-ionized plasma into the jet experiment chamber. The plasma transitioned from an ionizing phase with depressed neutral emission to a recombining phase with enhanced emission during the course of the experiment, causing fast camera images to be a poor indicator of the density distribution. Under certain conditions, the total visible and infrared brightness and the downstream ion density both increased after the RF power was turned off. The time-dependent emission patterns were used for an indirect measurement of the neutral gas pressure.

The low-mass jets formed with the aid of the pre-ionization system were extremely narrow and collimated near the electrodes, with peak density exceeding that of jets created without pre-ionization. The initial neutral gas distribution prior to plasma breakdown was found to be critical in determining the ultimate jet structure. The visible radius of the dense central jet column was several times narrower than the axial current channel radius, suggesting that the outer portion of the jet must have been force free, with the current parallel to the magnetic field. The studies of non-equilibrium flows and plasma self-organization being carried out at Caltech are relevant to astrophysical jets and fusion energy research.

Observation and interpretation of tectonic strain release mechanisms

In four chapters various aspects of earthquake source are studied.

Chapter I

Surface displacements that followed the Parkfield, 1966, earthquakes were measured for two years with six small-scale geodetic networks straddling the fault trace. The logarithmic rate and the periodic nature of the creep displacement recorded on a strain meter made it possible to predict creep episodes on the San Andreas fault. Some individual earthquakes were related directly to surface displacement, while in general, slow creep and aftershock activity were found to occur independently. The Parkfield earthquake is interpreted as a buried dislocation.

Chapter II

The source parameters of earthquakes between magnitude 1 and 6 were studied using field observations, fault plane solutions, and surface wave and S-wave spectral analysis. The seismic moment, M_O, was found to be related to local magnitude, M_L, by log M_O = 1.7 M_L + 15.1. The source length vs magnitude relation for the San Andreas system found to be: M_L = 1.9 log L - 6.7. The surface wave envelope parameter AR gives the moment according to log M_O = log AR₃₀₀ + 30.1, and the stress drop, τ, was found to be related to the magnitude by τ = 0.54 M - 2.58. The relation between surface wave magnitude M_S and M_L is proposed to be M_S = 1.7 M_L - 4.1. It is proposed to estimate the relative stress level (and possibly the strength) of a source-region by the amplitude ratio of high-frequency to low-frequency waves. An apparent stress map for Southern California is presented.

Chapter III

Seismic triggering and seismic shaking are proposed as two closely related mechanisms of strain release which explain observations of the character of the P wave generated by the Alaskan earthquake of 1964, and distant fault slippage observed after the Borrego Mountain, California earthquake of 1968. The Alaska, 1964, earthquake is shown to be adequately described as a series of individual rupture events. The first of these events had a body wave magnitude of 6.6 and is considered to have initiated or triggered the whole sequence. The propagation velocity of the disturbance is estimated to be 3.5 km/sec. On the basis of circumstantial evidence it is proposed that the Borrego Mountain, 1968, earthquake caused release of tectonic strain along three active faults at distances of 45 to 75 km from the epicenter. It is suggested that this mechanism of strain release is best described as "seismic shaking."

Chapter IV

The changes of apparent stress with depth are studied in the South American deep seismic zone. For shallow earthquakes the apparent stress is 20 bars on the average, the same as for earthquakes in the Aleutians and on Oceanic Ridges. At depths between 50 and 150 km the apparent stresses are relatively high, approximately 380 bars, and around 600 km depth they are again near 20 bars. The seismic efficiency is estimated to be 0.1. This suggests that the true stress is obtained by multiplying the apparent stress by ten. The variation of apparent stress with depth is explained in terms of the hypothesis of ocean floor consumption.

Heterometallic Complexes as Models of Enzymatic Active Sites

This dissertation describes studies on two multinucleating ligand architectures: the first scaffold was designed to support tricopper complexes, while the second platform was developed to support tri- and tetrametallic clusters.

In Chapter 2, the synthesis of yttrium (and lanthanide) complexes supported by a tripodal ligand framework designed to bind three copper centers in close proximity is described. Tricopper complexes were shown to react with dioxygen in a 1:1 [Cu₃]/O₂ stoichiometry to form intermediates in which the O–O bond was fully cleaved, as characterized via UV-Vis spectroscopy and determination of the reaction stoichiometry. Pre-arrangement of the three Cu centers was pivotal to cooperative O₂ activation, as mono-copper complexes reacted differently with dioxgyen. The reactivity of the observed intermediates was studied with various substrates (reductants, O-atom acceptors, H-atom donors, Brønsted acids) to determine their properties. In Chapter 3, the reactivity of the same yttrium-tricopper complex with nitric oxide was explored. Reductive coupling to form a trans-hyponitrite complex (characterized by X-ray crystallography) was observed via cooperative reactivity by an yttrium and a copper center on two distinct tetrametallic units. The hyponitrite complex was observed to release nitrous oxide upon treatment with a Brønsted acid, supporting its viability as an intermediate in nitric oxide reduction to nitrous oxide.

In Chapter 4, a different multinucleating ligand scaffold was employed to synthesize heterometallic triiron clusters containing one oxide and one hydroxide bridges. The effects of the redox-inactive, Lewis acidic heterometals on redox potential was studied by cyclic voltammetry, unveiling a linear correlation between redox potential and heterometal Lewis acidity. Further studies on these complexes showed that the Lewis acidity of the redox-inactive metals also affected the oxygen-atom transfer reactivity of these clusters. Comparisons of this reactivity with manganese systems, collaborative efforts to reassign the structures of related manganese oxo-hydroxo clusters, and synthetic attempts to access related dioxo clusters are also described.

In Appendix A, ongoing efforts to synthesize new clusters supported by the same multinucleating ligand platform are described. Studies of novel approaches towards ligand exchange in tetrametallic clusters and incorporation of new supporting and bridging ligand motifs in trinuclear complexes are presented.