A study of protein targeting reveals insights into mitigating protein aggregation

A unique chloroplast Signal Recognition Particle (SRP) in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll-a/b binding (LHC) proteins. Our study of the thermodynamics and kinetics of the GTPases of the system demonstrates that GTPase complex assembly and activation are highly coupled in the chloroplast GTPases, suggesting they may forego the GTPase activation step as a key regulatory point. This reflects adaptations of the chloroplast SRP to the delivery of their unique substrate protein. Devotion to one highly hydrophobic family of proteins also may have allowed the chloroplast SRP system to evolve an efficient chaperone in the cpSRP43 subunit. To understand the mechanism of disaggregation, we showed that LHC proteins form micellar, disc-shaped aggregates that present a recognition motif (L18) on the aggregate surface. Further molecular genetic and structure-activity analyses reveal that the action of cpSRP43 can be dissected into two steps: (i) initial recognition of L18 on the aggregate surface; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. We also tested the adaptability of cpSRP43 for alternative substrates, specifically in attempts to improve membrane protein expression and inhibition of amyloid beta fibrillization. These preliminary results attest to cpSRP43’s potential as a molecular chaperone and provides the impetus for further engineering endeavors to address problems that stem from protein aggregation.

An economic approach to consumer product ratings

In three essays we examine user-generated product ratings with aggregation. While recommendation systems have been studied extensively, this simple type of recommendation system has been neglected, despite its prevalence in the field. We develop a novel theoretical model of user-generated ratings. This model improves upon previous work in three ways: it considers rational agents and allows them to abstain from rating when rating is costly; it incorporates rating aggregation (such as averaging ratings); and it considers the effect on rating strategies of multiple simultaneous raters. In the first essay we provide a partial characterization of equilibrium behavior. In the second essay we test this theoretical model in laboratory, and in the third we apply established behavioral models to the data generated in the lab. This study provides clues to the prevalence of extreme-valued ratings in field implementations. We show theoretically that in equilibrium, ratings distributions do not represent the value distributions of sincere ratings. Indeed, we show that if rating strategies follow a set of regularity conditions, then in equilibrium the rate at which players participate is increasing in the extremity of agents' valuations of the product. This theoretical prediction is realized in the lab. We also find that human subjects show a disproportionate predilection for sincere rating, and that when they do send insincere ratings, they are almost always in the direction of exaggeration. Both sincere and exaggerated ratings occur with great frequency despite the fact that such rating strategies are not in subjects' best interest. We therefore apply the behavioral concepts of quantal response equilibrium (QRE) and cursed equilibrium (CE) to the experimental data. Together, these theories explain the data significantly better than does a theory of rational, Bayesian behavior -- accurately predicting key comparative statics. However, the theories fail to predict the high rates of sincerity, and it is clear that a better theory is needed.

Sensor networks for geospatial event detection - theory and applications

This thesis presents theories, analyses, and algorithms for detecting and estimating parameters of geospatial events with today's large, noisy sensor networks. A geospatial event is initiated by a significant change in the state of points in a region in a 3-D space over an interval of time. After the event is initiated it may change the state of points over larger regions and longer periods of time. Networked sensing is a typical approach for geospatial event detection. In contrast to traditional sensor networks comprised of a small number of high quality (and expensive) sensors, trends in personal computing devices and consumer electronics have made it possible to build large, dense networks at a low cost. The changes in sensor capability, network composition, and system constraints call for new models and algorithms suited to the opportunities and challenges of the new generation of sensor networks. This thesis offers a single unifying model and a Bayesian framework for analyzing different types of geospatial events in such noisy sensor networks. It presents algorithms and theories for estimating the speed and accuracy of detecting geospatial events as a function of parameters from both the underlying geospatial system and the sensor network. Furthermore, the thesis addresses network scalability issues by presenting rigorous scalable algorithms for data aggregation for detection. These studies provide insights to the design of networked sensing systems for detecting geospatial events. In addition to providing an overarching framework, this thesis presents theories and experimental results for two very different geospatial problems: detecting earthquakes and hazardous radiation. The general framework is applied to these specific problems, and predictions based on the theories are validated against measurements of systems in the laboratory and in the field.

Intramolecular conflict : conformation and self-assembly of architecturally complex macromolecules in solution

The solution behavior of linear polymer chains is well understood, having been the subject of intense study throughout the previous century. As plastics have become ubiquitous in everyday life, polymer science has grown into a major field of study. The conformation of a polymer in solution depends on the molecular architecture and its interactions with the surroundings. Developments in synthetic techniques have led to the creation of precision-tailored polymeric materials with varied topologies and functionalities. In order to design materials with the desired properties, it is imperative to understand the relationships between polymer architecture and their conformation and behavior. To meet that need, this thesis investigates the conformation and self-assembly of three architecturally complex macromolecular systems with rich and varied behaviors driven by the resolution of intramolecular conflicts. First we describe the development of a robust and facile synthetic approach to reproducible bottlebrush polymers (Chapter 2). The method was used to produce homologous series of bottlebrush polymers with polynorbornene backbones, which revealed the effect of side-chain and backbone length on the overall conformation in both good and theta solvent conditions (Chapter 3). The side-chain conformation was obtained from a series of SANS experiments and determined to be indistinguishable from the behavior of free linear polymer chains. Using deuterium-labeled bottlebrushes, we were able for the first time to directly observe the backbone conformation of a bottlebrush polymer which showed self-avoiding walk behavior. Secondly, a series of SANS experiments was conducted on a homologous series of Side Group Liquid Crystalline Polymers (SGLCPs) in a perdeuterated small molecule liquid crystal (5CB). Monodomain, aligned, dilute samples of SGLCP-b-PS block copolymers were seen to self-assemble into complex micellar structures with mutually orthogonally oriented anisotropies at different length scales (Chapter 4). Finally, we present the results from the first scattering experiments on a set of fuel-soluble, associating telechelic polymers. We observed the formation of supramolecular aggregates in dilute (≤0.5wt%) solutions of telechelic polymers and determined that the choice of solvent has a significant effect on the strength of association and the size of the supramolecules (Chapter 5). A method was developed for the direct estimation of supramolecular aggregation number from SANS data. The insight into structure-property relationships obtained from this work will enable the more targeted development of these molecular architectures for their respective applications.

Geochemistry and resonance ionization of platinum-group elements

Experimental studies were conducted with the goals of 1) determining the origin of Pt- group element (PGE) alloys and associated mineral assemblages in refractory inclusions from meteorites and 2) developing a new ultrasensitive method for the in situ chemical and isotopic analysis of PGE. A general review of the geochemistry and cosmochemistry of the PGE is given, and specific research contributions are presented within the context of this broad framework.

An important step toward understanding the cosmochemistry of the PGE is the determination of the origin of POE-rich metallic phases (most commonly εRu-Fe) that are found in Ca, AJ-rich refractory inclusions (CAI) in C3V meteorites. These metals occur along with γNi-Fe metals, Ni-Fe sulfides and Fe oxides in multiphase opaque assemblages. Laboratory experiments were used to show that the mineral assemblages and textures observed in opaque assemblages could be produced by sulfidation and oxidation of once homogeneous Ni-Fe-PGE metals. Phase equilibria, partitioning and diffusion kinetics were studied in the Ni-Fe-Ru system in order to quantify the conditions of opaque assemblage formation. Phase boundaries and tie lines in the Ni-Fe-Ru system were determined at 1273, 1073 and 873K using an experimental technique that allowed the investigation of a large portion of the Ni-Fe-Ru system with a single experiment at each temperature by establishing a concentration gradient within which local equilibrium between coexisting phases was maintained. A wide miscibility gap was found to be present at each temperature, separating a hexagonal close-packed εRu-Fe phase from a face-centered cubic γNi-Fe phase. Phase equilibria determined here for the Ni-Fe-Ru system, and phase equilibria from the literature for the Ni-Fe-S and Ni-Fe-O systems, were compared with analyses of minerals from opaque assemblages to estimate the temperature and chemical conditions of opaque assemblage formation. It was determined that opaque assemblages equilibrated at a temperature of ~770K, a sulfur fugacity 10 times higher than an equilibrium solar gas, and an oxygen fugacity 10⁶ times higher than an equilibrium solar gas.

Diffusion rates between -γNi-Fe and εRu-Fe metal play a critical role in determining the time (with respect to CAI petrogenesis) and duration of the opaque assemblage equilibration process. The diffusion coefficient for Ru in Ni (D^Ru_Ni) was determined as an analog for the Ni-Fe-Ru system by the thin-film diffusion method in the temperature range of 1073 to 1673K and is given by the expression:

D^Ru_Ni (cm² sec^-1) = 5.0(±0.7) x 10^-3 exp(-2.3(±0.1) x 10¹² erg mole^-1/RT) where R is the gas constant and T is the temperature in K. Based on the rates of dissolution and exsolution of metallic phases in the Ni-Fe-Ru system it is suggested that opaque assemblages equilibrated after the melting and crystallization of host CAI during a metamorphic event of ≥ 10³ years duration. It is inferred that opaque assemblages originated as immiscible metallic liquid droplets in the CAI silicate liquid. The bulk compositions of PGE in these precursor alloys reflects an early stage of condensation from the solar nebula and the partitioning of V between the precursor alloys and CAI silicate liquid reflects the reducing nebular conditions under which CAI were melted. The individual mineral phases now observed in opaque assemblages do not preserve an independent history prior to CAI melting and crystallization, but instead provide important information on the post-accretionary history of C3V meteorites and allow the quantification of the temperature, sulfur fugacity and oxygen fugacity of cooling planetary environments. This contrasts with previous models that called upon the formation of opaque assemblages by aggregation of phases that formed independently under highly variable conditions in the solar nebula prior to the crystallization of CAI.

Analytical studies were carried out on PGE-rich phases from meteorites and the products of synthetic experiments using traditional electron microprobe x-ray analytical techniques. The concentrations of PGE in common minerals from meteorites and terrestrial rocks are far below the ~100 ppm detection limit of the electron microprobe. This has limited the scope of analytical studies to the very few cases where PGE are unusually enriched. To study the distribution of PGE in common minerals will require an in situ analytical technique with much lower detection limits than any methods currently in use. To overcome this limitation, resonance ionization of sputtered atoms was investigated for use as an ultrasensitive in situ analytical technique for the analysis of PGE. The mass spectrometric analysis of Os and Re was investigated using a pulsed primary Ar⁺ ion beam to provide sputtered atoms for resonance ionization mass spectrometry. An ionization scheme for Os that utilizes three resonant energy levels (including an autoionizing energy level) was investigated and found to have superior sensitivity and selectivity compared to nonresonant and one and two energy level resonant ionization schemes. An elemental selectivity for Os over Re of ≥ 10³ was demonstrated. It was found that detuning the ionizing laser from the autoionizing energy level to an arbitrary region in the ionization continuum resulted in a five-fold decrease in signal intensity and a ten-fold decrease in elemental selectivity. Osmium concentrations in synthetic metals and iron meteorites were measured to demonstrate the analytical capabilities of the technique. A linear correlation between Os⁺ signal intensity and the known Os concentration was observed over a range of nearly 10⁴ in Os concentration with an accuracy of ~ ±10%, a millimum detection limit of 7 parts per billion atomic, and a useful yield of 1%. Resonance ionization of sputtered atoms samples the dominant neutral-fraction of sputtered atoms and utilizes multiphoton resonance ionization to achieve high sensitivity and to eliminate atomic and molecular interferences. Matrix effects should be small compared to secondary ion mass spectrometry because ionization occurs in the gas phase and is largely independent of the physical properties of the matrix material. Resonance ionization of sputtered atoms can be applied to in situ chemical analysis of most high ionization potential elements (including all of the PGE) in a wide range of natural and synthetic materials. The high useful yield and elemental selectivity of this method should eventually allow the in situ measurement of Os isotope ratios in some natural samples and in sample extracts enriched in PGE by fire assay fusion.

Phase equilibria and diffusion experiments have provided the basis for a reinterpretation of the origin of opaque assemblages in CAI and have yielded quantitative information on conditions in the primitive solar nebula and cooling planetary environments. Development of the method of resonance ionization of sputtered atoms for the analysis of Os has shown that this technique has wide applications in geochemistry and will for the first time allow in situ studies of the distribution of PGE at the low concentration levels at which they occur in common minerals.

How resources control aggression in Drosophila

How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner in which food and females promotes aggression.

In the first chapter, we explore how food controls aggression. As in many other species, food promotes aggression in flies, but it is not clear whether food increases aggression per se, or whether aggression is a secondary consequence of increased social interactions caused by aggregation of flies on food. Furthermore, nothing is known about how animals evaluate the quality and quantity of food in the context of competition. We show that food promotes aggression independently of any effect to increase the frequency of contact between males. Food increases aggression but not courtship between males, suggesting that the effect of food on aggression is specific. Next, we show that flies tune the level of aggression according to absolute amount of food rather than other parameters, such as area or concentration of food. Sucrose, a sugar molecule present in many fruits, is sufficient to promote aggression, and detection of sugar via gustatory receptor neurons is necessary for food-promoted aggression. Furthermore, we show that while food is necessary for aggression, too much food decreases aggression. Finally, we show that flies exhibit strategies consistent with a territorial strategy. These data suggest that flies use sweet-sensing gustatory information to guide their decision to fight over a limited quantity of a food resource.

Following up on the findings of the first chapter, we asked how the presence of a conspecific female resource promotes male-male aggression. In the absence of food, group-housed male flies, who normally do not fight even in the presence of food, fight in the presence of females. Unlike food, the presence of females strongly influences proximity between flies. Nevertheless, as group-housed flies do not fight even when they are in small chambers, it is unlikely that the presence of female indirectly increases aggression by first increasing proximity. Unlike food, the presence of females also leads to large increases in locomotion and in male-female courtship behaviors, suggesting that females may influence aggression as well as general arousal. Female cuticular hydrocarbons are required for this effect, as females that do not produce CH pheromones are unable to promote male-male aggression. In particular, 7,11-HD––a female-specific cuticular hydrocarbon pheromone critical for male-female courtship––is sufficient to mediate this effect when it is perfumed onto pheromone-deficient females or males. Recent studies showed that ppk23⁺ GRNs label two population of GRNs, one of which detects male cuticular hydrocarbons and another labeled by ppk23 and ppk25, which detects female cuticular hydrocarbons. I show that in particular, both of these GRNs control aggression, presumably via detection of female or male pheromones. To further investigate the ways in which these two classes of GRNs control aggression, I developed new genetic tools to independently test the male- and female-sensing GRNs. I show that ppk25-LexA and ppk25-GAL80 faithfully recapitulate the expression pattern of ppk25-GAL4 and label a subset of ppk23⁺ GRNs. These tools can be used in future studies to dissect the respective functions of male-sensing and female-sensing GRNs in male social behaviors.

Finally, in the last chapter, I discuss quantitative approaches to describe how varying quantities of food and females could control the level of aggression. Flies show an inverse-U shaped aggressive response to varying quantities of food and a flat aggressive response to varying quantities of females. I show how two simple game theoretic models, “prisoner’s dilemma” and “coordination game” could be used to describe the level of aggression we observe. These results suggest that flies may use strategic decision-making, using simple comparisons of costs and benefits.

In conclusion, male-male aggression in Drosophila is controlled by simple gustatory cues from food and females, which are detected by gustatory receptor neurons. Different quantities of resource cues lead to different levels of aggression, and flies show putative territorial behavior, suggesting that fly aggression is a highly strategic adaptive behavior. How these resource cues are integrated with male pheromone cues and give rise to this complex behavior is an interesting subject, which should keep researchers busy in the coming years.

Part I. Electric birefringence studies of deoxyribonucleic acids. Part II. Selective dissociation of nucleohistone complexes

Part I

The electric birefringence of dilute DNA solutions has been studied in considerable detail and on a large number of samples, but no new and reliable information was discovered concerning the tertiary structure of DNA. The large number of variables which effect the birefringence results is discussed and suggestions are made for further work on the subject.

The DNA molecules have been aligned in a rapidly alternating (10 to 20 kc/sec) square wave field confirming that the orientation mechanism is that of counterion polarization. A simple empirical relation between the steady state birefringence, Δn_st, and the square of the electric field, E, has been found: Δn_st = E²/(a E² + b), where a = 1/Δn_s and b = (E²/Δn_st)_E→o. Δn_s is the birefringence extrapolated to infinite field strength.

The molecules show a distribution of relaxation times from 10^-4 to 0.2 sec, which is consistent with expectations for flexible coil molecules. The birefringence and the relaxation times decrease with increasing salt concentrations. They also depend on the field strength and pulse duration in a rather non-reproducible manner, which may be due in part to changes in the composition of the solution or in the molecular structure of the DNA (other than denaturation). Further progress depends on the development of some control over these effects.

Part II

The specificity of the dissociation of reconstituted and native deoxyribonucleohistones (DNH) by monovalent salt solutions has been investigated. A novel zone ultracentrifugation method is used in which the DNH is sedimented as a zone through a preformed salt gradient, superimposed on a stabilizing D₂O (sucrose) density gradient. The results, obtained by scanning the quartz sedimentation tubes in a spectrophotometer, were verified by the conventional, preparative sedimentation technique. Procedures are discussed for the detection of microgram quantities of histones, since low concentrations must be used to prevent excessive aggregation of the DNH.

The data show that major histone fractions are selectively dissociated from DNH by increasing salt concentrations: Lysine rich histone (H I) dissociates gradually between 0.1 and 0.3 F, slightly lysine rich histone (H II) dissociates as a narrow band between 0.35 and 0.5 F, and arginine rich histone (H III, H IV) dissociates gradually above 0.5 F NaClO₄.

The activity of the partially dissociated, native DNH in sustaining RNA synthesis, their mobility and their unusual heat denaturation and renaturation behavior are described. The two-step melting behavior of the material indicates that the histones are non-randomly distributed along the DNA, but the implications are that the uncovered regions are not of gene-size length.

Biochemical and Biophysical Characterization of Huntingtin

Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disease. HD has no cure, and patients pass away 10-20 years after the onset of symptoms. The causal mutation for HD is a trinucleotide repeat expansion in exon 1 of the huntingtin gene that leads to a polyglutamine (polyQ) repeat expansion in the N-terminal region of the huntingtin protein. Interestingly, there is a threshold of 37 polyQ repeats under which little or no disease exists; and above which, patients invariably show symptoms of HD. The huntingtin protein is a 350 kDa protein with unclear function. As the polyQ stretch expands, its propensity to aggregate increases with polyQ length. Models for polyQ toxicity include formation of aggregates that recruit and sequester essential cellular proteins, or altered function producing improper interactions between mutant huntingtin and other proteins. In both models, soluble expanded polyQ may be an intermediate state that can be targeted by potential therapeutics.

In the first study described herein, the conformation of soluble, expanded polyQ was determined to be linear and extended using equilibrium gel filtration and small-angle X-ray scattering. While attempts to purify and crystallize domains of the huntingtin protein were unsuccessful, the aggregation of huntingtin exon 1 was investigated using other biochemical techniques including dynamic light scattering, turbidity analysis, Congo red staining, and thioflavin T fluorescence. Chapter 4 describes crystallization experiments sent to the International Space Station and determination of the X-ray crystal structure of the anti-polyQ Fab MW1. In the final study, multimeric fibronectin type III (FN3) domain proteins were engineered to bind with high avidity to expanded polyQ tracts in mutant huntingtin exon 1. Surface plasmon resonance was used to observe binding of monomeric and multimeric FN3 proteins with huntingtin.

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.

Structural Design under Seismic Risk Using Multiple Performance Objectives

Structural design is a decision-making process in which a wide spectrum of requirements, expectations, and concerns needs to be properly addressed. Engineering design criteria are considered together with societal and client preferences, and most of these design objectives are affected by the uncertainties surrounding a design. Therefore, realistic design frameworks must be able to handle multiple performance objectives and incorporate uncertainties from numerous sources into the process.

In this study, a multi-criteria based design framework for structural design under seismic risk is explored. The emphasis is on reliability-based performance objectives and their interaction with economic objectives. The framework has analysis, evaluation, and revision stages. In the probabilistic response analysis, seismic loading uncertainties as well as modeling uncertainties are incorporated. For evaluation, two approaches are suggested: one based on preference aggregation and the other based on socio-economics. Both implementations of the general framework are illustrated with simple but informative design examples to explore the basic features of the framework.

The first approach uses concepts similar to those found in multi-criteria decision theory, and directly combines reliability-based objectives with others. This approach is implemented in a single-stage design procedure. In the socio-economics based approach, a two-stage design procedure is recommended in which societal preferences are treated through reliability-based engineering performance measures, but emphasis is also given to economic objectives because these are especially important to the structural designer's client. A rational net asset value formulation including losses from uncertain future earthquakes is used to assess the economic performance of a design. A recently developed assembly-based vulnerability analysis is incorporated into the loss estimation.

The presented performance-based design framework allows investigation of various design issues and their impact on a structural design. It is a flexible one that readily allows incorporation of new methods and concepts in seismic hazard specification, structural analysis, and loss estimation.